Research Report 2012 | 2013

Transcription

Leibniz Institute
for Natural Product Research and Infection Biology
Hans Knöll Institute
Research Report 2012 | 2013
HKI Research Report 2012/2013
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As in the previous years, the Leibniz Institute for Natural Product Research and Infection Biology – Hans Knöll Institute (HKI)
continuously developed, networked and expanded further in the years 2012 and 2013.
In the previous years, the number of staff and
research groups increased to such an extent
that we had to look for more office and laboratory space. Our new research building that
we opened up in 2012, offers not only sufficient space but also features state-of-the art
technology in stunning architecture. Since
2012, the team of the “Centre for Innovation Competence (ZIK) Septomics” has been
performing their research in a new research
building adding distinction to the profile of
the Beutenberg Campus.
In the years 2012 and 2013 we were pleased
to welcome two new associated research
groups originating from the Jena University
Hospital. These include the working group
“Infections in haematology / oncology” managed by Prof. Marie von Lilienfeld-Toal that
studies infectious diseases on patients suffering from malign neoplasia. The working
group “Network modelling”, headed by Prof.
Rainer König, investigates different clinical
progressions of infections on a molecular
level by means of network analyses.
In addition to that, we were proudly able to
win two young scientists from the Harvard
Medical School in Boston as the heads of junior research groups. Dr. Christine Beemelmanns and her group “Chemical biology of
microbe – host interactions” researches into
the interconnectivities between bacteria,
fungi and insects together with the involved
natural products. Finding out about the communication between bacteria and other organisms through chemical signal molecules
I N T R O D U C T I O N | VO RWO R T
Auch in den Jahren 2012 und 2013 hat sich
das Leibniz-Institut für Naturstoff-Forschung
und Infektionsbiologie – Hans-Knöll-Institut
(HKI) kontinuierlich weiter entwickelt, vernetzt und vergrößert. In den vergangenen
Jahren ist die Zahl der Mitarbeiter und Forschungsgruppen gar so weit angestiegen,
dass wir uns eine neue Bleibe suchen mussten. In unserem 2012 eingeweihten, neuen
Forschungsgebäude haben wir nicht nur ausreichend Raum, sondern auch optimal ausgestattete Arbeitsplätze verpackt in reizvoller
Architektur gefunden. Das Team des „Zentrums für Innovationskompetenz Septomics“
forscht seit 2012 ebenso in einem neuen
Forschungsbau, der das Gesicht des Beutenberg-Campus mitprägt.
In den Jahren 2012 und 2013 durften wir zwei
neue assoziierte Forschungsgruppen vom Universitätsklinikum Jena bei uns begrüßen: Die
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HKI Research Report 2012 / 2013
Arbeitsgruppe „Infektionen in der Hämatologie / Onkologie“ unter der Leitung von Prof.
Marie von Lilienfeld-Toal beschäftigt sich mit
der Erforschung von Infektionskrankheiten
bei Patienten mit malignen Neoplasien. Prof.
Rainer Königs Gruppe „Netzwerkmodellierung“ klärt unter Zuhilfenahme von Netzwerken verschiedene klinische Verläufe von
Infektionen auf molekularer Ebene auf.
Des Weiteren konnten wir zwei junge Wissenschaftler von der Harvard Medical School
in Boston als Nachwuchsgruppenleiter gewinnen: Dr. Christine Beemelmanns forscht
mit ihrer Gruppe „Chemische Biologie der Mikroben-Wirt Interaktionen“ an den Wechselwirkungen zwischen Bakterien, Pilzen und
Insekten und den daran beteiligten Naturstoffen. Wie Bakterien und andere Organismen durch chemische Signalmoleküle miteinander kommunizieren, möchte Dr. Pierre
is the focus of Dr. Pierre Stallforth’s group
“Chemistry of microbial communication”. PD
Ilse Jacobsen, PhD has now started to head
an independent research group – “Microbial
Immunology”.
What is becoming noticeably more important for us is the information exchange with
colleagues across Germany and also with
representatives from other disciplines. Within the scope of the Collaborative Research
Center / Transregio 124 “Pathogenic fungi
and their human hosts: networks of interaction – FungiNet”, which is funded by the
Deutsche Forschungsgemeinschaft, scientists
from Jena and Würzburg work together to understand the complex mechanisms of fungal
infections in order to develop a virtual infection model. We expand our horizons by means
of the “InfectoGnostics research campus
Jena”, which is funded by the Federal Minis-
Stallforth mit seiner Gruppe „Chemie Mikrobieller Kommunikation“ herausfinden. Eine
eigene Forschungsgruppe leitet nun PD Ilse
Jacobsen, PhD, – „Mikrobielle Immunologie“.
Zusehends wichtiger wird für uns der Austausch mit Kollegen in ganz Deutschland und
Vertretern anderer Disziplinen. Im Sonderforschungsbereich / Transregio 124 „Pathogene Pilze und ihr menschlicher Wirt: Netzwerke der Interaktion – FungiNet“, gefördert
von der Deutschen Forschungsgemeinschaft,
arbeiten Wissenschaftler aus Jena und Würzburg daran, die komplexen Mechanismen
von Pilzinfektionen zu verstehen und ein
virtuelles Infektionsmodell zu entwickeln.
Über den Tellerrand der Disziplingrenzen
hinaus blicken wir im „InfectoGnostics Forschungscampus Jena“, der vom Bundesministerium für Bildung und Forschung unterstützt wird. Gemeinsam mit Partnern aus
try for Education and Research. Together with
partners from science, medicine and industry
we develop point-of-care tests for acute infections. We even proceed one step further in the
project “InfectControl 2020 – New Anti-Infective Strategies – Science • Society • Economy”,
which is also funded by the Federal Ministry
for Education and Research. Especially East
German research institutions, companies
and interest groups from several disciplines
work together in this medically and socially
relevant enterprise aiming at preventing and
fighting infectious diseases effectively in the
future.
Thanks to the extensive global networking of
our scientists, we were able to host internationally renowned conferences and meetings
in 2012 and 2013 that helped us to foster relations with scientists from all over the world.
Prof. Bernhard Hube hosted the ISHAM-con-
Wissenschaft, Medizin und Wirtschaft entwickeln wir Vor-Ort-Analysen akuter Infektionen. Noch einen Schritt weiter gehen wir
mit „InfectControl 2020 – Neue Antiinfektionsstrategien – Wissenschaft – Gesellschaft –
Wirtschaft“, das ebenfalls vom Bundesministerium für Bildung und Forschung gefördert
wird. Größtenteils ostdeutsche Forschungsinstitute, Unternehmen und Interessengruppen
verschiedenster Disziplinen arbeiten hier an
dem medizinisch und gesellschaftlich relevanten Vorhaben, Infektionskrankheiten in
Zukunft umfassend zu vermeiden und effektiv zu bekämpfen.
Dank der guten weltweiten Vernetzung unserer Wissenschaftler, konnten wir auch
2012 und 2013 international renommierte
Kongresse und Tagungen ausrichten sowie
Wissenschaftler aus aller Welt bei uns begrüßen. Prof. Bernhard Hube richtete den
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ference and provided an insight into the work
of our institute to young academics within
the scope of the FEBS Advanced Course. Prof.
Peter F. Zipfel co-operated in organising the
specialist conference “14th European Meeting on Complement in Human Disease”.
Since exceptional research results can only
be achieved in a team that enjoys their working environment, it is in our utmost interest
to promote gender equality between men and
women as well as balancing work and family
life. We proudly signed the „Charta der Vielfalt“ (“Charta of Diversity”) and we are a partner of the „Jenaer Bündnis für Familie“ (“Jena
Association for Families”). This commitment
was honoured by the „Total E-Quality“.
ISHAM-Kongress aus und bot innerhalb des
FEBS Advanced Courses Nachwuchswissenschaftlern Einblicke in die Arbeit unseres
Instituts. Prof. Peter F. Zipfel organisierte
die Fachtagung „14th European Meeting on
Complement in Human Disease“ mit.
Da herausragende Forschungsergebnisse nur
mit einem Team erreicht werden können, das
seinen Arbeitsplatz als angenehm empfindet, setzen wir uns verstärkt für die Gleichstellung von Frauen und Männern sowie die
Vereinbarkeit von Familie und Beruf ein. So
unterzeichneten wir die „Charta der Vielfalt“
und sind Teil des „Jenaer Bündnisses für Familie". Quittiert wurde dieses Engagement
mit dem Prädikat „Total E-Quality“ vom Bundesministerium für Familie, Senioren, Frauen und Jugend.
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I would like to thank all colleagues at the
HKI for their excellent cooperation. It is
this outstanding collaboration that makes
it possible to maintain and further develop
the high quality of the scientific work at
the HKI. I would like to express my special
thanks to Dr. Michael Ramm and Dr. Christine Vogler for their contribution to this Report 2012/2013.
Jena, June 2014
Axel Brakhage
Director of the HKI
Für die ausgezeichnete Kooperation möchte
ich mich bei allen Kolleginnen und Kollegen
des HKI sehr herzlich bedanken. Nur durch
die hervorragende Zusammenarbeit ist es
möglich, die hohe Qualität der wissenschaftlichen Leistung des HKI zu halten und weiter zu steigern. Für die Mitarbeit an diesem
Report 2012/13 danke ich allen Mitarbeitern
des HKI, insbesondere Dr. Michael Ramm
und Dr. Christine Vogler.
Jena, June 2014
Axel Brakhage
Director of the HKI
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Contents | Inhalt
2
Introduction | Vorwort
6
Contents | Inhalt
8
Organization of the HKI | Organisation des HKI
Departments | Abteilungen
11
Biomolecular Chemistry | Biomolekulare Chemie
27
Infection Biology | Infektionsbiologie
43
Microbial Pathogenicity Mechanisms | Mikrobielle Pathogenitätsmechanismen
61
Molecular and Applied Microbiology | Molekulare und Angewandte Mikrobiologie
79
Cell and Molecular Biology | Zell-und Molekularbiologie
Research Groups | Forschungsgruppen
95
Applied Systems Biology | Angewandte Systembiologie
105
Fungal Septomics | Fungal Septomics
115
Microbial Immunology | Mikrobielle Immunologie
125
Systems Biology and Bioinformatics | Systembiologie und Bioinformatik
Independent Junior Research Group | Unabhängige Nachwuchsgruppe
133
Secondary Metabolism of Predatory Bacteria | Sekundärmetabolismus räuberischer Bakterien
Cross-sectional Units | Querschnittseinrichtungen
143
Bio Pilot Plant | Biotechnikum
157
ILRS – International Leibniz Research School
165
JSMC – Jena School for Microbial Communication
169
JMRC – Jena Microbial Resource Collection
177
CRC/Transregio FungiNet | SFB/TR FungiNet
Associated Groups | Assoziierte Gruppen
181
Infections in Hematology / Oncology | Infektionen in der Hämatologie / Onkologie
189
Microbial Biochemistry and Physiology | Mikrobielle Biochemie und Physiologie
199
Pharmaceutical Microbiology | Pharmazeutische Mikrobiologie
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Appendix | Anhang
208
Peer Reviewed Articles 2012 | Originalarbeiten 2012
213
Peer Reviewed Articles 2013 | Originalarbeiten 2013
218
Reviews, Monographs, Book chapters 2012/2013 | Übersichtsarbeiten, Monographien, Sammelwerke
220
Memberships in Editorial Boards 2012/2013 | Mitgliedschaften in Editorial Boards
220
Lectures at the HKI 2012/2013 | Kolloquien am HKI
221
Scientific Awards 2012/2013 | Preise und Auszeichnungen
222
Meetings, Workshops, Symposia 2012/2013 | Wissenschaftliche Veranstaltungen
222
Participation in Research Networks 2012/2013 | Beteiligung an Netzwerken und Verbundprojekten
224
Calls for Appointments 2012/2013 | Rufe
224
Postdoctoral Lecture Qualifications 2012/2013 | Habilitationen
224
Graduations 2012/2013 | Promotionen
225
Bachelor / Master / Diploma Theses 2012/2013 | Bachelor- / Master- / Diplomarbeiten
228
Inventions and Patents 2012/2013 | Erfindungen und Schutzrechte
229
External Funding 2012/2013 | Drittmittel
230
Maps | Lagepläne
7
Organization of the HKI
Board of Trustees | Kuratorium
Dr. W. Eberbach
Thuringian Ministry of Education, Science and Culture (Head)
N. Graf zu Stolberg
Medac GmbH, Wedel (Deputy Head)
Prof. Dr. G. Diekert
Friedrich Schiller University Jena
Prof. Dr. F. Laplace
Federal Ministry of Education and Research, Berlin
Prof. Dr. T. Heinzel
Vice-Rector of Friedrich Schiller University Jena
Prof. Dr. W. Rosenthal
Max Delbrück Center for Molecular Medicine Berlin-Buch
Dr. T. Maier
Wacker Biotech Jena GmbH
Scientific Council | Wissenschaftlicher Beirat
Prof. Dr. W. Rosenthal
Max Delbrück Center for Molecular Medicine Berlin-Buch (Head)
Prof. Dr. G. Diekert
Friedrich Schiller University Jena (Deputy Head)
Prof. Dr. E. Dittmann
University Potsdam
Prof. Dr. B. Fleischer
Bernhard Nocht Institute for Tropical Medicine, Hamburg
Prof. Dr. H. Haas
Innsbruck Medical University
Prof. Dr. J. Hacker
German National Academy of Sciences Leopoldina, Halle (Saale)
Prof. Dr. P. Neubauer
Technical University Berlin
Prof. Dr. G. Pohnert
Prof. Dr. J. Selbig
University Potsdam
Executive Board | Vorstand
Prof. Dr. Axel A. Brakhage Scientific director
Elke Jäcksch
Administrative director
Scientific Coordination | Wissenschaftliche Koordination
Dr. Michael Ramm
Scientific coordination
Dr. Christine Vogler
Scientific coordination
Angelika Rauchmaul
Assistant of the scientific director
Administration | Verwaltung
Elke Jäcksch
Administrative director
Renate Becher
Sandra Ertel
Julia Große (since 07/2013)
Fabian Grunert (since 08/2013)
Nils Haußner (since 08/2013)
Eckhard Hemme
Carmen Henze
Michael Hetz
Michael Kind
Britta Kammer-Rattey
Jan Kösling-Wettstaedt
Ilona Lux
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Andrea Matthies
Frank Schebitz
Reinald Schorcht
Michael Schunk
Christine Serfling
Kerstin Siegmund
Ekkehard Tittelbach
Jens Trotzer (since 05/2013)
Scientific Advisory Board | Board of Trustees | General Meeting
Administration
Elke Jäcksch
Scientific Coordination
Public Relations
Dr. Michael Ramm
Departments
Biomolecular Chemistry
Prof. Christian Hertweck
Executive Board
Prof. Axel Brakhage
Elke Jäcksch
Research Groups
Independent
Junior Research Groups
Applied Systems Biology
Prof. Marc Thilo Figge
Chemistry of Microbial
Communication
Dr. Pierre Stallforth
Ombudsman
Prof. Reinhard Guthke
Equal Opportunities
Graduate Schools
Cross Sectional Units
Associated Groups
Bio Pilot Plant
Dr. Uwe Horn
Infections in
Hematology / Oncology
Prof. Marie von Lilienfeld-Toal
(UKJ)
(since 12/2013)
Infection Biology
Prof. Peter F. Zipfel
Fungal Septomics
Prof. Oliver Kurzai
(since 04/2013)
Chemical Biology of
Microbe-Host Interactions
Dr. Christine Beemelmanns
International Leibniz
Research School
Prof. Peter F. Zipfel
Microbial Biochemistry
and Physiology
Dr. habil. Matthias Brock
(FSU)
Jena Microbial
Resource Collection
PD Dr. Kerstin Voigt
Network Modeling
Prof. Rainer König
(UKJ)
(since 12/2013)
Microbial Pathogenicity
Mechanisms
Prof. Bernhard Hube
Microbial Immunology
PD Ilse Jacobsen, PhD
Molecular and Applied
Microbiology
Prof. Axel Brakhage
Systems Biology and
Bioinformatics
Prof. Reinhard Guthke
(since 08/2013)
Secondary Metabolism
of Predatory Bacteria
Dr. habil. Markus Nett
National Reference
Center for Invasive Fungal
Infections
Prof. Oliver Kurzai
(since 01/2014)
Cell and Molecular Biology
Prof. Hans Peter Saluz
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Pharmaceutical
Microbiology
Prof. Dirk Hoffmeister
(FSU)
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Department of Biomolecular Chemistry
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Department of Biomolecular Chemistry
The Department of Biomolecular Chemistry, headed by Christian Hertweck, focuses
on various aspects of microbial secondary
metabolism. The group covers the chemical
and biochemical expertise in modern natural product research (structure elucidation,
biosynthesis, enzymology, synthesis, interactions through biomolecules). In addition
to the basic research projects, the department operates the institute’s state-of-the-art
analytical facilities (NMR, MS-techniques,
MALDI-TOF) and supports other HKI departments and FSU groups with metabolite and
proteome analyses.
The department is specialised in the genomebased isolation and structural elucidation of
natural products. This work is funded by the
Pakt für Forschung und Innovation, the DFG
and BMBF, and supported by various collaborations with industrial partners. For the
discovery of new potential therapeutics we
investigate primarily neglected microorganisms (endophytes, symbionts, little explored
microorganisms) and apply genome-mining
strategies (activation of silent gene clusters).
A major research area of the department is
the investigation of biosynthetic pathways
by a combination of chemical and biological methods. In nature, structural and functional diversity is governed by multienzyme
processing lines that lead to highly complex compounds. To gain insights into the
I N T R O D U C T I O N | E I N LE I T U N G
Die Abteilung Biomolekulare Chemie befasst
sich unter der Leitung von Prof. Christian
Hertweck mit der Chemie und Biologie des
facettenreichen Sekundärmetabolismus von
Mikroorganismen – Bakterien und Pilzen.
Die Abteilung integriert die chemischen und
biochemischen Arbeitsgebiete der modernen Naturstoff-Forschung integriert (Strukturaufklärung, Biosynthese, Enzymmechanismen, Synthese und Naturstoff-basierte
Interaktionen). Die für das Institut essenzielle Hochleistungsanalytik (NMR, MS,
MALDI-TOF) wird in der Abteilung Biomolekulare Chemie betrieben. Dort ist ein breites
Methodenspektrum zur Strukturaufklärung von niedermolekularen Verbindungen
und zur Proteomanalyse verfügbar, durch
das insbesondere die HKI-Abteilungen und
FSU-Arbeitsgruppen unterstützt werden.
Head
Prof. Dr. Christian Hertweck
Die Abteilung Biomolekulare Chemie ist auf
die Genom-basierte Isolierung und Struk-
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turaufklärung von Naturstoffen spezialisiert. Diese Arbeiten werden vom Pakt für
Forschung und Innovation, der DFG und dem
BMBF gefördert und in zahlreichen Industriekooperationen durchgeführt.
Zur Suche nach neuen Naturstoffen werden
vorwiegend neue Organismentypen (Endophythen, Symbionten, wenig untersuchte
Mikroorganismen) verwendet und genome
mining-Strategien eingesetzt. Ein Schwerpunkt der Abteilung Biomolekulare Chemie
liegt in der Aufklärung von mikrobiellen
Biosynthesewegen über biologische und
chemische Methoden. Die strukturelle und
funktionelle Diversität entsteht in der Natur über Multi-Enzym-Prozesslinien, die zu
hochkomplexen Verbindungen führen. Um
die Mechanismen und die Evolution der Synthesemaschinerien biologischer Systeme zu
verstehen, studiert die Arbeitsgruppe die
Biosynthese-Gencluster und ausgewählte
HKI Research Report 2012 / 2013 Department of Biomolecular Chemistry
mechanisms and the evolution of the synthetic machineries the group investigates
the functions of biosynthesis gene clusters
and selected intriguing enzymes. For this
purpose, we also do structural studies in
collaboration. The majority of our projects is
conducted in national and international scientific networks. In addition to understanding natural evolution of metabolic diversity
we aim at harnessing the biosynthetic potential to produce novel natural product derivatives. In particular, biosynthetic pathways
of pharmacologically relevant polyketides
are investigated. Modern organic synthetic
methods complement the microbes’ biosynthetic capabilities.
Natural products represent mediators of biological communication, and their specific
functions have developed during evolution.
They play a key role in the interaction of
microorganisms, in symbiosis and also in
pathogenesis, e.g. in fungal infections. The
group also specialises on fungal-bacterial
interactions and the molecular basis of selected symbiotic and pathogenic relationships.
The biomolecular interactions in fungalbacterial associations are jointly investigated
with research groups at the HKI, the FSU,
and the Max Planck Institute for Chemical
Ecology in the context of the graduate schools
ILRS and JSMC.
Enzyme. In Kollaboration werden hierzu
auch strukturbiologische Projekte durchgeführt. Die meisten dieser interdisziplinären
Arbeiten werden in überregionalen Netzwerken bearbeitet. Die Projekte haben nicht
nur zum Ziel, ein besseres Verständnis von
der Evolution metabolischer Diversität zu
bekommen, sondern auch, das natürliche
Biosynthesepotential zu nutzen, um neue
Naturstoff-Derivate darzustellen. Bei den
hierzu angewandten biokombinatorischen
Techniken stehen Biosynthesewege pharmakologisch relevanter Polyketide im Mittelpunkt. Moderne Synthesemethoden und
Biotransformationen komplementieren die
Biosyntheseleistung der Mikroorganismen.
als Virulenzdeterminanten, zum Beispiel bei
Pilzinfektionen eine wichtige Rolle.
Die Abteilung Biomolekulare Chemie hat
sich auf Pilz-Bakterien-Interaktionen spezialisiert und studiert die molekulare Basis
von symbiotischen und pathogenen Beziehungen. Die biomolekularen Interaktionen
in Pilz-Bakterien-Symbiosen sind Themen
stark vernetzter Gemeinschaftprojekte der
Arbeitsgruppen am HKI, der FSU und des
Max-Planck-Instituts für chemische Ökologie im Rahmen der Graduiertenschulen ILRS
und JSMC.
Naturstoffe sind Mediatoren der biologischen
Kommunikation, die im Laufe der Evolution
auf biologische Aktivität selektioniert worden sind. Sie spielen in der Interaktion von
Mikroorganismen, bei Symbiosen und auch
Department of Biomolecular Chemistry HKI Research Report 2012 / 2013
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Scientific Projects
Figure 1
Activation of a silent biosynthesis gene
cluster in Burkholderia thailandensis.
Figure 2
Structure and siderophore action
of malleobactin.
1
Genomics-driven
natural product discovery
Group Leaders: Keishi Ishida, Sacha Pidot,
Christian Hertweck
The current crop of antibiotics in clinical use are either natural products or their
derivatives. However, the rise of a multitude of different antibiotic resistant human
pathogens means that new antibiotics are
urgently needed. Unfortunately, the search
for new antibiotics from traditional bacterial
sources often results in a high rediscovery
rate of known compounds and a low chance
of identifying truly novel chemical entities.
To overcome this, previously unexplored
(or underinvestigated) bacterial sources
are being tapped for their potential to produce novel compounds with new activities.
Furthermore, in the postgenomic era it has
become increasingly obvious that the biosynthetic potential of microorganisms has
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been greatly underexplored. In many organisms the majority of genes potentially coding for the biosynthesis of biologically active
compounds remain silent, and new methods
are required to bring such ‘cryptic natural
products’ to light. Our aim is to develop new
ways to specifically activate such cryptic
gene clusters.
Manipulation of the promotor for the quorum
sensing regulator gene in the model organism Burkholderia thailandensis caused a significant change in the metabolic profile. This
genetic manipulation led to the activation of
the thailandamide biosynthesis gene cluster.
We succeeded in a stereochemical analysis
to unravel the structure of the short-lived
polyene, which likely is a signalling molecule. Using a similar genetic approach we
unearthed the product of a cryptic NRPSPKS gene cluster that seems to be a hallmark
of well-studied pathogenic bacteria belonging to the B. mallei group. Notably, Burkhold-
eria mallei and Burkholderia pseudomallei
are infamous animal and human pathogens.
Overall, surprisingly little is known about
the biosynthetic potential and small-molecule virulence factors of these pathogens.
Using the phylogenetically related albeit nonvirulent Burkholderia thailandensis strain as
model organisms for pathogenesis, we succeeded in the isolation and structure elucidation of burkholderic acid, a highly oxygensensitive metabolite featuring an unusual
polyketide scaffold. Using the same model organism, we have elucidated the structure of
malleobactin A, the long searched for siderophore of animal and human pathogenic bacteria of the B. mallei complex. Through gene
cluster analyses and targeted knock-outs we
have unequivocally correlated siderophore
activity to the mba gene cluster in B. thailandensis, the less-virulent model organism
of the B. mallei family. In addition to disclosing an extraordinary siderophore variant featuring a nitro group, we report the unusual
nitro amino acid ANPA for the first time as
part of a natural product.
With respect to natural products, one group
of highly neglected bacteria are those that
grow without oxygen: the anaerobes. Anaerobic bacteria are ubiquitous in the environment and play major roles in the natural
cycling of both carbon and sulfur, and in
the degradation of organic matter, are important in biofuel manufacture, and certain
species are also well known pathogens. A
major obstacle to the investigation of natural products in anaerobes comes from the
long held view amongst secondary metabolite researchers that these bacteria are incapable of producing secondary metabolites.
However, the isolation of the first antibiotic
from an anaerobe, closthioamide from Clostridium cellulolyticum in the BMC, has turned
this supposition on its head. Encouraged by
this finding, a total of 211 complete and published genomes from anaerobic bacteria are
analysed for the presence of secondary metabolite biosynthesis gene clusters, in particular those tentatively coding for polyketide
synthases (PKS) and non-ribosomal peptide
Figure 3
Cover art for Nat Prod Rep showing a
summary of the genome analysis of
anaerobic bacteria.
Figure 4
Structure of the antibiotic closthioamide from Clostridium cellulolyticum
and crystal structure of the Cu(I)
complex; background: visialised Cu
depletion in plate culture.
synthetases (NRPS). We investigate the distribution of these gene clusters according to
bacterial phylogeny and, if known, correlate
these to the type of metabolic pathways they
encode. Interestingly, we noted that PKS
gene clusters were silent in representative
strains. However, we successfully employed
an antiterminator gene, nusG, for the induction of a cryptic biosynthetic pathway in
Clostridium cellulolyticum. In this way we
generated a mutant that can be harnessed
for the sustainable production of the potent
polythioamide antibiotic. Using this mutant,
we also succeeded in the isolation of seven
novel thioamide metabolites, which by far
exceed the number of currently known natural products featuring this rare functional
group. We also found that closthioamide is
a selective metal chelator that forms a binuclear Cu(I) complex and disclosed an unparalleled architecture of the complex.
15
Figure 5
Structure of aureothin (1) and multifactorial control of chain assembly.
2
Natural product biosynthesis and
pathway engineering
Group Leader: Christian Hertweck
Complex bacterial polyketides such as polyenes, macrolides and polyethers are a rich
source of clinically used therapeutics. Despite their highly diverse structures, these
valuable compounds are assembled from
simple acyl and malonyl building blocks in
an enzymatic assembly line. The polyketide
synthases (PKS) in charge of selecting, fusing and processing the building blocks are
organised in modules that consist of individual catalytic domains. Typically, there is
a unidirectional progress of chain elongation, where each domain is only used once
in the biosynthesis of one polyketide molecule. Thus, for most synthases there is a
strict co-linearity between the number and
architecture of the modules and the number of elongations and degree of β-keto pro-
16
cessing. However, there are more and more
deviations from this textbook knowledge;
in various modular PKS single domains or
entire modules are skipped and individual
modules may be used more than once. Despite the growing number of non-colinear
PKS, to date the mechanisms that control
the iteration processes in non-colinear
modular PKS have remained obscure. We
have obtained first insights into the factors
governing the iteration processes in a noncolinear PKS involved in the biosynthesis
of aureothin. Through gene deletion and
complementation with a synthetic surrogate we found that the KS1 of the iterating
module is exclusively in charge of priming
the PKS. This is a prerequisite to warrant a
vacant position for the retrotransfer of the
diketide. The model of a kinetic control is
fully in line with the observed complete loss
of iteration in an engineered AurA variant
harboring the designated loading module of
the avermectin PKS. Finally, through dele-
Figure 6
Enzymatic chain branching reaction
to install a pharmacophoric group
of rhizoxin (left) and structure of
the module (right, x-ray: G. Zocher,
Tübingen).
tion of the TE domain and the use of synthetic reference compounds we succeeded
in detecting the polyketide intermediates
of iterative and non-iterative aur PKS modules. Whereas this experiment confirmed a
high fidelity of the programmed events, the
iterative module alone produces polyketides
with varying lengths that correspond to one
to four rounds of elongations. Thus, we provide strong experimental support for a scenario where multiple factors are responsible
for the rare iteration process. It is clearly
an inherent quality of the KS1 to mediate
priming, chain propagation and intermediate retrotransfer, but the downstream KS2
functions as a gatekeeper that excludes the
propagation of intermediates that are either
too short or too long. Beyond new insights
into the hidden programme of a non-canonical multimodular assembly line our findings
also provide valuable information for the rational design of complex polyketides by pathway engineering.
A hallmark of all polyketide synthases
studied to date is the head-to-tail fusion of
acyl and malonyl building blocks, exclusively yielding linear chains. We recently
discovered an unprecedented, non-canonical
PKS module that mediates a chain bifurcation, thus enabling the formation of novel
branched metabolites. This particular chain
branching plays a key role in the biosynthesis of rhizoxin, one of the strongest antimitotic agents known. Notably, the biological
activity of the molecule critically depends on
the branch, and also in numerous other compounds similar pharmacophores are found.
We experimentally support our findings by
the successful full in vitro reconstitution of
a non-canonical PKS module and infer the
exact course of the reaction from protein
structure data, point mutagenesis, isotope
labelling, transformation of synthetic surrogates and NMR/MS analysis of ACP-bound
and liberated products. We unveiled a novel
role for a ketosynthase (KS) in polyketide
17
Figure 7
Unusual malonyl transfer reaction
by the ketosynthase CerJ
(X-ray: G. Zocher, Tübingen).
biosynthesis. In light of the countless known
KS domains that uniformly produce linear
carbon chains, our finding of a KS that generates a branch is a rare case for a substantially different KS-mediated reaction channel. The crystal structure of the branching
module provides new structural insight into
the functioning of these important assembly
lines. Moreover, we pioneered a new NMRbased method for analyzing ACP-bound enzyme products. This method will be of great
value for many other studies as it allows the
unequivocal assignment of hard-to-detect
intermediates or products.
In a different study we discovered another
non-canonical ketosynthase homologue, CerJ,
which unexpectedly forms malonyl esters
during the biosynthesis of the anti-MRSA
glycoside antibiotic cervimycin from the
cave-derived bacterium Streptomyces tendae.
Through a series of experiments we found
that CerJ is uniquely capable of transferring
18
malonyl-, methylmalonyl-, and dimethylmalonyl units onto the glycoside. Elucidation of
the protein crystal structure of CerJ revealed
that this unusual enzyme is functionally and
structurally positioned between KS catalyzing Claisen condensations and acyl-ACP
shuttles. A non-canonical catalytic triad has
been assigned to the aberrant malonyl transfer. Furthermore, we generated a mutant that
produces a significantly more active cervimycin variant lacking the malonyl side chain.
Chartreusin is a well-known antibacterial
and cytotoxic agent equipped with a DNAintercalating aglycone. As to date, there has
been a complete lack of information on the
impact of the aglycone (chartarin) ring substitution on antitumoral and antibacterial
activities. Notably, there are no reported efficient synthetic methods available for altering the ring substitution pattern, and to date
the total synthesis of chartreusin has not
been achieved. We have rationally designed
Figure 8
Mutasynthesis of chartreusin derivatives.
improved chartreusin analogs by merging
the strengths of chemical synthesis with biosynthesis. Specifically, we deleted the PKS
genes and cross-complemented the mutant
with anthracycline biosynthesis genes or
with fully synthetic aglycones. In this way
we succeeded in generating a library of chartreusin derivatives with altered aglycone
structures. The blend of biological and synthetic methods not only granted access to a
library of chartreusins that are otherwise
not accessible, but also yielded promising
candidates for further development as antitumoral or antibacterial agents. Specifically,
we synthesised a novel vinyl-substituted
chartreusin analogue, which forms covalent links to DNA upon mild photoactivation
with visible light and has a markedly higher
antitumoral potency than the parent compound. The novel apoptosis-inducing photoreactive DNA intercalator can be activated
by visible light, a lead that holds promise for
curing skin cancers and tumors that are ac-
Figure 9
Cover art for Angew Chem illustrating photoactivation of a chartreusin
derivative.
cessible with light probes. Furthermore, we
found that the lack of the methyl substituent
causes a dramatic decrease in cytotoxicity,
but strongly enhances antibacterial activities. Specifically, the desmethyl variant is ac-
19
Figure 10
Biosynthesis gene cluster and structures of a family of bacterial indoloterpenes.
tive against mycobacteria, which are in the
focus of antibiotic research due to the rise of
tuberculosis and other life-threatening infectious diseases.
Indole terpenoids encompass a highly diverse group of natural products, including
infamous psychotropic agents such as lysergic acid derivatives, the aphrodisiac yohimbine, and the potassium channel blockers
paxilline and lolitrem. What is remarkable
about this multifarious class is that practically all indole terpene alkaloids have been
isolated from plants and fungi. However,
to date nothing is known about the biosynthesis of indole terpenes in bacteria. We have
unveiled the molecular basis for the first
indole terpenoid pathway in a bacterium.
Through heterologous reconstitution of the
xiamycin pathway we elucidated the set of
xia genes that are sufficient for xiamycin biosynthesis and demonstrated the biogenetic
relationship of the diverse alkaloids. Since
20
genetic engineering of the xia gene cluster
resulted in increased metabolite production,
we succeeded in the characterisation of yet
overlooked, structurally unique N-N- and
C-N-coupled xiamycin dimers. Through in
silico studies, mutational analysis of all pathway genes and metabolite analyses we succeeded in dissecting an unparalleled bacterial alkaloid pathway. Our results reveal the
involvement of two prenyl transferases, XiaK
and XiaM for FPP formation and indole prenylation, respectively, and suggest concomitant epoxidation by a novel hybrid enzyme,
XiaL. On biosynthetic grounds the most important discovery were two novel cyclases
that play key roles in indole terpenoid cyclisation: XiaE, which promotes the cyclisation
of the epoxidized farnesyl side chain, and
the indole oxygenase XiaF, which paves the
way for diverging cyclisation schemes. Because of the unusual IS architectures and the
novel mechanisms involved, the xia biosynthesis enzymes are an important addition
to the emerging family of bacterial terpene
synthases and modifying enzymes. Thus,
this study not only sets the stage for investigating mechanistically intriguing reactions,
but also is a prerequisite for engineering indoloterpenoids in bacteria.
3
Natural products in microbial
interactions
Group Leaders: Kirstin Scherlach, Christian
Hertweck
Natural products play a key role in symbiotic
interactions between microorganisms and
higher organisms, covering all kingdoms of
life. In many cases the interactions involve
multiple partners and thus the biogenetic
basis of chemical mediators can be quite complex. This is well exemplified by the unparalleled tripartite relationship between the rice
seedling blight fungus Rhizopus microsporus,
its host plant Oryza sativa and endosymbiotic bacteria that reside in the fungal cytosol. The bacterial symbionts (Burkholderia
spp.) produce a phytotoxin complex (rhizoxin
and congeners) to assist the phytopathogenic
fungus in colonising rice seedlings. Whereas
the biosynthesis of the macrolide backbone
has been decoded by mutational analyses,
polyketide tailoring mechanisms and the
biological role of the bis-epoxidation have
remained elusive. Through a combination of
genetic and chemical analyses we have now
solved the riddle of the dual epoxidation in
rhizoxin biosynthesis. Sequencing and comparison of rhizoxin biosynthesis gene clusters and engineering of a mutant producing
didesepoxy variants of rhizoxin revealed
that the macrolide is first epoxidized by the
cytochrome P450 monooxygenase RhiH. By
whole-cell biotransformation and cross-infection experiments we could unequivocally
demonstrate that the 2,3-oxirane ring is introduced by the fungal host to specifically
tailor the rhizoxin scaffold. According to the
rice seedling swelling assays, the additional epoxide moiety substantially increases
phytotoxic potency. From an ecological point
of view, this finding is fully plausible since
the second epoxidation is a specific trait of
fungi belonging to the clade of rice seedling
blight fungi. We therefore report for the first
time on symbiotic synergism in the biosynthesis of a secondary metabolite that has a
biological significance.
A related bacteria-fungi interaction plays a
key role in the context of food fermentations.
The respiratory toxin bongkrekic acid (BA)
caused countless health-threatening and even
lethal intoxications after consumption of the
Southeast-Asian specialty tempe bongkrek.
Whereas it was long known that the toxin producer is a contaminant of the fungus used for
fermentation, the genetic basis for BA biosynthesis has remained obscure. Through whole-genome sequencing of B. gladioli pv. cocovenenans we identified the gene locus for a giant
trans-AT PKS and accessory enzymes catalysing assembly of the polyunsaturated tricarboxylic acid. Considering that BA is one of the
most potent respiratory toxins known, these
insights are of significance for pinpointing toxinogenic B. gladioli pathovars in the context
of food processing, agriculture and medicine.
On pharmacological grounds knowledge of
the molecular basis for BA biosynthesis paves
the way for engineering specific ligands for
adenine nucleotide translocator, a promising
target for antiapoptosis and chemotherapy.
Figure 11
Symbiotic cooperation in the biosynthesis of the phytotoxin rhizoxin.
21
Figure 12
Structure of bongkrekic acid (BA),
micrograph of fungal-bacterial coculture and formation of BA at the
bacterial-fungal interface.
Soft rot diseases caused by a variety of bacteria account for severe losses in agriculture, devastating fruits, vegetables and cultivated mushrooms. After bacterial infection,
often due to direct contact or transmission
by insects, factors and lytic enzymes cause
degradation of plant and mushroom tissues,
turning crop into mush. In many cases, the
chemical mediators of soft rot diseases are
unknown, as in the long-known mushroom
pathogen Janthinobacterium agaricidamnosum. We have discovered a new antifungal
virulence factor, jagaricin, from the mushroom pathogen. The isolation proved to be
challenging because the jag biosynthesis
genes are downregulated or silenced under
regular cultivation conditions. In the pathobiological context jagaricin production is
triggered upon contact of the bacterium (J.
agaricidamnosum) with the mushroom (A.
bisporus). To elucidate the cryptic natural
product we employed a combination of imaging mass spectrometry and genome mining.
22
Imaging mass spectrometry has proven to be
a valuable tool for the discovery of microbial
and plant natural products, in particular in
symbioses. Coupling this technique to genome mining is a new approach, and to the
best of our knowledge we describe the first
case where this combination enabled the systematic discovery and full characterisation
of a cryptic natural product. Furthermore,
the revelation of a virulence factor by imaging mass spectrometry and genomics is unprecedented. For the rigorous elucidation of
the antifungal principle the combination of
physicochemical methods, chemical derivatisation and bioinformatic analysis of the encoded NRPS assembly line was most effective. Herein, predicting the lactone ring size
by a TE phylogeny proved to be a particularly
valuable, new approach that will certainly aid
in solving related structural riddles. Furthermore, comparison of the jagaricin structure
and the thiotemplate system revealed several
unusual features of the jag NRPS.
Figure 13
Gene cluster and structure of jagaricin, a virulence factor of mushroom
soft rot and illustration of imaging
mass spectrometry.
On ecological and agricultural grounds the
discovery of the virulence factor and the corresponding genes is significant as it helps
to understand the pathobiology of bacteriainduced soft rot. From a chemical point of
view, our study highlights the impact of
blending modern analytics with genetics to
unveil cryptic natural products from neglected microorganisms. The general approach of
combining imaging mass spectrometry with
genome mining holds promise to be generally
applicable to the discovery of cryptic natural
products including chemical mediators such
as virulence factors that are only produced in
a particular (patho)biological context.
pathogens. Its high pathogenicity combined
with an almost worldwide spread constitutes
serious ecological and economical problems.
Thus, deciphering the molecular mechanisms
Fire blight is one of the most devastating
plant diseases that can eradicate entire orchards. It mainly affects apple and pear
trees, among other members of the Rosaceae
family. The causative agent of this rapidly
spreading disease, the bacterium Erwinia
amylovora, ranks among the top ten plant
Figure 14
Cover art for Angew Chem showing
the structure of 6-thioguanine from
Erwinia amylovora and effect on
apple plants.
23
Figure 15
Biosynthesis of gliotoxin, a virulence
factor of the human pathogen Aspergillus fumigatus.
responsible for the success of this bacterium
is of utmost importance.
Interestingly, E. amylovora is capable of producing 6-thioguanine (6TG). Thioguanine,
initially discovered and synthesised in the
1950s by Elion and Hitchings (Nobel Prize
in Physiology or Medicine for their discovery
of antimetabolite drugs) is a cytotoxic agent
in clinical use. The natural role of 6TG and
its biosynthesis in E. amylovora, however,
have remained enigmatic until now. We
have unveiled the molecular basis of 6TG
biosynthesis in the plant pathogen, gained
first insights into the formation of a peculiar
enzymatic carbon-sulfur bond formation and
demonstrate for the first time the crucial role
of 6TG in fire blight pathogenesis.
sulfide bridge. This signature residue for epidithiodiketopiperazines (ETPs) inactivates
various proteins by thiol conjugation and
generates reactive oxygen species by redox
cycling. Gliotoxin, the archetypal ETP, has
been in the focus of much research endeavor
as it has been implicated as a virulence factor of Aspergillus fumigatus, a human fungal
pathogen causing life-threatening systemic
mycoses. In collaboration with the department MAM we have elucidated intriguing
enzymatic steps for sulfur introduction in
the gliotoxin biosynthetic pathway (for details see report MAM).
A broad range of toxic fungal secondary metabolites that have been implicated in human,
animal and plant diseases share a diketopiperazine scaffold that is overarched by a di-
24
Group members
Head
Prof. Dr. Christian Hertweck
Phone +49 3641 532-1100
Fax
+49 3641 532-0804
[email protected]
Trainees
Laura Broschat (since 08/2013)
Secretary
Hiltrud Klose (until 5/2012)
Lab Manager/Executive Assistant
Caroline Rückert (since 10/2012)
Visiting Scientists
Scientists
Dr. Emma Barnes (since 04/2013)
Dr. Daniela Böttger-Schmidt (until 12/2013)
Dr. Sebastien Coyne (until 12/2012)
Dr. Ling Ding (maternity leave since 08/2013)
Dr. Andreas Habel (until 12/2013)
Dr. Keishi Ishida
Dr. Mie Ishida-Ito
Dr. Juangjun Jumpathong (since 12/2012)
Dr. Sacha Pidot (until 07/2013)
Dr. Severin Sasso (until 02/2012)
Dr. Kirstin Scherlach
Dr. Zhongli Xu (until 01/2013)
Ph.D. Students
Martin Baunach (since 11/2012)
Swantje Behnken (until 02/2012)
Nicole Brendel (until 11/2012)
Sabrina Boldt (since 01/2012)
Daniela Böttger-Schmidt
Tom Bretschneider (until 11/2013)
Pranatchareeya Chankhamjon
Jakob Franke
Katharina Graupner (maternity leave since
10/2013)
Daniel Heine
Florian Kloss
Anne-Catrin Letzel
Agnieszka Litomska
Florian Meyer (since 01/2013)
Nadine Möbius (until 03/2013)
Claudia Roß
Gülmire Shabuer (since 08/2012)
Yuki Sugimoto
Srividhya Sundaram (since 07/2012)
Nico Ueberschaar
Zerrin Üzüm (since 10/2012)
Christoph Zähle (until 12/2012)
Diploma / Bachelor / Master Students
Philipp Balzer (since 02/2012)
Tim Baumeister (04–07/2013)
Martin Baunach (until 11/2012)
Marco Böttger (03/2012–03/2013)
Jan Dworschak (since 01/2013)
Elena Geib (09/2012–12/2013)
Stefanie Groening (since 2/2012)
Florian Meyer (until 12/2012)
Anja Hartmann (03–09/2013)
Eric Helfrich (since 6/2012)
Toni Neuwirth (since 1/2012)
Viktoria Opel (04–07/2013)
Elena Seibel (04–07/2013)
Gülmire Shabuer (until 08/2012)
Zerrin Üzüm (until 09/2012)
Research Assistants
Heike Heinecke
Carmen Karkowski
Dipl.-Troph. Barbara Urbansky
Ing. Andrea Perner
Ing. Ulrike Valentin (until 7/2012)
Dr. Songsak Wattanachaisaereekul
Bangkok University, Thailand
01/2012
Dr. Anton Liaimer
University of Tromsoe, Norway
07/2012; 12/2012
Irshad ul Haq
Universty of Groningen, The Netherlands
10/2013
Dr. Katherine Watts
Standford University, USA
10/2013–11/2013
External funding
Bundesministerium für Bildung und Forschung
GenBioCom – Genombasierte Produktion
bioaktiver Verbindungen aus Aktinomyceten:
Komplexe Polyketide aus Aktinomyceten:
Genom-Analyse, Gensynthese und Aktivitätsbestimmung
Christian Hertweck
Deutsche Forschungsgemeinschaft
Jena School for Microbial Communication
Project: Endosymbiosis – Molecular Basis of
Bacterial-Fungal Symbioses
Christian Hertweck
Selected publications
Baunach M, Ding L, Bruhn T, Bringmann G,
Hertweck C (2013) Regiodivergent N,C- and
N,N-Aryl couplings of indoloterpenes and
cycloether formation mediated by a single
bacterial flavoenzyme. Angew Chem Int Ed 52
9040-9043.
Bretschneider T, Zocher G, Kusebauch B,
Heine D, Winkler R, Stehle T, Hertweck C
(2013) Vinylogous chain branching catalyzed
by a dedicated polyketide synthase module.
Nature 502, 124-128.
Coyne S, Chizzali C, Khalil MNA , Litomska A,
Richter K, Beerhues L, Hertweck C (2013) Biosynthesis of the antimetabolite 6-thioguanine in
Erwinia amylovora plays a key role in fire blight
pathogenesis. Angew Chem Int Ed 52 1056410568.
Franke J, Ishida K, Ishida-Ito M, Hertweck C
(2013) Nitro versus hydroxamate in siderophores of pathogenic bacteria: effect of missing hydroxylamine protection in malleobactin
biosynthesis. Angew Chem Int Ed 52, 8271-8275.
Scharf D, Chankhamjon P, Scherlach K, Heinekamp T, Willing K, Brakhage AA, Hertweck C
(2013) Epidithiodiketopiperazine biosynthesis:
a four-enzyme cascade converts glutathione
conjugates into transannular disulfide bridges.
Angew Chem Int Ed 52, 11092-11095.
Collaborations
Centers for Disease Control and Prevention,
Atlanta, USA
Prof. Dr. Jay E. Gee
Chalmers University of Technology,
Göteborg, Sweden
Prof. Dr. Jens Nielsen
Project: Symbiosis – Bacterial endosymbionts
in plant-pathogenic fungi
Christian Hertweck
Friedrich Schiller University Jena, Germany
Dr. Helmar Görls
Prof. Dr. Maria Mittag
Prof. Dr. Georg Pohnert
Prof. Dr. Severin Sasso
Microviridine – Ribosomale Biosynthese trizyklischer Microviridine in Cyanobakterien: Biochemie, Evolution und Funktion
Christian Hertweck
INSA Rouen, Mont Saint Aignan, France
Dr. Michael DePaolis
Prof. Dr. Jacques Maddaluno
Freistaat Thüringen
ProExzellenz Initiative
NanoConSens – Nanocontainer und nanostrukturierte Trägermaterialien für Sensorik und
Wirkstofftransport
Axel Brakhage, Christian Hertweck
Pakt für Forschung und Innovation
Genome Mining – Genome Mining zur Wirkstoffsuche in Mikroorganismen
Christian Hertweck
Max Planck Institute for Chemical Ecology Jena,
Germany
Prof. Dr. Jonathan Gershenzon
Dr. Martin Kaltenpoth
Dr. Bernd Schneider
Dr. Aleš Svatoš
John Hopkins University, Balitmore, USA
Prof. Dr. Craig A. Townsend
Medical University Innsbruck, Austria
Prof. Dr. Hubertus Haas
25
Oncotest GmbH, Freiburg, Germany
Prof. Dr. Heinz-Herbert Fiebig
Dr. Armin Maier
TU Dortmund University, Germany
Prof. Dr. Michael Spiteller
UC San Diego Skaggs School of Pharmacy
and Pharmaceutical Sciences, USA
Prof. Dr. Bradley S. Moore
University of Bonn, Germany
Prof. Dr. Jörn Piel
University of Kentucky, USA
Prof. Dr. Jurgen Rohr
University of Lausanne, Switzerland
Prof. Dr. Uta Paszkowski
University of Napoli, Italy
Prof. Dr. Anna Molinaro
University of Potsdam, Germany
Prof. Dr. Elke Dittmann
University of Tromsoe, Norway
Dr. Antong Liaimer
University Tübingen, Germany
Prof. Dr. Thilo Stehle
Prof. Dr. Georg Zocher
26
Department of Infection Biology
27
The fungal pathogen Candida albicans uses
an arsenal of immune inhibitors to control
the hostile immune attack generated by the
immunocompetent human host. Upon infection the activated human immune system
generates potent effector molecules that attack the invader C. albicans. However as a
pathogen C. albicans controls, modulates
and blocks the damaging and inflammatory
actions of the host immune response. The
aim of the Department of Infection Biology
is to identify the microbial and human molecules that act on this fungal-host immune
interface. To better understand the infection
process we identify fungal immune evation
proteins and also characterise the human
immune proteins that participate in this
immune battle. The identification and functional characterisation of fungal immune
evasion proteins is a prerequisite to understand fungal evasion strategies in molecular
detail, to define new targets and ultimately
to develop new antifungal strategies.
Four major topics are followed in the individual project groups in the Department of
Infection Biology:
Die human-pathogene Hefe C. albicans nutzt
ein breites Arsenal von Immuninhibitoren
und Regulatoren um die schädigende Immunantwort des menschlichen Wirts zu umgehen. Im Laufe einer Infektion versucht C. albicans die Immunantwort des Menschen
direkt und sofort zu kontrollieren, zu modulieren, und zu blockieren. Ziel der Arbeiten
in der Abteilung Infektionsbiologie des HKI
ist es, Immunevasionsproteine der pathogenen Hefe zu identifizieren, welche die humane Immunantwort steuern. Die funktionelle
Charakterisierung dieser Immunevasionsproteine und die Beschreibung ihrer Inter-
Head:
Prof. Dr. Peter F. Zipfel
28
aktion mit den entsprechenden Wirtsproteinen erlaubt die detaillierte Analyse der
Immuninteraktion auf molekularer Ebene.
Die Identifizierung und Charakterisierung
von Immunevasionsproteinen des Pathogens C. albicans bei der Immuninteraktion
ist ein wichtiger Ansatz um neue Targets zu
identifizieren und um eine C. albicans Infektion präventiv zu verhindern.
Vier zentrale Themen werden in den Projektgruppen der Abteilung Infektionsbiologie bearbeitet:
HKI Research Report 2012 / 2013 Department of Infection Biology
1) Identification of multifunctional C. albicans
virulence factors,
2) Characterisation of general immune escape principles that are used by fungi, and
other infectious pathogenic microbes
3) A n indepth understanding of the human
innate immune response and its influence
on the adaptive immunity during the infection process
This approach allows to define general principles of microbial immune escape which is
shared by pathogenic fungi, Gram-negative
as well as Gram-positive bacteria, protozoa
and viruses. Understanding the detailed action of human immune regulators also provides a platform to identify disease causing
mechanisms of severe human disorders and
to translate these results into novel diagnostic and therapeutic strategies.
4) Characterisation of protection mechanisms
for self-structures against toxic immune
reaction products
1) die Identifizierung von multifunktionalen
Virulenzfaktoren von C. albicans
2) die Entschlüsselung eines allgemeinen
Immunevasion-Prinzips, das auch von
anderen Pilzen und weiteren infektiösen
pathogenen Erregern genutzt wird
3) ein vertieftes Verständnis der angeborenen humanen Immunantwort sowie der
Interaktion zwischen angeborener und
erworbener Immunität
Mit diesem Vorgehen werden zentrale Prinzipien der mikrobiellen Immunevasion definiert, die sowohl von C. albicans, als auch von
weiteren pathogenen Pilzen, von Gram-negativen und Gram-positiven bakteriellen Erregern, Protozoen und Viren genutzt werden.
Das detaillierte Verständnis der Immunreaktion hilft, Krankheitsmechanismen zu
verstehen und neue Strategien für die Diagnose und Therapie schwerer menschlicher
Erkrankungen zu entwickeln.
4) die Charakterisierung der Schutzmechanismen von körpereigenen Zellen vor toxischen Immunaktivierungprodukten
Department of Infection Biology HKI Research Report 2012 / 2013
29
Scientific Projects
1
Immune and complement escape
of the human pathogenic fungus
C. albicans: Gpd2 (Glycerin-aldehyde 3 phosphatase) is a novel
fungal moonlighting protein and
immune evasion factor
Group Leader: Peter F. Zipfel
C. albicans is an opportunistic and medically
important human fungal pathogen. Infections
with C. albicans can range from superficial
to systemic disorders. Resistant C. albicans
strains are continuously developing and thus
new prophylactic and therapeutic strategies
in treatment of fungal infections are urgently
needed. The identification of novel yeast virulence factors or determinants that substantially contribute to pathogenic growth inside
an infected host represent ideal targets to ultimately fight C. albicans infections.
Figure 1
C. albicans binds to human endothelial cells.
C. albicans upon co cultivation with
human endothelial cells (HUVEC)
binds to the surface of HUVEC cells.
C. albicans when opsonised with the
complement activation product C3b
binds strongly to the endothelial cells,
and in consequence the phagocytic
uptake of the pathogen by the human cells is increased.
30
C. albicans infections, similar to other microbial infections, activate the immune response
of their host. The immediately responding innate immune system of the infected host uses
pattern recognition molecules to recognise
and damage the surface of the foreign invader. Candida infections are a particular
challenge for immunocompromised individuals. A pathogenic microbe upon overcoming
host immune reactions can propagate and
bind to host cellular and extracellular matrices. The pathogens even degrade these tissue
matrices to disseminate into deeper tissue
layers.
Complement is a central part of the human
immune response and provides one of the
first defense lines of host immunity. The complement cascade, which is activated by three
major pathways, forms an immediate barrier
against invading microbes. The alternative
pathway is initiated spontaneously and is
activated strongly on foreign surfaces. The
lectin pathway is activated upon binding of
mannose-binding lectin to mannan and carbohydrate structures on microbial surfaces.
The classical pathway is activated via antigen-antibody complexes or by C-reactive protein. All three pathways merge on the level of
the C3 convertase, which is a newly formed
enzymatic complex that cleaves C3 into C3a
and C3b. The cleavage product C3b binds to
microbial surfaces where it acts as opsonin
and mediates recognition and phagocytic
uptake by human immune effector cells. C3a
has antifungal, antimicrobial, chemotactic
as well as inflammatory activities.
C. albicans and also other infectious pathogens express a series of immune escape
proteins, including complement inhibitors,
proteases as well as adhesion proteins. Furthermore C. albicans recruits and exploits
protective human plasma proteins with the
purpose of controlling the innate immune attack, as well as mediating adhesion and tissue interaction and degradation (Fig. 1).
C. albicans expresses four immune evasion
proteins that bind the central human complement inhibitor Factor H (i.e. CRASP1/Gpm1,
CRASP2/Pra1, CRASP3/Gpd2 and Hgt) and
exposes a total of 12 surface proteins that
bind the human coagulation zymogen plasminogen. Notably these fungal immune
evasion proteins control many host immune
reactions and represent multifunctional innate immune inhibitors. A single C. albicans
immune escape protein can bind different
human complement components or regula-
Figure 2
Pra1, the pH regulated antigen of
C. albicans is a multifarious fungal
immune escape protein.
Pra1 binds and interacts with many
human complement components,
complement regulators Factor H,
FHL1, C4BP, C3 and the C3 activation
fragments C3b, iC3b, C3d as well
as C3a. In addition Pra1 binds the
extracellular matrix components
lamin, fibrinogen and the proenyzme
plasminogen. Plasminogen attached
via Pra1 to the surface of C. albicans
is accessible for the activator and
is converted to the active protease
which in consequence cleaves fibrinogen. Pra1 also binds to cellular integrin
receptors, i.e. CD11b/CD18 (CR3)
and to CD11c/CD18 (CR4) which are
expressed by human macrophages
and neutrophils. In addition Pra1 is a
zinc chelator and assists in fungal zinc
metabolism. Pra1 acts at many locations: the immune regulator is present
and exposed at the fungal surface, is
secreted by the pathogen, binds back
to the fungal surface and also interacts with specific receptors that are
expressed on human immune cells.
tors, adhesion proteins or coagulation factors. CRASP2 of C. albicans, also termed Pra1
(pH regulated antigen 1), is a prime example
for such a multifunctional immune evasion
protein. Pra1 binds many soluble human immune proteins, including the complement
components and regulators Factor H, FHL1,
C4BP, C3, the C3 activation fragments, C3b,
iC3b, C3d and C3a. Furthermore Pra1 binds
the adhesion components fibrinogen, laminin
as well as plasminogen. Plasminogen when
bound to the fungal surface via Pra1 is accessible for activators and is converted to the
active protease plasmin. In addition Pra1 is
a zinc chelator and binds to the human integrin receptors i.e. CD11b/CD18 (CR3) as well
as CD11c/CD18 (CR4) (Fig. 2). Upon binding
to these human integrin receptors at the surface of human macrophages and neutrophilic
granulocytes Pra1 modulates the response of
these human innate immune effector cells.
The Department of Infection Biology is interested in defining the whole immune escape
repertoire that the fungal pathogen C. albicans uses to control human complement attack and to direct the host immune response.
Therefore we aimed to identify new fungal
complement escape proteins. Using a systematic screening approach C. albicans cell
extract was adsorbed to a matrix to which the
human plasma protein Factor H was attached.
A 52 kDa C. albicans protein was eluted and
identified by mass spectrometry as Glycerol3-phosphate dehydrogenase 2, Gpd2. Consequently the Gpd2 gene from C. albicans was
amplified by PCR, cloned and the protein was
recombinantly expressed in Pichia pastoris.
Recombinant Gpd2 binds the three human
immune regulators, Factor H, FHL-1 and
also plasminogen. Each of the three human
regulators when attached to Candida Gpd2
is functionally active and assists in complement inactivation as well as fibrinogen degradation. FHL1 and FHL1 inhibit the comple-
31
Gpd2
candida
Figure 3
Gpd2 (Glycerinaldehyde 3 phosphatase) is expressed at the surface of C. albicans yeast cells and
hyphae.
Gpd2 is a novel fungal complement
regulator binding protein of the
human pathogenic fungus C. albicans.
A new polyclonal antiserum was
generated by immunising rabbits
with recombinant Gpd2. This antiserum identified Gpd2 on the surface of
C. albicans yeast cells (top panel) and
hyphae (bottom panel).
References
Luo S, Hoffmann R, Skerka S, Zipfel PF
(2013) Glycerol-3-phosphate Dehydrogenase 2 Is a Novel Factor H, FHL-1
and Plasminogen Binding Surface
Protein of C. albicans albicans. J Infect
Dis 207, 594-603.
hyphae
ment cascade at the level of the C3 convertase
by acting as cofactors for the serine protease
Factor I, which cleaves and degrades C3b into
iC3b. In addition Factor H and FHL1 compete
with Factor B for C3b binding and both proteins also accelerate the decay of a preformed
alternative pathway C3 convertase.
Native Gpd2 is identified at the surface of
C. albicans yeast cells, as well as hyphae
by a polyclonal anti-serum to Gpd2 (Fig.
3). Recombinant Gpd2 also binds to human
non – phagocytic cells including endothelial
HUVEC cells and human keratinocytes (HACAT) (Fig. 4). This specific binding to human
endothelial cells and to keratinocytes indicates an additional role of the fungal protein
for directing the host cellular response. In
summary the C. albicans surface protein
Gpd2 is a new fungal immune escape protein
which binds several human immune regulators and also binds to the surface of human
endothelial cells and keratinocytes.
32
Altogether evasion strategies to control complement action used by C. albicans are very
similar to those used by certain other microorganisms and thus reflect rather general
principles which are shared by pathogens
derived from different species.
2
Identification and characterisation
of human complement regulators as
microbial evasion molecules (Group
Immunoregulation)
Group Leader: Christine Skerka
Infectious microorganisms are immediately
recognised and attacked by the innate immune system of the human host. This innate
immune response results in the generation
of antimicrobial peptides, the generation of
complement activation products as well as
the activation of immune cells. The complement system is a central part of the immune
Figure 4
The C. albicans Gpd2 protein
binds to the surface of human endothelial cells and keratinocytes.
Gpd2 binding to human endothelial
cells and keratinocytes was analysed
by flow cytometry (A) and by confocal laser scanning microscopy (B).
Gpd2 binds to the surface of nonphagocytic endothelial cells and
keratinocytes, but not to the surface
of monocytic U937 cells.
system and upon activation generates C3b,
which then deposits onto nearby surfaces. On
the surface of human cells, this newly generated C3b is immediately inactivated by regulators, but on microbial surfaces C3b deposition is amplified, leading to opsonisation.
Such C3b covered foreign microbial surfaces
are recognised and their uptake by phagocytes is enhanced. The complement activation
split products C3a and C5a act as anaphylatoxins which recruit additional effector cells
and initiate the adaptive immune response.
In order to evade these potent and damaging
host immune reactions C. albicans and other
pathogenic microbes exploit human complement regulators to control the host response.
In addition, these human regulators play a
major role in the complement mediated noninflammatory phagocytosis and clearance of
modified and dead cells from the circulation.
Therefore many human complement regulators harbour a surface recognition region
and a complement regulatory region which
enables inhibition of different levels of activation on certain surfaces. As these proteins
regulate important steps of the human immune response they represent attractive targets for infectious microbes like C. albicans
(Fig. 1).
The aim of the Group of Immunoregulation
is to identify and characterise novel human
complement regulators and to investigate the
role of these regulators in the infection process with C. albicans. The group focuses on
three human proteins: CFHR2 (complement
factor H related protein 2), CFHR3, as well as
plasminogen. The role of the individual proteins in complement activation and regulation is poorly defined and genetic variations
in individual genes are associated with several immune diseases, including the retinal
disorder AMD (Age Related Macular Degeneration).
33
Figure 5
CFHR2, the human plasma protein
is a complement inhibitor.
(A) Structure and domain composition of CFHR2, CFHR1 and Factor H
and of the two deletions mutants
CFHR2/1-2 and CFHR2/3-4. The numbers between the proteins indicate
amino acid homology. (B-D) CFHR2
forms dimers in solution. Static light
scattering in combination with size
exclusion chromatography was used
to determine the molecular mass of
the complex in solution. The molar
mass of each protein is indicated, as
well as the status of dimerisation, in
the panel above each experimental
figure. The light scattering signals
(LS) and the mass distribution (Mw)
in each of the peaks in the elution
profiles monitored by the absorbance at 280 nm (UV) and changes of
the refractive index (dRI). (E) CFHR2
inhibits the alternative complement
pathway in NHS which was activated
on foreign surfaces. Factor H was
used a positive control for alternative
pathway inhibition. (F) CFHR2 did not
affect classical pathway activation in
NHS. Terminal complement complex
(TCC) deposition in untreated NHS
was set as 100 %. C4BP the classical
pathway inhibitor was used as a positive control.
CFHR2 and CFHR3 circulate in human
plasma as differently glycosylated variants
and both proteins bind the central activated
complement component S3b and C3d. CFHR2
and CFHR3 also bind via specific binding domains to glycosaminoglycans on human cell
surfaces, indicating a complement regulatory role of CFHR2 and CFHR3 on human cell
surfaces (Fig. 5).
In addition CFHR2 is bound by C. albicans
and also by the Gram negative bacterium Borrelia burgdorferi, thus suggesting that these
pathogenic microbes use the human protein
for immune escape. The hypothesis that
CFHR2 and CFHR3 are important immune
modulators is further supported by the fact
that mutations or deletions of the encoding
genes enhance the risk to develop autoantibodies and kidney damages.
34
Based on the recruitment of CFHR2 to the
surface of C. albicans and an association of
genetic mutations in the CFHR2 gene with
kidney diseases, we were interested to define the function of CFHR2. To this end
the CFHR2 gene was amplified, cloned and
cloned and the protein recombinantly expressed in Pichia pastoris. The secreted protein was purified by chromatography and
analysed by multi-angle laser scattering.
Interestingly recombinant CFHR2 exists
exclusively in a dimeric form. To determine
the dimerisation domain, two fragments of
CFHR2 were recombinantly expressed and
analysed accordingly. Dimers were formed
via the N-termini and the C-termini bound
to C3b and C3d. Thus one CFHR2 dimer can
bind via the two C-termini two C3b or C3d
molecules at the same time.
The complement regulatory activity of CFHR2
was evaluated in hemolysis assays, where the
effect of CFHR2 on the complement mediated
lysis of erythrocytes was assayed. CFHR2
inhibited hemolysis when complement was
activated via the alternative, but not via the
classical pathway. Further studies revealed
that CFHR2 inhibits the C3 convertase, the
central enzyme of complement. The CFHR2
homodimer binds to the C3 convertase and
blocks convertase function(s). Thereby the
CFHR2 homodimer likely complexes the C3
convertases so that the substrate C3 is no
longer cleaved and no activation products, i.e.
C3b and C3a, are formed.
In addition CFHR2 inhibits a further activation step of the complement cascade: the proteine blocks the terminal complement pathway. Each complement pathway generates the
terminal complement complex, TCC, which
forms a lytic pore, composed of polymerised
C9 molecules and which integrates into the
target cell membrane. CFHR2 binds to C9 and
inhibits TCC assembly and pore formation.
Thus CFHR2 is a complement regulator and
protects self surfaces from complement damages. Pathogenic microbes like C. albicans and
B. burgdorferi bind CFHR2 to their surfaces
and thereby recruit this human complement
regulator which can inhibit C3 convertase activity and TCC formation. Complement inhibition helps C. albicans and B. burgdorferi to
survive the strong innate immune response
of the human immune system.
Plasminogen is another human plasma protein which is very frequently recruited by
pathogens. Human plasminogen is a proenzyme that is proteolytically processed and
cleaved to the active protease plasmin. Plasmin controls several relevant biological reactions, including fibrinolysis, wound healing
and tissue remodelling. C. albicans, similar to
many other pathogenic microbes, binds plasminogen to its surface and the inactive zymogen when attached to the fungal surface can
be converted to the active protease plasmin.
Plasmin in consequence degrades components of the extracellular matrix (ECM) and
fibrinogen which allows pathogens to disseminate into deeper tissue layers. Besides the
fibrinolytic activity, plasmin also influences
the complement cascade, as newly generated
plasmin cleaves native C3, via the intermediates C3b and C3a. However, plasmin further
processes and degrades both complement
activation fragments, thereby blocking their
effector functions. This degrading activity is
of interest for pathogens and explains why
many pathogens recruit plasminogen to their
surface.
The pathogenic microbe Staphylococcus aureus as a master of immune evasion binds
C3 to its surface via the microbial proteins
Sbi and Efb. Whether S. aureus binds plasminogen to target the host C3 molecule
for complement evasion was still unclear.
To identify new plasminogen binding proteins of S. aureus we analysed whether the
staphylococcal surface proteins Sbi and
Efb also bind plasminogen. Plasminogen
bound to both Sbi and Efb. Bound plasminogen is subsequently converted to plasmin by S. aureus secreted staphylokinase and
newly generated plasmin is proteolytically
active and cleaves the synthetic substrate
and also the complement components C3 and
C3b. This proteolytic activity is modulated
and enhanced by the conformational changes
induced of C3 and C3b upon interaction with
Sbi or Efb. Plasmin generated on the surface
of S. aureus cleaves and degrades additional
substrates, like C3a and thereby reduces the
chemoattractant and antimicrobial activities
of this complement activation product. Thus,
recruitment and activation of plasminogen by
S. aureus is highly coordinated to maximise
complement inhibition at the level of C3.
3
References
Koch TK, Reuter M, Barthel D, Böhm
S, van den Elsen J, Kraiczy P, Zipfel PF,
Skerka C (2012) Staphylococcus aureus
proteins Sbi and Efb recruit human
plasmin to degrade complement C3
and C3b. PLoS One 7, e47638.
Eberhardt HU, Buhlmann D,
Hortschansky P, Chen Q, Böhm S,
Kemper MJ, Wallich R, Hartmann A,
Hallström T, Zipfel PF, Skerka C (2013)
Human factor H-related protein
2 (CFHR2) regulates complement
activation. PLoS One, 8:e78617.
The role of complement regulators
in human kidney diseases
Group Leader: Peter F. Zipfel
Human complement regulators are essential
for the protection of self-cells and tissues
against toxic complement activation prod-
35
Figure 6
C. albicans expresses proteins to
bind and exploit human regulators which play a central role in
self-cell protection and prevention of diseases.
Aim of the human immune studies is
to define new complement regulators
that C. albicans recruits for the fungal
surface and uses immune evasion
and for dissemination. The acquired
human proteins are linked to human
diseases.
ucts and for the non-inflammatory phagocytosis of modified or damaged self-cells.
Such non-inflammatory action of the human
regulators is apparently highly attractive for
pathogenic microbes and another reason why
such host immune regulators are acquired by
pathogens. Pathogenic microbes express specific surface proteins or secrete moonlighting
proteins to recruit human regulators. However, the exact role of recruited individual
human proteins in the immune response is
often not precisely defined. Even for known
human regulators like Factor H new functions are postulated and identified. Genetic
variations in form of mutations, polymorphic
variations, as well as gene copy number variations often affect or disrupt the function of
the individual proteins and are frequently
associated with human diseases. In consequence such genetic variations interfere
with the fine-tuned action and regulation of
innate immunity and of the complement cascade and cause pathology in single organs.
36
The pathological effects of the genetic variations confirm the important role of single
complement proteins or regulators for host
immune homeostasis and for restricting inflammation. In this context defining how
single gene mutations, genetic copy number
variations or even genetic polymorphisms infuence the action of a single regulator and in
consequence how variant proteins affect the
tightly controlled innate immune and complement network helps to understand disease
pathology. Ultimately this also allows us to
understand why pathogenic microbes exploit
the regulators of their human host and how
the acquired regulators are used for microbial immune escape (Fig. 6). The human kidney disorder C3 Glomerulopathy with Dense
Deposit Disease pattern (C3G-DDD) is a renal
disease that is associated with mutations in
complement regulatory genes. C3G-DDD primarily affects children and young adults, is
difficult to treat and at present no established
and safe therapy exists.
Figure 7
Kidney biopsy of a patient with C3
Glomerulopathy with Dense Deposit Disease pattern (C3G-DDD).
Hematoxylin and eosin staining in a
kidney biopsy of patient #635 reveals
the presence of foam cells (asterix)
and shows hypercellularity and mesangial cell proliferation (arrow).
C3G-DDD patients present with nephrotic or
nephritic syndrome or with asymptomatic
renal disease. The disease often progresses
to end-stage renal disease (ESRD) and tends
to recur in the transplanted kidney. Diagnosis of C3G-DDD is primarily based on a histological evaluation of a kidney biopsy with
prominent C3 staining, the absence of immunoglobulins and the presence of dense deposits along the glomerular basement membrane
(GBM) combined with mesangial hypercellularity by electron microscopy. Numerous diverse genetic, as well as autoimmune causes
are reported for C3G-DDD patients, including mutations of single complement genes,
genetic deficiencies, copy number variations.
Also autoimmune forms exist which include
various autoantibodies to the central complement enzyme C3 convertase.
In collaboration with M. Wiesener from the
University Hospital Erlangen we analysed
two related C3G-DDD patients who presented
with low C3 plasma levels, end stage renal
disease and who have received dialysis for
over 10 years (Fig. 7). Genetic analyses were
performed by MLPA (multiplex ligation dependent probe amplification) and revealed a
new heterozygous chromosomal deletion in
the cluster of complement regulators on chromosome 1q32. Subsequently, genomic DNA
isolated from both patients was amplified by
nested PCR in order to identify and localise
the chromosomal breakpoint. Both patients
had the same 24804 nucleotide fragment in
the CFHR gene cluster deleted and this deleted chromosomal fragment spans from exon
II of the CFHR2 gene to the promoter region
of CFHR5 gene (Fig. 8).
This chromosomal deletion resulted in expression of a hybrid transcript that had the
signal peptide and SCRs1-2 of the CFHR2
gene fused to the CFHR5 coding exons.
This novel transcript encoded a CFHR2CFHR5 hybrid protein which was secreted
37
Figure 8
A new genetic deletion in the
CFHR gene cluster on human
chromosomes 1q32 results in expression of a CFHR2-CFHR5 hybrid
protein.
Two related C3G - DDD patients #635
and #638 showed the same deletion.
The breakpoint was identified by
sequencing and a 24805 bp region is
deleted in the patients. The deletion
brings the cFHR2 and cFHR5 gene
in close proximity, results in a hybrid
transcript that links exon I to exon III of
the CFHR2 gene to the whole CFHR5
transcript. The protein product is a
hybrid CFHR2-CFHR5 protein that is
expressed, secreted and identified in
plasma of both patients.
into plasma and was identified as two bands
with mobilities of ca. 65 and 70 kDa. The
CFHR2-CFHR5 hybrid was composed of 11
SCR domains comprising the two N terminal domains of CFHR2, i.e. SCRs1-2 linked to
SCRs1-9 of the CFHR5 gene.
To allow a detailed characterisation a recombinant CFHR2-CFHR5 hybrid protein was
generated, expressed in HEK 293 cells and
purified. This recombinant hybrid protein
bound to C3b and C3 activation fragments
iC3b and C3d. Given this C3 binding profile
and upon intensive discussions with the clinicians to obtain appropriate treatment options, one patient first received a plasma
infusion. The rational was to dilute the hybrid protein in plasma, to reconstitute complement components like C3 and also to
increase the levels of the CFHR2, CFHR5
regulators. Unexpectedly this treatment resulted in enhanced complement activation,
as upon plasma addition the levels of C3
38
activation fragments and terminal complement complexes increased. In consequence
this problem was moved back to the laboratory, with the goal to identify why and how
the hybrid protein deregulated complement.
The subsequent functional characterisation
revealed that the purified recombinant hybrid protein deregulated the C3 convertase
of complement in fluid phase. CFHR2-CFHR5
stabilised the C3 convertase, reduced the
dissociation of the enzymatic complex and
blocked the access or binding of the natural
inhibitor Factor H. Now we can explain how
the novel hybrid protein overactivates complement in plasma of both patients.
This functional characterisation combined
with the understanding how the hybrid protein deregulated complement on the level of
the C3 convertase resulted in a treatment
switch to plasma exchange. Plasma exchange
reduced the levels of the deregulating, hybrid
protein and this resulted in lower comple-
Figure 9
Evaluating complement inhibitors
as treatment option for patients
who express a novel CFHR2-CFHR5
hybrid protein.
The inhibitory effect of the complement inhibitors Eculizumab, the
tagged version of the C3 inhibitor
Compstatin and soluble CR1 on the
enhanced complement activation
in substituted serum of patient
#635 was evaluated. C3b surface
deposition was monitored. sCR1 and
tagged Compstatin inhibited C3 convertase action and C3b deposition,
Eculizumab which acts downstream
of the C3 convertase had no effect.
ment activation, as revealed by reduced levels of the activation marker Ba. However this
effect was short lived. Already 12 hours after
treatment the activation marker Ba increased
and reached previous levels after 24 hours.
Thus complement activation correlated directly with the levels of the damaging hybrid
protein in the patient’s plasma.
In order to evaluate additional treatment options complement inhibitors were tested in
vitro in the laboratory. Various complement
inhibitors were screened for their effect on
complement activation in plasma of patient
#635 which was substituted with normal
plasma. Eculizumab the established complement and C5 convertase inhibitor did not
block the damaging action of the hybrid protein (Fig. 9). Therefore additional inhibitors
were tested, including the complement inhibitor Compstatin, which binds directly C3
and blocks C3 activation. Compstatin when
equipped with a surface anchoring domain
blocked the defective complement action by
the hybrid protein in patient’s plasma. Similarly sCR1, the soluble complement inhibitor
also overruled the CFHR2-CFHR5 mediated
defective complement regulation. In consequence this soluble complement inhibitor
provides an option for therapy of the patient.
The identification of a new genetic cause that
resulted in C3 glomerulopathy with DDD pattern provides a basis to understand how the
newly generated CFHR2-CFHR5 hybrid protein deregulates the complement convertase
C3 in fluid phase and allows to evaluate
and to develop therapeutic approaches for
these patients. These findings also provide
insights into the role of CFHR proteins that
in general are frequently recruited by pathogenic microbes including C. albicans.
References
Chen Q, Wiesener M, Eberhardt HU,
Hartmann A, Uzonyi B, Kirschfink M,
Amann K, Buettner M, Goodship T,
Hugo C, Skerka C, Zipfel PF (2014)
Complement factor H-related hybrid
protein deregulates complement in
dense deposit disease. J Clin Invest
124, 145-155.
39
Group members
Prof. Christoph Licht
Hospital for Sick Children, Toronto, Canada
03/2008, 03/2009, 10/2009
Head
Prof. Dr. Peter F. Zipfel
Phone +49 3641 532-1300
Fax
+49 3641 532-0807
[email protected]
Prof. Kristian Riesbeck
University Malmö, Sweden
07/2008
Secretary
Heike Gäbler
Prof. Richard Smith
The University of Iowa, USA
10/2009
Scientists
Dr. Hans-Martin Dahse
Dr. Prasad Dasari (since 10/2013)
Dr. Teresia Hallström
Dr. Nadine Lauer (until 02/2012)
Dr. Shanshan Luo (until 07/2012)
Dr. Michael Reuter (until 08/2012)
Dr. Malik J. Reza (since 02/2013)
PD Dr. Christine Skerka
External funding
Verbundvorhaben Medizinische Infektionsgenomik: Wirt-Pathogen-Interaktion: Wirkung sekretierter Proteine von Staphylococcus aureus auf
Zellen und Komponenten des Immunsystems
Peter F. Zipfel
AMD des Auges – Rolle der Komplementregulatoren Faktor H, CFHL1, CFHR1 und CFHHR3 bei der
Altersabhängigen Makuladegeneration (AMD)
des Auges
Christine Skerka
AMD des Auges II – Rolle der Komplementregulatoren Faktor H, FHL1, CFHR1 und CFHR3 bei der
Altersabhängigen Makuladegeneration (AMD)
des Auges
Christine Skerka
Research Assistants
Nadine Flach
Steffi Hälbich
Andrea Hartmann
Monika von der Heide
Ina Löschmann
Susanne Lorenz
Eva-Maria Neumann
Silvya Spielmann
SFB/TR FungiNet
Project C04: Mechanismen der Adhäsion und
Modulation humaner Immunzellen durch Candida
albicans
Christine Skerka
SFB/TR FungiNet
Project C06: Einfluss sekretierter Proteine von
Candida albicans bei der Immunevasion und
der Pathogenität
Peter F. Zipfel
Project: Immunevasion of the Gram positive
bacterium Streptococcus pneumoniae
Peter F. Zipfel
Molekulare Genetik IV – Molekulare Genetik und
Pathogenese des Hämolytisch-Urämischen Syndroms im Erwachsenenalter
Peter F. Zipfel
Trainee
Alexandra Rülke (since 08/2013)
Visiting Scientists
Punet Juneja
Himachal Pradesh University, India
07/2008–09/2008
40
Pro Retina Stipendium
Die Rolle des ARMS2 Proteins in der Immunregulation bei der AMD
Christine Skerka
Anika Westphal
Ernst Moritz Arndt University Greifswald, Germany
01/2012–02/2012
Ph.D. Students
Susanne Ackermann (since 10/2012)
Sascha Böhm (until 06/2013)
Denise Buhlmann (since 08/2012)
Qian Chen
Nadia Döring (until 08/2012)
Hannes Eberhardt (until 06/2013)
Selina Icke (until 06/2012)
Emeraldo Jo (since 10/2013)
Isabell Kopka (until 06/2013)
Justus Linden (since 08/2013)
Susanne Lindner
Crisanto Lopez (until 06/2013)
Christian Meinel (since 10/2013)
Sven Micklisch
Christiane Pilz
Sarbani Sarkar
Anika Westphal (since 10/2013)
Fei Zhao (since 09/2012)
Florian Bredy (until 08/2013)
Denise Buhlmann (until 07/2012)
Karolin Hess (until 08/2013)
Susanne Jacksch (until 09/2012)
Tom Kretzschmar (until 08/2013)
Melanie Manzke (until 08/2013)
Iris Koske (10/2012–12/2013)
Christian Meinel (10/2012–09/2013)
Marina Raguse (until 09/2012)
Kristin Schult (09/2012–09/2013)
Rajiuddin Sheikh MD (until 06/2012)
Corinna Weist (until 09/2012)
Jana Ziereisen (until 09/2012)
National Institutes of Health / University of Iowa
Collaborative Study of Membranoproliferative
Glomerulonephritis Type II
Peter F. Zipfel
European Union
Framework Programme 7: Large-scale Integrating
Project
EURenOmics – European Consortium for HighThroughput Research in Rare Kidney Diseases;
WP 05: Complement Disorders
Peter F. Zipfel
Chen Q, Wiesener M, Eberhardt HU, Hartmann
A, Kirschfink M, Uzonyi B, Amann K, Buettner M,
Goodship T, Hugo C, Skerka C, Zipfel PF (2014)
Complement factor H-related hybrid protein deregulates complement in dense deposit disease. J
Clin Invest 124, 145-155.
Hallström T, Siegel C, Mörgelin M, Kraiczy P,
Skerka C, Zipfel PF (2013) CspA from Borrelia
burgdorferi inhibits the terminal complement
pathway. MBio 4, e00481-13.
Luo S, Hoffmann R, Skerka C, Zipfel PF (2013)
Glycerol-3-phosphate dehydrogenase 2 is a novel
factor H-, factor H-like protein 1-, and plasminogen-binding surface protein of Candida albicans. J
Infect Dis 207, 594-603.
Simon N, Lasonder E, Scheuermayer M, Kuehn A,
Tews S, Fischer R, Zipfel PF, Skerka C, Pradel G
(2013) Malaria parasites co-opt human factor H to
prevent complement-mediated lysis in the mosquito midgut. Cell Host Microbe 13, 29-41.
Ueberschaar N, Dahse HM, Bretschneider T, Hertweck C (2013) Rational design of an apoptosisinducing photoreactive DNA intercalator. Angew
Chem Int Ed 52, 6185-6189.
Collaborations
Albert Ludwigs University Freiburg, Germany
PD Dr. Eva Decker
Prof. Dr. Hans-Hartmut Peter
Prof. Dr. Ralf Reski
Centro de Investigaciones Biologicas (CSC),
Madrid, Spain
Prof. Dr. Santiago R. de Córdoba
Charité Universitätsmedizin Berlin, Germany
PD Dr. Dominik Müller
Prof. Dr. Antonia Joussen
Prof. Dr. Olaf Strauß
Ernst Moritz Arndt University Greifswald, Germany
Prof. Dr. Sven Hammerschmidt
Prof. Dr. Susanne Engelmann
Technische Universität Braunschweig, Institut für
Mikrobiologie, Germany
University Hospital Jena, Germany
Prof. Dr. Dagmar Barz
PD Dr. Uta-Christina Hipler
PD Dr. Ulrike John
Prof. Dr. Iver Petersen
Prof. Dr. Gunter Wolf
Heinrich Heine University Düsseldorf, Germany
Prof. Dr. Joachim F. Ernst
Hôpital Européen Georges-Pompidou,
Paris, France
Prof. Dr. Veronique Fremeaux-Bacchi
University of Lübeck, Germany
Prof. Dr. med. Jörg Köhl
Prof. Dr. Marc Ehlers
Prof. Dr. Rolf Hilgenfeld
Instituto di Recherche Farmacologoche “Mario
Negri”, Bergamo, Italy
Prof. Dr. Marina Noris
Prof. Dr. Giuseppe Remuzzi
University of Massachussetts, USA
Prof. Dr. Sanjay Ram
University of Pennsylvania, USA
Prof. Dr. John Lambris
Prof. Dr. Wenchao Song
Johann Wolfgang Goethe University Frankfurt,
Germany
Prof. Dr. Peter Kraiczy
University of Regensburg, Germany
Prof. Dr. Bernhard Weber
Dr. Diana Pauly
Lund University Malmö, Sweden
Prof. Dr. Anna Blom
Prof. Dr. Kristian Riesbeck
Prof. Dr. Matthias Mörgelin
University of Rostock, Germany
Prof. Dr. Marianne Wigger
Medical University of Graz, Austria
Prof. Dr. Christoph Mache
Western General Hospital Edinburgh,
Edinburgh, UK
Prof. Dr. Alan Wright
Medical University of Vienna, Austria
Prof. Dr. Christoph Binder
West Viginia University Morgantown, USA
Prof. Dr. Slawomir Lukomski
Queen's University Belfast, Ireland
Prof. Anne E. Hughes
Dr. Declan T. Bradley
University Uppsala, Sweden
Bo Nilsson
Rheinisch-Westfaelische Technische Hochschule
Aachen, Germany
PD Dr. Gabriele Pradel
University Kalmar, Sweden
Kristina Nilsson-Eckdahl
Rupprechts Karl University Heidelberg, Germany
Prof. Dr. Michael Kirschfink
Prof. Dr. Reinhard Wallich
Sir William Dunn School of Pathology, Oxford, UK
Prof. Dr. Christoph M. Tang
The Hospital for Sick Children, Toronto, Canada
Prof. Dr. Christoph Licht
The Johns Hopkins University, School of Medicine,
Baltimore, Maryland, USA
Prof. Dr. Hendrik Scholl
University of Aarhus, Denmark
Prof. Dr. Gregers R. Andersen
Universitäts-Augenklinik Bonn, Germany
Prof. Dr. Frank G. Holz
University Hospital Heidelberg, Germany
Prof. Dr. Franz Schäfer
University Medical Center/Institute for
Cardiovascular Research, Amsterdam, Netherland
Prof. Dr. Hans W. M. Niessen
University Newcastle upon Tyne, U.K.
Prof. Dr. Tim Goodship
University of Colorado Denver, Denver, USA
Dr. Nirmal K. Banda
University of Erlangen Nürnberg, Germany
Prof. Dr. Michael Wiesener
Prof. Dr. Kerstin Amann
University of Iowa, USA
Prof. Dr. Richard Smith
University of Kentucky, USA
Prof. Dr. Brian Stevenson
41
42
Department of
Microbial Pathogenicity Mechanisms
HKI Research Report 2012/ 2013
43
Department of Microbial Pathogenicity Mechanisms
The Department of Microbial Pathogenicity
Mechanisms (MPM) is investigating infections caused by human pathogenic fungi.
The research is focussed on the most common pathogenic yeasts Candida albicans, and
C. glabrata, which has been recognised as an
emerging pathogenic yeast. In addition, projects deal with C. dubliniensis, C. tropicalis
and C. parapsilosis.
We investigate all aspects of pathogenicity
mechanisms of these yeasts using genomics,
in silico analyses, microbiology, cell biology,
biochemistry, infection biology and immunology approaches.
Projects deal with (1) infection associated
genes of Candida, including unknown function genes, genes required for osmolarity and
encoding damage factors, and genes unique
for C. albicans, C. glabrata or C. dubliniensis;
(2) macro- and micronutrients of Candida,
including nitrogen, iron and zinc sources
within the host; (3) evolution, adaptation
and pathogenicity of Candida, including microevolution, flexibility of transcriptional
networks and transcriptional profiling of
Candida within blood; and (4) interaction of
Candida with immune cells, including neutrophils and macrophages.
The research group Microbial Immunology,
headed by Ilse D. Jacobsen, which carries out
Head:
Prof. Dr. Bernhard Hube
Die Abteilung Mikrobielle Pathogenitätsmechanismen (MPM) erforscht die Ursache und
Entwicklung von Infektionen durch humanpathogene Pilze.
Die Forschung konzentriert sich auf die häufigsten humanpathogenen Hefen Candida
albicans und C. glabrata. Bei weiteren Projekten werden auch Aspekte der Pathogenität von C. dubliniensis, C. parapsilosis und
C. tropicalis bearbeitet.
Wir untersuchen alle Aspekte der Pathogenitätsmechanismen dieser Hefen mit Hilfe von
genomischen, molekularbiologischen, mikrobiologischen, zellbiologischen, biochemischen, infektionsbiologischen und immunologischen Methoden, sowie in silico-Analysen.
44
Die Projekte der Abteilung bearbeiten folgende Themen: (1) infektionsassoziierte Gene
von Candida (Gene mit unbekannter Funktion, für Osmolarität, gewebeschädigende
Faktoren sowie Gene, die spezifisch für C. albicans, C. glabrata oder C. dubliniensis sind);
(2) Makro- und Mikronährstoffe von Candida
(einschließlich Stickstoff-, Eisen- und Zinkquellen innerhalb des Wirts); (3) Evolution,
Adaptation und Pathogenität von Candida
(einschließlich Mikroevolution, Flexibilität von transkriptionellen Netzwerken und
Transkriptionsprofile im Blut); und (4) Interaktionen von Candida mit Immunzellen (zum
Beispiel Neutrophile und Makrophagen).
Die Arbeitsgruppe von Ilse D. Jacobsen, die
experimentelle Pilzinfektionen durchge-
HKI Research Report 2012 / 2013 Department of Microbial Pathogenicity Mechanisms
experimental fungal infections and investigates the pathogenesis of systemic candidiasis and other fungi, was integrated into
the department of MPM. Since August 2013,
this group is an independent research group
of the HKI (see report Microbial Immunology).
From 2012 to 2013, the research of MPM was
supported by the Jena School for Microbial
Communication (JSMC), the International
Leibniz Research School (ILRS), the Deutsche
Forschungsgemeinschaft (DFG), the Studienstiftung des Deutschen Volkes, the Federal
Ministry of Education and Research (BMBF),
the European Union and Bayer Vital GmbH.
Projects are integrated into the Center for
Sepsis Control and Care (CSCC), the DFG Pri-
führt hat und welche die Pathogenese von
systemischen Candida-Infektionen and Infektionen mit anderen Pilzen untersuchte,
war in die Abteilung MPM integriert. Seit
August 2013 arbeitet diese Arbeitsgruppe
am HKI selbständig (siehe Bericht „Mikrobielle Immunologie“).
In den Jahren 2012 bis 2013 wurde die Forschung der Abteilung MPM durch folgende
Institutionen unterstützt: Jena School for
Microbial Communication (JSMC), International Leibniz Research School (ILRS), Deutsche
Forschungsgemeinschaft (DFG), Studienstiftung des Deutschen Volkes, Bundesministeriums für Bildung und Forschung (BMBF),
Europäische Union und Bayer Vital GmbH.
Mehrere Projekte waren in die folgenden
ority Programmme SPP1580 “Intracellular
compartments as places of pathogen-host-interactions”, the DFG Collaborative Research
and their human host: Networks of interaction – FungiNet”, the project “Microevolution
of pathogenic yeasts during interactions with
the host” within the DFG DACH programme,
the European CandiCol consortium within
the ERA-Pathogenomics programme and the
European Marie Curie Research Training
Network FINSysB.
Netzwerke integriert: Center for Sepsis Control and Care (CSCC), DFG-Schwerpunktprogramm SPP1580 „Intracellular compartments
as places of pathogen-host-interactions“,
DFG-Sonderforschungsbereich / Transregio
124 „Pathogenic fungi and their human host:
Networks of interaction – FungiNet“, das Verbundprojekt „Microevolution of pathogenic
yeasts during interactions with the host”
innerhalb des DFG DACH-Programms, das
Europäische CandiCol-Consortium innerhalb
des ERA-Pathogenomics Programmes und
das European Marie Curie Research Training
Netzwerks FINSysB.
Department of Microbial Pathogenicity Mechanisms HKI Research Report 2012 / 2013
45
Scientific Projects
1
Infection-associated genes of
Candida albicans
Group Leader: Duncan Wilson
Transcriptional profiling of fungal pathogens
during interactions with their hosts can provide important insights into the biology of
infection. We have performed global gene expression analysis of Candida albicans as it infects a range of anatomical niches: oral tissue
(representing superficial mucosal infection);
blood and blood components (reflecting dissemination of the fungus throughout the host)
and invasion of internal organs, such as the
liver (representing deep seated infections).
The expression patterns of known genes have
provided detailed information on the environmental conditions faced by the pathogen in
these diverse niches. However, a large proportion of genes, upregulated during infection
encode proteins with no known function. To
address this, we have systematically deleted
over 40 such “infection associated genes”
and, from these, identified a number of novel
C. albicans pathogenicity mechanisms.
With these approaches, we are elucidating,
at the molecular level, the mechanisms employed by C. albicans to survive, grow within
and damage its host.
Evolutionary adaptation: C. albicans
unique genes
The major fungal pathogens of humans are
distantly related to each other and often
more closely related to non-pathogenic species. Therefore, fungal pathogenicity is a
trait which has been selected independently,
multiple times during evolution. To test the
hypothesis that species-specific genes represent candidate virulence factors, we have performed genome subtraction to identify C. albicans unique genes. Subsequent functional
analysis of seven C. albicans unique genes
46
demonstrated that they are dispensable for
in vitro growth, but play specific roles during
host pathogen interactions. Detailed analysis
of one gene demonstrated a role in both superficial and invasive candidiasis.
Survival: counteracting the stressful
environment during infection
Immune cells are known to impose significant stress on engulfed microbes (see “Interaction with immune cells”). We have deleted
a set of genes which are transcriptionally upregulated upon interactions with phagocytes.
These genes are required for resisting attack
by environmental stresses. For example, the
small heat shock protein Hsp21 governs tolerance to fluctuations in the environment
by modulating MAP kinase signalling and
controlling homeostasis of the stress protectant, trehalose (Fig. 1). We found that Hsp21
was not only required for C. albicans growth
under elevated temperatures and in the presence of oxidants, but also for surviving attack
by human neutrophils and for virulence.
We have also demonstrated that C. albicans
synthesises lipid droplets. These intracellular
organelles act as storage depots for neutral
lipids and contain high levels of triacylglycerols. These esters consist of three fatty acids joined to a glycerol backbone. As glycerol
is the major cellular protectant against osmotic stress, we reasoned that lipid dropletassociated triacylglycerides may serve as a
novel glycerol source and osmoprotectant
pathway. Indeed, salt stress resulted in the
rapid degradation of lipid droplets in wild
type C. albicans cells. We identified and deleted a putative acylglyceride hydrolase. Interestingly, the hydrolase deficient mutant
was unable to degrade lipid droplets in response to cationic stress, defective in osmotic
stress-induced accumulation of intracellular
glycerol and unable to grow in the presence
of cationic stressors.
Figure 1
The novel small heat shock protein,
Hsp21, is required for regulating MAP
kinase signalling and growth under
environmental stress.
Figure 2
A fungal zincophore system. Invasive
C. albicans hyphae secrete a zincbinding protein, Pra1, which sequesters this essential metal from host
cells before reassociating with the
fungus via a cognate receptor, Zrt1.
Growth: feeding on the host with an
extracellular micronutrient scavenger,
the zincophore
The ability of pathogenic microorganisms
to assimilate essential nutrients from their
hosts is critical for pathogenesis. We have
demonstrated endothelial zinc sequestration by Candida albicans. We hypothesised
that, analogous to siderophore-mediated
iron acquisition, C. albicans utilises an extracellular zinc scavenger for acquiring this
essential metal. We postulated that such a
“zincophore” system would consist of a secreted factor with zinc-binding properties,
which can specifically reassociate with the
fungal cell surface. In silico analysis of the
C. albicans secretome for proteins with zinc
binding motifs identified the pH-regulated
antigen 1 (Pra1). Three-dimensional modelling of Pra1 indicated the presence of at least
two zinc coordination sites. Indeed, recombinantly expressed Pra1 exhibited specific
zinc binding properties in vitro. Deletion of
PRA1 in C. albicans prevented fungal sequestration and utilisation of host zinc, and
specifically blocked host cell damage in the
absence of exogenous zinc. Phylogenetic
analysis revealed that PRA1 arose in an ancient fungal lineage and developed synteny
with ZRT1 (encoding a zinc transporter) before divergence of the Ascomycota and Basidiomycota. Structural modelling indicated
physical interaction between Pra1 and Zrt1
and this was confirmed experimentally by
demonstrating that Zrt1 was essential for
binding of soluble Pra1 to the cell surface
of C. albicans. Therefore, we have identified
a novel metal acquisition system consisting
of a secreted zinc scavenger (“zincophore”),
which reassociates with the fungal cell (Fig.
2). Furthermore, functional similarities with
phylogenetically unrelated prokaryotic systems indicate that syntenic zinc acquisition
loci have been independently selected during evolution.
47
Figure 3
Model of Ece1 peptide pore formation
by a novel C. albicans toxin in host cell
membranes.
48
Damage: a dual substrate transporter
and the first fungal pore-forming peptide toxin
Although hypha formation is known to play
a crucial role in pathogenicity at epithelial
surfaces, the exact molecular mechanisms
by which C. albicans destroys these host
cells has remained enigmatic. We have made
recent progress in characterising epithelial
damage at both the molecular and cellular
level.
amines from the environment and functions
in the extracellular alkalinisation pathway.
Importantly, this gene was essential for epithelial damage, resisting attack by immune
effector cells and virulence. Intriguingly,
Dur31 also imports the deadly antimicrobial
peptide, histatin-5. Therefore, a single gene
product can serve to both promote virulence
and kill the pathogen, depending on the host
niche in question. This represents a novel example of pathogen-host co-evolution.
The import and subsequent metabolism of
amine-containing compounds by C. albicans
allows the release of ammonia from the cell
and active alkalinisation of the surrounding environment. This sequence of events
is known as the hyphal auto-induction pathway because an alkaline environmental pH
serves as a strong inducer of C. albicans
hyphal filamentation. We have shown that a
previously unknown function gene, DUR31,
encodes a transporter, which imports poly-
Although hypha formation is known to play a
crucial role in pathogenicity at epithelial surfaces, the exact molecular mechanisms by
which C. albicans destroys these host cells
has remained enigmatic. We have identified a
peptide toxin, secreted by C. albicans, which
has remarkable similarities with mellitin –
the major component of bee venom. By deleting the encoding gene and in vitro synthesis
of the fragment, we have shown that this
peptide is, in itself, essential and sufficient
for lysis of host cells. In collaboration with
Julian Naglik, Kings College London/UK,
and Thomas Gutsmann, Forschungszentrum
Borstel/Germany, we are now examining the
mechanism by which this potent C. albicans
cytolysin disrupts host cell integrity (Fig. 3).
These projects were supported by the Federal
Ministry of Education and Research (BMBF)
within the ERA-Pathogenomics programme
(CandiCol 0315901B), the European Marie
Curie Research Training Network FINSysB
(MCRTN FP7-214004), and the International
Leibniz Research School (ILRS).
2
Nutrient acquisition in infections
Group Leader: Sascha Brunke
The ability to acquire nutrients during infection is essential for the survival and proliferation of pathogenic microbes. In return,
the host fights back by limiting access to
these nutrients, in a process termed “nutritional immunity”. We are interested in how
Candida species still manage to obtain their
essential nutrients in the host, and how this
process is regulated via the transcriptional
networks of the fungi.
One focus of our work is the acquisition of
trace metals, especially iron and zinc. As
transition metals, both play indispensable
roles in a plethora of biological processes, especially as co-factors in many essential enzymes and transcription factors. We mainly
use in vitro models of single metal limitation
to obtain transcriptome data from zinc or
iron starved Candida cells. In close cooperation with the research group Systems Biology / Bioinformatics, we were able to infer
gene regulatory networks from these conditions and define possible gene-gene interactions.
Regulation of C. glabrata and C. albicans
metal acquisition systems
Interestingly, C. glabrata has the ability to
survive and even replicate inside the phago-
some of macrophages (Seider, Brunke et al.
2011), which is generally considered to be
strongly depleted for iron and other metals.
We are therefore especially interested in the
mechanisms C. glabrata employs to obtain
metals in this organelle. Our data indicated
that C. glabrata is able to actively manipulate the pH in the phagosome, which has a
direct influence on the solubility and hence
accessibility of iron. Experimental manipulation of the phagosomal pH indeed inhibits
growth effectively, and C. glabrata mutants
with disrupted iron homeostasis show a reduced survival in macrophages (Seider, Gerwien et al. report 2012/2013). Furthermore,
transcription data suggest that C. glabrata
has iron available inside the phagosome.
Detailed investigations into the type of iron
source and the C. glabrata factors needed for
both the phagosomal manipulation and the
iron uptake are underway.
For C. albicans, we focus on the interconnection of metal acquisition systems, exemplified by zinc and iron. Data obtained with deletion mutants and transcriptome analyses
indicate strong links in detecting the levels
of these two metals. In fact, many transcriptional pathways are shared between the two
metal homeostasis systems, but the precise
response is modulated depending on the specific absent metal. The molecular basis for
this regulation is under investigation using
deletion mutant libraries and transcriptome
analyses (Citiulo, Jacobsen et al. 2012). This
effort to investigate the regulation of C. albicans metal acquisition in the host is a joint
project with the team “Infection Associated
Genes” (see below for more details).
Regulation of nitrogen and carbon
acquisition
Furthermore, in a joint project with the research groups Microbial Biochemistry and
Physiology and Systems Biology / Bioinformatics, we investigate the signal transduction pathways which allow C. albicans to
adapt to available nitrogen sources in the
host. Using the network inference approach,
we investigated the transcriptional response
49
Figure 4
Network of iron homeostasis in
C. albicans. Extensive transcriptional
data was used to infer a regulatory
network model for C. albicans under
iron starvation. The hub-like function
of the HAP complex is clearly visible,
and several novel gene-gene connections were detected.
of C. albicans to typical host-associated nitrogen availability scenarios: nitrogen starvation, presence of free amino acids, and
presence of proteins. We obtained transcription network models of selected genes for all
these conditions (Ramachandra, Linde et al.
2014), which indicate novel network connections involved in nitrogen sensing and uptake. Furthermore, we found that the C. albicans transcription networks themselves are
flexible. Depending on the available nitrogen
source, gene-gene transcriptional interactions were present or absent, shaping the
overall response of the fungus to nutrient
availability.
In a related project, we have analysed the
function of a transcription factor with presumed roles both in iron and carbon uptake.
Here, we were able to describe new functions
for this factor, which connect the use of alternative (non-glucose) carbon sources with
50
available oxygen. Under conditions which resemble growth in the human gut, this transcription factor evidently plays a central role
in carbon source uptake and utilisation.
Altogether, our data indicate a complex and
interconnected transcriptional regulation
in nutrient acquisition. Deciphering these
networks will help to understand how the
fungus survives and multiplies inside the
host during infection. This knowledge will
be helpful in developing novel antifungal
strategies aimed at the metabolism of the
pathogens.
These projects were supported by the Jena
School for Microbial Communication (JSMC)
and by the Deutsche Forschungsgemeinschaft (Hu528/15-1 and 16-1) within the DFG
Priority Progamme SPP1580: Intracellular
compartments as places of pathogen-host interaction (Fig. 4).
3
Evolution, adaptation and
pathogenicity
The ability to adapt to the different niches
in the host is realised in the short term
mainly by changes in the transcriptional
programme of the infecting microbe. In the
long term, however, an optimal adaptation to
the host requires evolutionary processes to
take place. In fact, the host is a highly stressful environment, with few freely available
resources and many immune-related antimicrobial activities. In addition, antifungal
treatments in the clinical setting provide
even more selection pressures on the fungus. We are interested in the ability of Candida species to cope with these stresses by
microevolutionary changes, and how these
adaptive mechanisms can shed light on
pathogenicity factors of the fungus.
Several researchers have previously shown
the ability of fungi to adapt to antifungal
stresses both in vitro and in vivo. Often, a few
or a single nucleotide mutations are sufficient to render a fungus comparatively resistant to a specific class of antifungals – commonly via increased export of the drug or
subtle changes in the target macromolecules.
These strong, but simple abiotic selection
pressures are evidently counteracted by fast
microevolutionary adaptations. Therefore, to
simulate biologically relevant, directly hostrelated stresses, we performed long-term
co-incubation experiments of Candida species with macrophages. These phagocytes
provide a complex selection pressure on the
fungi, which were essentially replicating inside the phagosome for several weeks during
the experiment. Analogous to the antifungal
experimental regimes, we expected adaptations to cope with the prevailing stresses.
C. glabrata and C. albicans adaptation
to macrophage exposure
In a first round, we continuously exposed
C. glabrata to macrophages (Fig. 7). After
several months of constant coincubation, the
fungus changed from its typical yeast growth
to a highly unusual filament-like form. In
this form, it was able to damage the hosting
macrophages quickly and escape. We tested
this evolved variant in our infection models
and found an increased virulence in mice,
with a surprisingly high fungal burden in
the brain and a concomitant increase in inflammatory markers in this organ. By whole
genome sequencing we were able to detect
all mutations in the evolved strain. Via genetic manipulation of the parental wild type,
we finally determined a single nucleotide
exchange as the basis of the novel phenotype. Hence, the complex selection pressure
of macrophages can be circumvented by a
single mutation event, with striking consequences for the growth morphology.
In a similar experiment, we used a non-filamentous mutant of C. albicans which is missing two central transcription regulators
of hyphal formation. Normally, C. albicans
can pierce macrophages from the inside by
hyphae production (Fig. 5), and hence this
strain was unable to escape. After several
weeks of coincubation, this strain regained
its ability to filament, and formed hyphae under most of the tested classically hyphae inducing conditions. Evidently, the need for the
deleted transcription factors was abolished
via a transcriptional rewiring event.
Consequently, the evolved strain regained
most of its virulence, which had been nearly
lost in the parental deletion mutant strain.
Like for C. glabrata, whole genome sequencing in combination with RNAseq approaches
to determine the transcribed alleles allowed
us to determine the mutation events during
the experiment. Again, a single nucleotide
could be found which is associated with the
striking phenotypic changes. We conclude
that C. albicans has robust ways to rewire the
signal transduction pathways of its hyphal
programme, and that filament formation
presents a huge selection advantage under
our experimental conditions.
51
Figure 5
Giemsa staining of a blood smear
after four hours of co-incubation.
Several C. albicans form hyphae to
escape from macrophages after
phagocytosis. The transcriptomes of
both host and fungus are analysed
during these events in our whole
blood model.
Evolution of pathogenicity and
patho-genicity-associated genes
To measure whether C. albicans can adapt
even further to the host during long-term infections, we performed a similar serial passage experiment with mice. After the initial
infection, we re-isolated C. albicans from the
target organ kidney and used these fungi for
the next round of experimental infections.
After eight passages, we detected no evident
changes in virulence or in the in vitro phenotypes, indicating that the fungus is already
well adapted to establish and maintain kidney infections (Lüttich, Brunke et al. 2013).
In another approach, we want to determine
the evolutionary events which allowed certain fungi to become pathogenic. As models,
we use the closely related pairs C. glabrata
and S. cerevisiae (a pathogenic and mostly
non-pathogenic yeast) and C. albicans and
C. dubliniensis (a frequent and an infrequent
human pathogen). In these systems, we
52
search for genes which are either unique to
the pathogen, or have different functions in
the two species.
For C. glabrata, genes were selected which
are a) unique to C. glabrata and not found in
other species like S. cerevisiae, C. albicans,
C. parapsilosis or S. pombe and b) expressed
under infection-simulating conditions, as determined by existing and specifically generated microarry and RNAseq transcriptome
data (Fig. 6). These analyses were performed
in cooperation with the research group Systems Biology / Bioinformatics. Unique, infection-associated genes are now being deleted,
and the resultant mutants are tested both
in vitro and in infection models to determine
their function in pathogenicity.
Similarly, for C. dubliniensis we used two
gene banks: one with C. albicans genome
fragments transferred into C. dubliniensis,
and the other with a reciprocal transfer of
Figure 6
Meta-analyses of published transcriptome data on
C. glabrata unique genes.
Genes which are specific for C. glabrata were selected,
and scored according to their expression under infection-simulating conditions. Heat map of the expression
profiles under different conditions created in cooperation with the Research Group Systems Biology / Bioinformatics.
C. dubliniensis to C. albicans. By selecting
the recipients for phenotypes characteristic for either donor, we can determine the
genetic basis for the strain differences. So
far, genes were found which are for example involved in chlamydospore formation, a
typical property of C. dubliniensis. Further
analyses are under way to solve the riddle
why these two very closely related species
differ in their pathogenicity potential.
These projects were supported by the Jena
School for Microbial Communication (JSMC),
the International Leibniz Research School
(ILRS), and by the Deutsche Forschungsgemeinschaft via the DACH programme grant
“Microevolution of pathogenic yeasts during
interactions with the host immune system”
(Hu 528 17-1).
Figure 7
In vitro microevolution regime.
Yeast cells (either C. glabrata or a C. albicans nonfilamenting mutant) were continuously exposed to
macrophages, which were replenished daily. Constant
selection inside the phagosome induced filamentous
growth in both strains, either as pseudohyphae-like
chains of yeasts (C. glabrata) or via recovery of hyphae
formation (C. albicans).
4
Joint CSCC project: Pathogen-host
interaction during infection with
Candida albicans and its progression to sepsis (Project D1.5)
The relevance of fungal pathogens in the
nosocomial setting is steadily increasing.
As causative agent, the opportunistic pathogenic yeast Candida albicans is the predominant species, with a high mortality due to
sepsis and organ failure despite antifungal
therapy. We aimed to analyse the initial steps
during fungal sepsis, after the pathogen has
entered the blood-stream and dispersed to its
target organs. To this end, we employed an
intravital microscopy model of murine systemic C. albicans infections to gain insight
into the early interaction between the fungus and the murine liver (Fig. 9).
53
References
Citiulo F, Jacobsen ID, Miramón P,
Schild L, Brunke S, Zipfel P, Brock M,
Hube B, Wilson D (2012) Candida
albicans scavenges host zinc via Pra1
during endothelial invasion. PLoS
Pathogens 8, e1002777.
Lüttich A, Brunke S, Hube B, Jacobsen
ID (2013) Serial passaging of Candida
albicans in systemic murine infection
suggests that the wild type strain
SC5314 is well adapted to the murine
kidney. PLoS One 8, e64482.
Ramachandra S, Linde J, Brock M,
Guthke R, Hube B, Brunke S (2014)
Regulatory networks controlling nitrogen sensing and uptake in Candida
albicans. PLoS One 9, e92734.
Seider K, Brunke S, Schild L, Jablonowski N, Wilson D, Majer O, Barz D,
Haas A, Kuchler K, Schaller M, Hube
B (2011) The facultative intracellular
pathogen Candida glabrata subverts
macrophage cytokine production
and phagolysosome maturation. J Immunol 187, 3072-3086.
Seider K, Gerwien F, Kasper L, Allert
S, Brunke S, Jablonowski N, Schwarzmüller T, Barz D, Rupp S, Kuchler
K, Hube B (2014) Immune evasion,
stress resistance, and efficient nutrient acquisition are crucial for intracellular survival of Candida glabrata
within macrophages. Eukaryot Cell
13, 170-183.
We developed a fluorescent C. albicans reporter strain suitable for use in vivo despite
the strong tissue autofluorescence, and modified an established murine intravital microscopy system for use with systemic fungal infections. With this system, we gained
first insight into the temporal and spatial kinetics of early C. albicans-liver interactions.
For this murine intravital microscopy model,
our main findings are: (1) The adhesion to
the murine liver endothelium occurs much
faster than expected. Adhesion of fluorescent
yeasts was observed tens of seconds after initial infection via a central venous catheter.
This adhesion explains the quick clearance
of the fungus from the blood-stream, which
is a well-known, but thus far not fully understood, event in C. albicans infection biology.
For the first time, we could show the fate of
these fungi in vivo (Fig. 9). (2) The binding of
C. albicans to the sinusoids is spatially distinct, as the yeasts bind preferentially in the
distal part of the hepatic lobule, and rarely
close to the central vein. (3) The morphological transition from yeast to hyphae, an
important virulence factor of C. albicans, is
still under investigation for its role in escape
from the blood-stream. To this end, fluorescent mutant strains were created which lack
the key regulators of hyphae formation.
This project was supported by the German
Federal Ministry of Education and Research
(BMBF) within the Integrated Research and
Treatment Center, Center for Sepsis Control
and Care (CSCC) (FKZ 01EO1002).
5
Interaction with immune cells
5.1 Survival of C. glabrata in macrophages
Group Leader: Katja Seider
Candida glabrata is a well-adapted fungal
species that establishes commensal relationships with its human host and beyond that
causes infections under certain predisposing conditions. Previous studies suggest that
immune evasion strategies may be key viru-
54
lence attributes of C. glabrata and that interactions with macrophages, important phagocytic cells of the host’s innate immunity,
may play a critical role in the pathogenesis of
C. glabrata infections (Jacobsen et al. 2010).
Macrophages ingest and destroy microbes
intracellularly, by exposing them to a mixture of acidic pH and highly toxic substances
such as oxidants, proteases and antimicrobial peptides in a specialised compartment
– the phago(lyso)some. However, C. glabrata
can resist killing by macrophages in vitro,
most probably by inhibiting antimicrobial
properties like phagosome maturation, oxidative burst and cytokine production (Seider
et al. 2011). Its intracellular replication results eventually in macrophage lysis, and is
thus a possible escape mechanism of C. glabrata from macrophages.
Screening a targeted gene deletion
library of C. glabrata for macrophage
survival
To gain more insights into the interactions of
C. glabrata with macrophages, we measured
survival rates of 433 C. glabrata deletion mutants, phagocytosed by human primary macrophages (Seider et al. 2013). 23 survivaldefective mutants, with putative functions in
response to stress, cell wall and membrane
architecture, glycosylation and nutritional
sensing and acquisition were identified.
Extensive phenotypic analysis of survivaldefective mutants revealed a relationship
between survival and growth under iron-,
but not carbon- or nitrogen-limitation. Furthermore, stress resistance is crucial for an
intra-phagosomal residence of C. glabrata.
Genes involved in cell surface integrity appeared to be of particular importance for
macrophage survival, too. In this context,
alterations of cell wall composition induced
both a higher production of reactive oxygen
species and the pro-inflammatory cytokine
TNFα by macrophages. These data highlight
the importance of the cell wall as a barrier
shielding yeasts from immune recognition
and substantiate immune evasion as a possible virulence strategy of C. glabrata.
Figure 8
Phases of C. albicans infection. After adhesion, C. albicans
penetrates the epithelial barrier by hyphae formation or
via injuries to the tissue. After dissemination via the bloodstream, fungal cells enter the target organs. In this project,
we investigate these early steps after dissemination.
Furthermore, detailed in vitro characterisation of three selected mutants (cch1Δ, slm1Δ,
vhr1Δ) point to a role of Ca2+/calcineurin- and
sphingolipid/TORC2-dependent signalling as
well as biotin-regulatory processes in coping
with macrophage-generated stresses.
Modulation of environmental and
phagosomal pH by C. glabrata
Because C. glabrata-containing phagosomes
lack acidification, the possibility of active pH
modulation by C. glabrata was analysed in vitro. In fact, C. glabrata is able to alkalinise
its extracellular environment, when growing
on amino acids as the sole carbon source. By
screening the C. glabrata mutant library we
identified genes important for extracellular
alkalinisation, which were further tested
for phagosomal pH. We found that the lack
of fungal mannosyltransferases resulted in
severely reduced alkalinisation in vitro and
in the delivery of C. glabrata to acidified
phagosomes. Therefore, protein mannosyl-
Figure 9
C. albicans in the murine liver.
Yeast cells attach to the sinusoids of the liver
lobule just seconds after injection into the
blood-stream. Using a fluorescent reporter
strain (mCherry), the fungus can be visualised
in the living animal via intra vital microscopy.
Blue, tissue autofluorescence; red, C. albicans
mCherry. Hepatic lobule at 63×.
ation may play a key role in alterations of
phagosomal properties caused by C. glabrata
(Fig. 10).
5.2 Interaction of Candida albicans with
neutrophils
Group Leader: Lydia Kasper
Neutrophils are key phagocytic cells, which
play an extremely important role in the innate
immune response and are the phagocytes
with the most potent candidacidal activity.
In contrast to C. albicans cells phagocytosed
by macrophages, C. albicans cells are unable
to form filaments inside the neutrophil. This
means that the fungus is unable to escape
from this phagocyte, hence it is effectively
killed. Antimicrobial mechanisms of neutrophils range from intracellular killing mechanisms, by exposing fungal cells to a mixture
of highly toxic substances such as oxidants,
55
Figure 10
C. glabrata (green) internalised by
macrophages (yellow). Red: LysoTracker staining showing acidic compartements within macrophages.
Blue: macrophage nuclei.
proteases and antimicrobial peptides, to extracellular inhibition of growth by releasing
the content of their granules to the extracellular milieu.
Single cell profiling
The contributions of the intra- and extracellular antifungal activities of human neutrophils
on the transcriptional response of C. albicans
were studied. For this purpose, C. albicans
strains expressing reporter gene fusions to
the Green Fluorescent Protein (GFP) were
used in a differential staining experiment
to determine the fungal transcriptional response at the single cell level of internalised
compared to non-phagocytosed cells.
It was found that a carbohydrate starvation response, indicated by up-regulation of
genes encoding proteins of the glyoxylate
cycle, was induced in C. albicans only in the
intracellular environment of the neutrophil.
Likewise, only internalised fungal cells were
56
exposed and responded to nitrosative stress.
On the other hand, the oxidative stress response was observed in both phagocytosed
and non-phagocytosed fungal cells, demonstrating that this type of stress is encountered inside and outside of the neutrophil.
Mutant analysis and oxidative stress
response of C. albicans
To assess the actual contributions of carbohydrate starvation, oxidative and nitrosative
stress in the killing of C. albicans, mutants
lacking key glyoxylate cycle, oxidative and
nitrosative stress genes were tested in their
susceptibility to neutrophil killing. The glyoxylate cycle was found to be crucial for normal survival of C. albicans cells exposed to
neutrophils.
In the context of oxidative stress, C. albicans
expresses genes coding for detoxifying enzymes, in order to cope with the oxidative
and nitrosative insult exerted by neutrophils
References
Fradin C, De Groot P, MacCallum D,
Schaller M, Klis F, Odds FC, Hube B
(2005) Granulocytes govern the transcriptional response, morphology
and proliferation of Candida albicans
in human blood. Mol Microbiol 56,
397-415.
Frohner IE, Bourgeois C, Yatsyk K,
Majer O, Kuchler K (2009) Candida
albicans cell surface superoxide
dismutases degrade host-derived
reactive oxygen species to escape
innate immune surveillance. Mol
Microbiol 71, 240-252.
Jacobsen ID, Brunke S, Seider K,
Schwarzmüller T, Firon A, d'Enfért
C, Kuchler K, Hube B (2010) Candida
glabrata persistence in mice does not
depend on host immunosuppression
and is unaffected by fungal amino
acid auxotrophy. Infect Immun 78,
1066-1077.
Figure 11
Microphotograph of a C. albicans reporter strain
expressing a GFP-tagged actin1 protein (green)
when incubated in RPMI medium supplemented
with 5 % FBS.
(Fradin et al. 2005). The oxidative stress
response effectors contribute differentially
to the normal outcome of the interaction.
Mutants lacking superoxide dismutase 5
(Sod5), glutathione reductase 2 (Grx2) or
a novel family of glutathione peroxidases
(Gpx31-33), rendered C. albicans hypersensitive to neutrophil killing (Miramon et al.
2013, Frohner et al. 2009). In contrast, catalase (Cta1) and thioredoxin (Trx1) seem to be
dispensable for coping with the neutrophilinduced oxidative stress. Using a C. albicans
mutant lacking the nitric oxide dioxygenase
Yhb1 it was demonstrated that nitrosative
stress generated by neutrophils is a relevant
stress that C. albicans faces as a killing
mechanism.
phil survival since mutants lacking this protein were clearly more susceptible to killing.
In addition, proteins involved in the synthesis of trehalose and glycerol, which expression is coordinated by Hog1, were needed for
full resistance of C. albicans to neutrophils.
These projects were financed by the Deutsche
Forschungsgemeinschaft (Hu528/15-1 and
16-1) within the DFG Priority Progamme
SPP1580: Intracellular compartments as
places of pathogen-host interaction; and the
European Commission through the FINSysB
Marie Curie Initial Training Network (PITNGA-2008-214004) (Fig. 11).
Moreover, the contribution of fungal stress
regulators to normal survival of C. albicans
upon neutrophil attack was assessed. The
stress regulator Hog1 and the oxidative stress
regulator Cap1 play a pivotal role in neutro-
Miramón P, Dunker C, Windecker H,
Bohovych IM, Brown AJ, Kurzai O,
Hube B (2012) Cellular responses of
Candida albicans to phagocytosis
and the extracellular activities of
neutrophils are critical to counteract
carbohydrate starvation, oxidative
and nitrosative stress. PLoS One 7,
e52850.
Miramón P, Dunker C, Kasper L,
Jacobsen ID, Barz D, Kurzai O, Hube B
(2014) A family of glutathione peroxidases contributes to oxidative stress
resistance in Candida albicans. Med
Mycol 52, 223-239.
Seider K, Brunke S, Schild L, Jablonowski N, Wilson D, Majer O, Barz D,
Haas A, Kuchler K, Schaller M, Hube
B (2011) The facultative intracellular
pathogen Candida glabrata subverts
macrophage cytokine production
and phagolysosome maturation.
J Immunol 187, 3072-3086.
Seider K, Gerwien F, Kasper L, Allert
S, Brunke S, Jablonowski N, Schwarzmüller T, Barz D, Rupp S, Kuchler K,
Hube B (2013) Immune evasion, stress
resistance, and efficient nutrient
acquisition are crucial for intracellular survival of Candida glabrata
within macrophages. Eukaryot Cell
13, 170-183.
57
Group members
Visiting Scientists
Head
Prof. Dr. Bernhard Hube
Phone +49 3641 532-1401
Fax
+49 3641 532-0810
[email protected]
Inês Correia
Universidad Complutense de Madrid, Spain
04/2013–07/2013
Josephine Fischer
Westfälische Wilhelms-Universität Münster
11/2013
Secretary
Petra Flemming
Scientists
Dr. Sascha Brunke
Dr. Francesco Citiulo (until 03/2012)
Dr. Betty Hebecker (joint project with MI, since
08/2013)
PD Ilse D. Jacobsen, PhD (Head MI, since 08/2013)
Dr. Lydia Kasper
Dr. François Mayer (since 10/2013)
Dr. Katja Seider
Dr. Duncan Wilson
Ph.D. Students
Stefanie Allert (since 11/2013)
Bettina Böttcher (since 01/2013)
Christine Dunker (MI, since 06/2013)
Daniel Fischer (since 04/2013)
Toni Förster (since 10/2013)
Franziska Gerwien
Katharina Große (until 06/2012)
Sarah Höfs
Philipp Kämmer (since 11/2012)
Anja Lüttich
François Mayer (until 12/2012)
Pedro Miramon (until 12/2013)
Tony Pawlik (joint project with MI, since
04/2013)
Melanie Polke (joint project with MI, since
08/2013)
Shruthi Ramachandra (until 09/2013)
Florian Roas (until 12/2012)
Volker Schwartze
Katja Seider
Volha Skrahina (since 09/2012)
María Cristina Albán Proaño (10/2012–11/2013)
Stefanie Allert (10/2012–10/2013)
Erik Borchert (10/2012–11/2013)
Christine Dunker (until 05/2013)
Daniel Fischer (01/2012–03/2013)
Alexa Lauer (since 10/2013)
Camilo Juan Molina Rodriguez (until 01/2012)
Christine Pöllath (since 11/2013)
Maria Schreiner (until 04/2012)
Marcel Sprenger (since 10/2013)
Elisabeth Weiß (10/2012–11/2013)
Cristina Zubiria Barrera (since 10/2013)
Research Assistants
Nadja Jablonowski
Daniela Schulz (since 04/2013)
Birgit Weber (MI, since 08/2013))
Stephanie Wisgott
Trainees
Dorothee Eckhardt (since 08/2013)
Madlen Peisker (09/2013–10/2013)
Jessica Pötschner (08/2013–08/2013)
58
Dr. Nayla Jbeily
University Hospital Jena
05/2013–12/2013
Jessica Quintin
Radboud University Nijmegen Medical Centre,
The Netherlands
03/2013–04/2013
Barbara Schaarschmidt
03/2013–05/2013
External funding
ERA-Net – CandiCol
Grundlagen zur Kolonisation und der
Dissemination pathogener CandidaSpezies: Entwicklung früher Diagnose- und
Therapieansätze
Bernhard Hube
Integriertes Forschungs- und
Behandlungszentrum (IFB) Sepsis und
Sepsisfolgen (CSCC)
Project: Pathogen-Wirt-Interaktion während
der Infektion mit Candida albicans und die
Entwicklung zur Sepsis
Bernhard Hube, Sascha Brunke
Integriertes Forschungs- und
Behandlungszentrum (IFB) Sepsis und
Sepsisfolgen (CSCC)
In-house Professorship Molekulare
Mechanismen der Candida-Sepsis
Bernhard Hube, Ilse Jacobsen
Project: MicroCom – Host pathogen
interactions of human pathogenic yeast
Bernhard Hube
Project. Infection-associated genes of
Candida glabrata
Bernhard Hube
SPP 1580: Intracellular compartments as
places of pathogen-host interaction
Phagosom – survival and proliferation of
human pathogenic Candida species within
phagocytes
Bernhard Hube
DACH Evolution – Microevolution of
pathogenic yeasts during interactions with
the host immune system
Bernhard Hube
Chlamydosporen – Identifizierung und Analyse
von Chlamydosporen-und Pathogenitätsassoziierten Genen in Candida albicans
Bernhard Hube
SFB/TR FungiNet
Project C01 – Charakterisierung molekularer
Mechanismen der Translokation von Candida
albicans durch Epithelgewebe
Bernhard Hube
European Union
Framework Programme 7 Marie Curie Action
Initial Training Network
FINSysB – Pathogenomics and Systems Biology
of Fungal Infections – An Integrative Approach
Bernhard Hube
Lüttich A, Brunke S, Hube B, Jacobsen ID
(2013) Serial passaging of Candida albicans in
systemic murine infection suggests that the
wild type strain SC5314 is well adapted to the
murine kidney. PLoS One 8, e64482.
Mayer FL, Wilson D, Hube B (2013) Hsp21
potentiates antifungal drug tolerance in
Candida albicans. PloS One 8, e60417.
Voigt J, Hünniger K, Bouzani M, Jacobsen ID,
Barz D, Hube B, Löffler J, Kurzai O (2013)
Human natural killer cells acting as phagocytes
against Candida albicans and mounting
an inflammatory response that modulates
neutrophil antifungal activity. J Infect Dis 209,
616-626.
Wilson D, Hebecker B, Moyes DL, Miramón P,
Jablonowski N, Wisgott S, Allert S, Naglik JR,
Hube B (2013) Clotrimazole dampens vaginal
inflammation and neutrophil infiltration
in response to Candida albicans infection.
Antimicrob Agents Chemother 57, 5178-5180.
Brunke S, Hube B (2013) Two unlike cousins:
Candida albicans and C. glabrata infection
strategies. Cell Microbiol 15, 701-708.
Collaborations
Bayer Vital GmbH, Leverkusen, Germany
Dr. Norbert-Heinz Becker
Prof. Dr. Alexander Berndt
Georg August University Göttingen, Germany
Prof. Dr. Uwe Groß
Heinrich Heine University Düsseldorf, Germany
Prof. Dr. Joachim Ernst
Humboldt University Berlin, Germany
Prof. Dr. Edda Klipp
INRA - Institut National de la Recherche
Agronomique, Paris, France
Dr. Mathias Richard
Institut Pasteur, Paris, France
Prof. Dr. Christophe d‘Enfert
Instituto de Agroquímica y Tecnología de
los Alimentos (CSIC), Departamento de
Biotecnología, Spain
Dr. Amparo Querol
University of Vienna, Kuchler Laboratory,
Austria
Prof. Dr. Karl Kuchler
VIB Department of Molecular Microbiology K.U.
Leuven Laboratory of Molecular Cell Biology,
Belgium
Prof. Dr. Patrick van Dijck
Westfälische Wilhelms University, Laboratory of
Molecular Stem Cell Biology, Münster, Germany
Prof. Dr. Frank Rosenbauer
ZIK Septomics, Research group Host Septomics,
Jena
Prof. Dr. Hortense Slevogt
King's College London Dental Institute, UK
Dr. Julian Naglik
Leibniz Institute for Age Research –
Fritz-Lipmann-Institute, Jena, Germany
Prof. Dr. Falk Weih
Max von Pettenkofer-Institut, Ludwig
Maximilians University, München, Germany
Prof. Dr. Frank Ebel
Radboud University Nijmegen Medical Centre,
Nijmegen, Netherlands
Prof. Dr. Mihai Netea
School of Dental Medicine, New York, USA
Prof. Dr. Mira Edgerton
Prof. Dr. Michael Bauer
Prof. Dr. Konrad Reinhart
Prof. Dr. Niels Riedemann
University Hospital Tübingen, Germany
Prof. Dr. Martin Schaller
University of Aberdeen, UK
Prof. Dr. Andrew Parker
University of Aberdeen & Aberdeen Fungal
Group, UK
Dr. Alexandra Brand
Prof. Dr. Alistair Brown
Prof. Dr. Neil Gow
University of Amsterdam, Netherlands
Prof. Frans Klis
University of Bonn, Institute for Cell Biology,
Germany
Prof. Dr. Albert Haas
University Dijon, France
Dr. Frédéric Dalle
University of Perugia, Italy
Prof. Dr. Anna Vecchiarelli
University of Szeged, Dept. of Microbiology,
Hungary
Dr. Attila Gacser
59
60
Department of
Molecular and Applied Microbiology
61
Department Molecular and Applied Microbiology
The Department of Molecular and Applied
Microbiology (MAM) is devoted to research
in the two main areas of the HKI, i.e. infection biology of fungi and natural product research (Fig. 1). The department studies the
ascomycetes Aspergillus fumigatus and Aspergillus nidulans but also zygomycetes. Furthermore, the department MAM sequenced
the genomes of dermatophytes, a group of
fungi which cause infections of the skin,
hair and nails by colonising keratin tissue.
Recently the department started research on
Streptomyces rapamycinicus, which induces
distinct silent gene clusters in A. fumigatus
and A. nidulans. Furthermore, the department is also producing compounds using
innovative techniques for further testing in
animal models. The departments is organised in seven subgroups reflecting the research areas (Fig. 2).
Aspergillus fumigatus has become the most
important airborne fungal pathogen in
humans. It causes different diseases ranging
from allergies to systemic, life-threatening
infections. Individuals at risk for developing
invasive aspergillosis are immunocompromised patients. This is particularly true for
patients with haematological malignancies
and patients undergoing bone marrow transplantation. On average, between 5 - 15 % of individuals of these patient groups acquire invasive aspergillosis during the course of their
underlying diseases. Invasive aspergillosis
Die Abteilung Molekulare und Angewandte
Mikrobiologie (MAM) widmet sich den beiden Forschungsschwerpunkten des HKI, d. h.
der Infektionsbiologie von Pilzen und der
Naturstoff-Forschung (Fig. 1). Die Abteilung
untersucht die beiden Ascomyzeten Aspergillus fumigatus und Aspergillus nidulans,
aber auch Zygomyzeten, ergänzt durch die
Untersuchung des Bakteriums Streptomyces rapamycinicus, welches spezifisch stille
Gencluster von A. nidulans und A. fumigatus
induziert. Die Abteilung MAM führte außerdem die Genomsequenzierung und biologische Charakterisierung von Dermatophyten
durch. Dermatophyten, sogenannte Hautpilze, können oberflächliche Infektionen der
Haut, der Haare und Nägel auslösen und stellen den häufigsten Erreger pilzlicher Erkrankungen beim Menschen dar. Die Abteilung
produziert mit Hilfe neuer Techniken Naturstoffe, die in verschiedenen Infektionsmodel-
Head:
Prof. Dr. Axel A. Brakhage
62
len getestet werden. Die Abteilung MAM ist
in sieben Untergruppen gegliedert (Fig. 2).
Aspergillus fumigatus ist medizinisch gesehen der wichtigste, über die Luft übertragbare human-pathogene Pilz. Er verursacht
verschiedenste Krankheitsbilder beim Menschen. Das Spektrum umfasst allergische
Symptome bis hin zu systemischen, lebensbedrohlichen Infektionen. Vor allem immunsupprimierte Patienten sind besonders
anfällig für eine invasive Aspergillus-Infektion. Dies trifft insbesondere auf Patienten
mit hämatologischen Tumoren und nach
Knochenmarkstransplantation zu. Im Durchschnitt erkranken 5 – 15 % dieser Patientengruppe an einer invasiven Aspergillose, die
in ca. 50 % der Fälle tödlich verläuft. Die
hohe Sterblichkeit liegt vor allem darin begründet, dass die Diagnose invasiver Aspergillosen schwierig ist und nur eine geringe
HKI Research Report 2012 / 2013 Department of Molecular and Applied Microbiology
is associated with high mortality because
diagnostic and therapeutic options are inefficient. Furthermore, the pathophysiology of
A. fumigatus infections is poorly understood.
Scientists of the department MAM study diverse aspects of the biology and virulence of
A. fumigatus including signal transduction,
the biosynthesis of secondary metabolites,
the improvement of genomic, transcriptomic
and proteomic tools and host-pathogen interaction.
Fungi produce numerous secondary metabolites including mycotoxins, antibiotics
such as penicillin, immunosuppressants
like cyclosporin and cholesterol lowering
drugs, e.g. lovastatin (Brakhage 2013). By
Zahl antimykotischer Therapeutika zur Verfügung steht. Zusätzlich sind die Pathogenitätsmechanismen von A. fumigatus bisher
kaum verstanden. Wissenschaftler der MAM
forschen an vielen Aspekten der Biologie
und Virulenz des Schimmelpilzes A. fumigatus, so. z. B. an der Signaltransduktion, der
Biosynthese von Sekundärmetaboliten und
deren Bedeutung für die Virulenz, der Anwendung und Verbesserung genomischer,
transkriptomischer und proteomischer Methoden sowie der Charakterisierung der Pathogen-Wirt Interaktion.
Pilze produzieren eine Vielzahl an Sekundärmetaboliten, z. B. Mykotoxine, Antibiotika
wie Penicillin, Immunsuppressiva und Cholesterin-senkende Wirkstoffe wie Lovastatin.
Mit Hilfe des Modellorganismus Aspergillus
nidulans versucht die Abteilung MAM aufzuklären, welche Regulationsmechanismen
using Aspergillus nidulans as a model organism the department uncovers the regulatory pathways and physiological conditions
which lead to the biosynthesis of secondary
metabolites (Fig. 3). In collaboration with
other departments of the HKI we focus on
the search and characterisation of new secondary metabolite gene clusters and their
molecular regulation. In this context, we
have discovered a novel principle of activation based on the interaction of A. nidulans
with a Gram-positive soil bacterium, Streptomyces rapamycinicus, which reprograms the
histone modification machinery of the fungus (Schroeckh et al. 2009, Nützmann et al.
2011, Nützmann et al. 2013).
und welche physiologischen Bedingungen
dazu führen (Fig. 3), dass Sekundärmetabolite produziert werden (Brakhage 2013). In
Zusammenarbeit mit anderen Abteilungen
sucht die MAM nach neuen, unbekannten
Sekundärmetaboliten, die auf genetischer
und biochemischer Ebene charakterisiert
werden. Mitarbeitern der Abteilung ist es
gelungen, ein neues Prinzip der Aktivierung
sogenannter stiller Gencluster zu entdecken:
Bei der direkten Interaktion des Gram-positiven Bodenbakteriums Streptomyces rapamycinicus werden spezifische stille Gencluster
sowohl in A. nidulans als auch A. fumigatus
aktiviert, die zur Produktion neuer Substanzen führen. Zumindest für A. nidulans konnte gezeigt werden, dass das Bakterium in der
Lage ist, die Histonmodifikationsmaschinerie im Pilz umzuprogrammieren (Schroeckh
et al. 2009, Nützmann et al. 2011, Nützmann
et al. 2013).
Department of Molecular and Applied Microbiology HKI Research Report 2012 / 2013
63
Scientific Projects
Figure 1
Research areas of the Department
of Molecular and Applied Microbiology.
The research areas combine various
aspects of infection biology and
natural product research with emphasis on microbial communication
and the importance of the produced
compounds as infochemicals.
1
Infection biology of
Aspergillus fumigatus
Group Leaders: Thorsten Heinekamp,
Axel A. Brakhage
In this project, we attempt to identify pathogenicity determinants of A. fumigatus and
their regulation. Also, the interaction between immune effector cells and the fungus
has been analysed. Below, a selection of research topics is summarised.
G protein-coupled receptors
of A. fumigatus
The capability to grow on a wide variety of
nutrients and its arsenal of virulence factors enable A. fumigatus to infect and colonise the human body. For the response to
changing environmental conditions during
infection, A. fumigatus contains a number of
sensing and signalling mechanisms including G protein-coupled receptors (GPCRs).
64
GPCRs form the largest group of membrane
receptors among eukaryotic organisms. Until
now, little is known about the stimuli and
signal transduction mechanisms associated
with the 15 predicted GPCRs in the genome
of A. fumigatus (Gehrke et al. 2009). Therefore, to understand their impact on fungal
growth, development and pathogenicity, all
GPCR-encoding genes were deleted. Various phenotypes of the deletion mutants were
observed such as a significant decrease in
spore formation and growth rate. A major
question concerning receptors of all organisms is the identification of agonists and
antagonists modulating their activity. Using
the “Biolog” System, we were able to identify
metabolites that led to significantly changed
growth of GPCR knock-out strains compared
to the wild type, suggesting that the recognition and/or corresponding signal transduction processes might be altered in these mutant strains. Interestingly, addition of excess
metabolites complemented the growth defect
Infec.on Biology of Aspergillus fumigatus Microbial Communica.on and Natural Products Thorsten Heinekamp Stress-‐ and Immunoproteomics Olaf Kniemeyer Volker Schroeckh Molecular and Applied Microbiology Transcrip.on Factors and Signal Transduc.on Molecular Biology of Natural Products Daniel Scharf Peter Hortschansky Zygomycetes – Pathogenicity and Biodiversity Molecular Biotechnology of Natural Products Kers:n Voigt Vito Valiante Special Methods:
Proteomics platform
Surface Plasmon Resonance Analyses
of the respective mutants, giving a strong
hint that the corresponding GPCRs are important for sensing these molecules. The ongoing analysis allows defining the function
of different GPCRs in this important opportunistic filamentous fungus.
Identification of a novel compound
regulated by protein kinase A in
A. fumigatus
After perception of environmental stimuli by
fungal receptors, intracellular signal transduction in A. fumigatus is mediated via several pathways. One of these signalling cascades is the cyclic AMP dependent protein
kinase A (PKA) pathway, which was shown
to be involved in virulence of A. fumigatus. In
a transcriptomics approach putative targets
of this signalling cascade were identified.
Among these were several transcriptional
regulators. The transcription factor gene,
which showed highest up-regulation by PKA
is located in a natural product gene cluster.
Figure 2
Research groups of the Department of Molecular and Applied
Microbiology.
Deletion of the corresponding gene and of
a non-ribosomal peptide synthetase (NRPS)
gene located in the cluster led to reduced
fungal growth and sporulation. Overproduction of the transcription factor resulted in
formation of a brown substance identified as
the novel compound fumipyrrole.
Interaction of A. fumigatus with macrophages – Interference of A. fumigatus
with phagolysosomal vATPase assembly
Human pathogenic microorganisms have
evolved a multitude of highly different immune evasion strategies for establishing
their pathogenic life style inside the host
(Brakhage et al. 2010). The pathogen mimics
or alters host structures thereby preventing
or diminishing the immune response. Especially professional phagocytes are the target
of manipulation by intruding microorganisms, e.g. by reprogramming the host endocytic pathway to prevent the formation of a
hostile phagolysosome and thereby generat-
65
Figure 3
Global regulatory proteins involved in the regulation of secondary metabolism gene clusters
in various fungi.
Environmental signals can influence
the regulation of various secondary
metabolism gene clusters through
regulatory proteins that respond to
these environmental stimuli and, in
turn, modulate the expression of the
clusters. Shown is a model secondary
metabolism gene cluster containing
a gene encoding a central non-ribosomal peptide synthetase (NRPS), a
polyketide synthase (PKS) or a hybrid
PKS–NRPS enzyme. CBC, CCAATbinding complex; CpcR1, cephalosporin C regulator 1; LaeA, loss of
aflR expression A; RsmA, restorer of
secondary metabolism A; SAGA-ADA,
Spt–Ada–Gcn5–acetyltransferaseADA (taken from Brakhage 2013, Nat
Rev Microbiol).
ing a compartment in which the pathogen
not only survives but also replicates until
it is released. However, only little is known
about how pathogenic filamentous fungi
avoid intracellular killing by phagocytes.
In lung alveoli, resident alveolar macrophages represent the first line of defence
against inhaled conidia of A. fumigatus. Recently, we showed that depending on dihydroxynaphthalene (DHN) melanin, the gray
green conidial pigment, A. fumigatus prevents acidification of phagolysosomes and
thereby counteracts degradation by phagozytes (Thywißen et al. 2011, Heinekamp
et al. 2013). We elucidated the mechanism
contributing to the regulation of the host
vATPase activity during phagosomal maturation. By interference of the DHN-melanin
polymer with the recruitment of lipid rafts,
small membrane microdomains rich in cholesterol and sphingolipids, A. fumigatus
prevents assembly of the vATPase at the
66
phagolysosomal membrane. Lipid rafts, but
not caveolae contribute as an intracellular
platform essential for maturation of phagolysosomes. We showed that pathogenic fungi
manipulate this system to generate a niche
allowing them to survive within the host.
Interaction of A. fumigatus with neutrophil granulocytes – Detoxification
of reactive nitrogen intermediates by
A. fumigatus and the role for nitric oxide
in regulation of neutrophil extracellular
trap formation
In the lung, spores (conidia) of A. fumigatus
are confronted with immune effector cells.
Macrophages and neutrophil granulocytes
phagocytose conidia and attack them intracellularly by various fungicidal mechanisms
including antimicrobial proteins. Neutrophils
also attack hyphae and employ extracellular
mechanisms like degranulation or neutrophil
extracellular trap (NET) formation (Bruns
et al. 2010). Additionally, macrophages and
neutrophils form nitric oxide (NO) and other
reactive nitrogen intermediates (RNI), whose
function in defence against A. fumigatus is
a matter of debate. Based on a dual RNAseq
approach including the analysis of the transcriptome of both the fungus and neutrophils, we showed that A. fumigatus produces
several enzymes potentially detoxifying RNI,
namely two flavohemoglobins, FhpA and
FhpB, and the S-nitrosoglutathione reductase
GnoA. To elucidate the role of these enzymes,
single and double deletion mutants of FhpA,
FhpB and GnoA encoding genes were generated.
Our data indicate that FhpA and GnoA are
the primarily responsible enzymes in A. fumigatus to counteract RNI. Interestingly,
based on the finding that human neutrophil
granulocytes showed a significant reduction
in NETosis when either confronted with hyphae of A. fumigatus mutants deficient for
RNI-detoxification or by addition of exogenous NO donors, we were able to attribute
NO a role as negative feedback regulator of
NET formation.
Interaction of Aspergillus fumigatus
with Pseudomonas aeruginosa
Cystic fibrosis (CF) is characterised by
chronic airway infections with opportunistic
pathogens. Pseudomonas aeruginosa is the
predominant bacterial pathogen in CF-lungs
and until now it is unclear how P. aeruginosa
interacts with the airborne fungal pathogen
A. fumigatus. To get first insights into putative inter-microbial communication processes between A. fumigatus and P. aeruginosa,
co-incubation experiments were performed.
A direct physical contact between both microorganisms was revealed by electron microscopy. Fungal growth was inhibited by
P. aeruginosa culture filtrate. Interestingly,
culture filtrate of the quorum sensing defective mutant ΔlasR was significantly less efficient in affecting fungal growth. Therefore,
P. aeruginosa molecules regulated by LasR
seem to be involved in fungal growth inhibition. In cell culture experiments the supernatant of the A. fumigatus-P. aeruginosa
co-culture revealed reduced cytotoxicity and
reduced anti-proliferative activity in comparison to the A. fumigatus control. These
findings led to the hypothesis that both microorganisms are able to detoxify each other
to enable prolonged coexistence during infection.
2
Stress and Immunoproteomics
Group Leader: Olaf Kniemeyer
The release of genome sequences of the filamentous fungi Aspergillus fumigatus, Aspergillus nidulans and the dermatophyte Arthroderma benhamiae (Burmester et al. 2011)
have paved the way for global studies on the
entire proteome and its dynamic changes
during specific developmental stages, stress
conditions or during the interaction with
immune effector cells (Kroll et al. 2014). In
the following, we report some exemplary research projects of the group “Stress and Immunoproteomics”.
Analysis of the Aspergillus fumigatus
response to low atmospheric oxygen
levels (hypoxia)
The ability to adapt to low oxygen levels
seems to be an important trait of A. fumigatus to be able to cause infections in humans
with impaired immune system. Therefore,
together with co-operation partners in the
USA (R. Cramer), we aimed at obtaining a
comprehensive overview about this process
on the transcript and protein level. We investigated the dynamic changes of the A. fumigatus transcriptome and proteome in hypoxia over a period of 24 hours utilising an
oxygen-controlled fermenter system.
Interestingly, significant increases in transcripts associated with iron and sterol metabolism, the cell wall and transcriptional regulators were observed in response to hypoxia.
On the protein level an increase of enzymes
involved in glycolysis was detected. Thus,
the hypoxic response of A. fumigatus is regu-
67
Figure 4
Light microscopic image (left) and
fluorescence emission (middle) from
mycelium of Aspergillus fumigatus
wild type and ΔhorA mutant strain
stained with MitoTracker Red, which
stains mitochondria in living cells. Its
accumulation is dependent on the
mitochondrial membrane potential.
The right pictures show an overlay
of the light microscopic and fluorescence microscopic image.
lated at both the transcriptional and posttranscriptional level. During the short term
response to hypoxic conditions, the observed
induction of ergosterol biosynthesis genes
may represent a strategy to compensate for a
temporary deficit of sterols, whose biosynthesis requires molecular oxygen. Because the
enzymes of the ergosterol pathway also need
iron as a co-factor, the increase of transcripts
of iron-uptake genes is consistent with an increased need for iron under hypoxia. In addition, the level of some proteins of the respiratory mitochondrial machinery increased in
abundance during hypoxia.
Besides the regulatory circuits controlling
the hypoxic response, our study provides candidate genes for further analysis of their role
in hypoxia adaptation and fungal virulence.
This includes a flavohemoprotein which is
known to be involved in the detoxification
of NO and which was clearly induced at the
early phase of hypoxia. To characterise its
68
role during hypoxic growth a deletion mutant
was created. The mutant showed increased
sensitivity towards reactive nitrogen species;
however, growth under hypoxic condition
was not reduced. This suggests that the level
of reactive nitrogen species increases during
hypoxia. However, the presence of the NOdetoxifying flavohemoprotein is not essential
at low oxygen levels.
A hypoxia-induced short-chain dehydrogenase HorA was another candidate protein,
which was investigated in more detail. Deletion of the corresponding gene revealed
interesting phenotypes: at atmospheric oxygen levels growth of the mutant was significantly reduced. Furthermore, the ∆horA mutant was attenuated in virulence, but showed
increased resistance towards antifungal
agents of the triazole group. These and further results along with the evidence of mitochondrial localisation suggest that HorA represents a new short-chain dehydrogenase
which is associated with the mitochondrial
respiratory complex. HorA is essential for
mitochondrial functionality (Fig. 4).
promoted the adhesion of fungal spores and
extracellular matrix production and led to an
eDNA-specific structure of the biofilm (Shopova et al. 2013).
Another hypoxia-induced gene was identified
as a member of the large protein family of
sensor globins. The function of these proteins
is largely unknown, but heterologous expression and purification gave evidence for a
functional heme-binding site. To characterise
the protein’s contribution to hypoxic survival
the corresponding gene was deleted in A. fumigatus. Interestingly, the mutant showed
impaired growth in low oxygen atmospheres
and hence, it may play role during adaptation
to hypoxic conditions.
In collaboration with the Aspergillus unit at
the Institut Pasteur, Paris, we compared the
different transcriptional and translational
signatures of biofilm-grown mycelium in
comparison to submerged A. fumigatus cultures. A. fumigatus biofilms produced more
antigens, the hydrophobic surface protein
RodB and mycotoxins. The overexpression
of several efflux pumps may explain the increased resistance of A. fumigatus biofilms
to antifungals.
Functional genomic profiling of
Aspergillus fumigatus biofilms
Besides disseminated forms of disease in
immunocompromised patients Aspergillus
fumigatus is able to cause local infections in
immunocompetent individuals. Particularly,
cystic fibrosis patients are at risk for developing locally restricted respiratory tract infections. Airway inflammation is the typical
symptom for this disease, which is referred
to as allergic bronchopulmonary aspergillosis (ABPA). To establish such an infection,
spores of A. fumigatus have to adhere to the
surface of epithelial cells and, as a second
step, start to germinate. Later, a biofilm-like
structure is formed comprising dense hyphal
networks embedded in an adhesive extracellular matrix built predominantly of polysaccharides (Bruns et al. 2010). Fungal biofilms
are likely to be of clinical relevance, as they
have been observed in patients. Interestingly, in vitro experiments also showed their
drastically reduced susceptibility to antifungal drugs. Little is known about the environmental factors which trigger the formation
of biofilms in A. fumigatus. Since extracellular DNA (eDNA) released from white
blood cells is an abundant molecule in the
mucus-rich airway surfaces of CF patients,
we investigated the effect of extracellular
DNA on A. fumigatus biofilm establishment.
Using an in vitro biofilm model eDNA was
identified as an efficient biofilm inducer. It
The immunoproteome of
2D-gel electrophoresis can be easily combined with Western blotting. The blotted proteins are then accessible for antibody binding and detection. Thus this method allows
screening for pathogen-specific anti-bodies,
which are produced in infected patients, in
comparison to healthy controls. Many allergenic A. fumigatus proteins have already
been detected in serum of patients with allergy-type Aspergillus diseases, such as asthma
and allergic broncho pulmonary aspergillosis
(ABPA). Only little is known about the A. fumigatus proteins produced during the course
of invasive aspergillosis. Furthermore, it has
not been investigated so far which of these
proteins trigger the production of Aspergillusspecific IgG anti-bodies. Therefore, we separated mycelial proteins by 2D-gel electrophoresis followed by Western blotting using
sera from patients with probable and proven
invasive aspergillosis, and risk patients
without evidence of invasive aspergillosis.
Altogether, 57 different protein spots representing 46 different proteins gave a positive
IgG antibody signal. Most of these protein
antigens play a role in primary metabolism
and the general stress response. In order to
examine whether some protein antigens may
be used as diagnostic or prognostic marker
or exhibit a protective activity we employed
a classification system based on supervised
69
References
Schroeckh V, Scherlach K, Nützmann
HW, Shelest E, Schmidt-Heck W,
Schuemann J, Martin K, Hertweck C,
Brakhage AA (2009) Intimate bacterial-fungal interaction triggers biosynthesis of archetypal polyketides in
Aspergillus nidulans. Proc Natl Acad Sci
U S A 106, 14558-14563.
Nützmann HW, Reyes-Dominguez
Y, Scherlach K, Schroeckh V, Horn
F, Gacek A, Schümann J, Hertweck
C, Strauss J, Brakhage AA (2011)
Bacteria-induced natural product
formation in the fungus Aspergillus
nidulans requires Saga/Ada-mediated
histone acetylation. Proc Natl Acad Sci
U S A 108, 14282-14287.
Nützmann HW, Fischer J, Scherlach
K, Hertweck C, Brakhage AA (2013)
Distinct amino acids of histone H3
control secondary metabolism in
Aspergillus nidulans. Appl Environ
Microbiol 79, 6102-6109.
Gehrke A, Heinekamp T, Jacobsen
ID, Brakhage AA (2010) Heptahelical
receptors GprC and GprD of Aspergillus fumigatus are essential regulators
of colony growth, hyphal morphogenesis, and virulence. Appl Environ
Microbiol 76, 3989-3998.
Brakhage AA, Bruns S, Thywißen A,
Zipfel PF, Behnsen J (2010) Interaction of phagocytes with filamentous
fungi. Curr Opin Microbiol 13, 409-415.
Thywißen A1, Heinekamp T, Dahse
HM, Schmaler-Ripcke J, Nietzsche S,
Zipfel PF, Brakhage AA (2011) Conidial
Dihydroxynaphthalene Melanin
of the Human Pathogenic Fungus
Aspergillus fumigatus Interferes with
the Host Endocytosis Pathway. Front
Microbiol 2, 96.
Heinekamp T, Thywißen A, Macheleidt J, Keller S, Valiante V, Brakhage AA
(2013) Aspergillus fumigatus melanins:
interference with the host endocytosis pathway and impact on virulence.
Front Microbiol 3, 440.
70
machine learning with decision tree as a predictive model. These analyses indicated that
this method is helpful to improve diagnosis of
IA and that some antigens may be protective
and may have the potential to be used for immunotherapy of invasive aspergillosis. Some
of these anti-gens have been used to test their
capacity to induce T cell expansion (in collaboration with Alexander Scheffold, Berlin)
(Bacher et al. 2013a, Bacher et al. 2013b).
3
Microbial communication and
natural products
Group Leaders: Volker Schroeckh,
Axel A. Brakhage
Distinct amino acids of histone H3
control secondary metabolism in
Aspergillus nidulans
For the important model fungus A. nidulans
we have shown that co-cultivation of the
fungus with the soil-dwelling actinomycete
Streptomyces rapamycinicus led to the specific activation of a cryptic polyketide gene
cluster that codes for the biosynthesis of the
archetypal polyketide orsellinic acid, as well
as for a number of polyphenolic compounds
with important pharmacological activities
(Schroeckh et al. 2009). Further studies revealed that the bacterium is able to reprogram the histone modifying SAGA/ADA machinery (Nützmann et al. 2011) (Fig. 5).
Our data showed that the histone H3 amino
acid lysine residues 9 and 14 at distinct secondary metabolism genes were specifically
acetylated during the bacterial fungal interaction, which furthermore was associated
with the activation of the otherwise silent
ors gene cluster. Lysine residues at this cluster were replaced by arginine or glutamine
residues. As a result, the lysine to arginine
exchange mimics a non-acetylatable lysine,
whereas the incorporation of glutamine displays a constitutive acetylation of histone H3.
Of all tested strains the exchange of lysine 14
and lysine 9/14 showed the strongest phenotype compared to the wild type. The mimic-
ry mutants of the non-acetylatable lysine 14
as well as those of the lysine 9/14 double exchange showed strongly reduced transcription of the penicillin, sterigmatocystin and
orsellinic acid gene clusters. These results
revealed a causal linkage between histone
acetylation, secondary metabolite gene cluster activation, and SAGA/ADA (Nützmann
et al. 2013). Further open questions concern
the identification and characterisation of upstream elements activating SAGA/ADA upon
a bacterial signal and downstream elements
regulated by SAGA/ADA including transcription factors and counteracting histone
deacetylases.
Bacteria-induced natural product formation in the fungus Aspergillus fumigatus
leads to fumicycline formation
Genome analyses indicated that bacteria and
fungi are able to produce a much broader
range of chemical entities than estimated
from previous chemical analyses of microorganisms grown under standardised laboratory conditions. To access this cryptic
metabolome it is necessary to elucidate the
physiological conditions under which the
compounds are produced. A strategy that
has proven highly successful in inducing the
expression of silent biosynthesis gene clusters is the simulation of a possible scenario
occurring in the habitat.
Interestingly, S. rapamycinicus has a similar impact on the human-pathogenic fungus
A. fumigatus. An intimate, physical interaction between both organisms resulted in the
activation of an otherwise silent polyketide
synthase (PKS) gene-containing cluster in
the fungus. We named the encoded compounds fumicyclines, which are unusual
prenylated polyphenols belonging to the
meroterpenoids (König et al. 2013). The biosynthesis of these compounds is regulated
by a pathway-specific activator as well as by
epigenetic factors. However, transduction of
the yet unknown bacterial signal seems to
be different in both Aspergillus species.
Figure 5
Intimate physical contact between S.
rapamycinicus and A. nidulans leads
to the activation of the previously
silent ors gene cluster in A. nidulans
and the formation of orsellinic acid
and derivatives thereof. Thereby, S.
rapamycinicus reprograms the histone-modifying SAGA/ADA complex
of the model fungus A. nidulans by a
yet unknown trigger.
Transposon mutagenesis of Streptomyces rapamycinicus – a tool to elucidate
how S. rapamycinicus targets Aspergillus
nidulans and Aspergillus fumigatus
To elucidate the activating principle of the
streptomycete, we generated a mutant library of S. rapamycinicus using random
Tnp(a) transposon insertion mutagenesis.
For this purpose, the standard protocols for
mutant library generation had to be adapted
for the genetically difficult to access S. rapamycinicus. We modified the transposon
vector pTNM. This plasmid contains a synthetic, codon usage-optimised transposase
gene tnp(a). Previously, it was shown that the
plasmid was randomly inserted into the genome of S. coelicolor. Successful conjugation
of the modified vector into S. rapamycinicus
was achieved. By analysing several mutants
of S. rapamycinicus, we verified both the selective induction of the transposase gene expression as well as the random transposon
insertion into the streptomycete genome.
Moreover, we optimised the mutant library
screening protocol, which is based on the
non-appearance of a brownish colour when
genes involved in the specific fungus-streptomycete interaction are ‘transposon’-mutated.
4
Transcription factors and signal
transduction
Group Leaders: Peter Hortschansky,
Axel A. Brakhage
CCAAT-binding complex structure reveals novel mode of DNA interaction by
minor groove sensing and widening
The CCAAT box is a frequent motif in eukaryotic promoters and its specific recognition by the heterotrimeric CCAAT-binding
complex (CBC), which is strictly conserved
among species, constitutes a key step in
promoter organisation and transcriptional
regulation of downstream-encoded genes.
71
Figure 6
Structure of the CBC:DNA complex.
(A) Ribbon plot of the CBC bound to the CCAAT box
in the cycA promoter. (B) Structural superposition of
the CBC and the first turn of the nucleosome. The high
similarity of the CBC to the histones H2A and H2B might
allow their mutual substitution in nucleosomes and enable interactions with the histones H3 and H4.
In Aspergillus nidulans the CBC consists of
the subunits HapB, HapC and HapE. A multitude of A. nidulans genes has been found to
be positively or negatively regulated by the
CBC, including developmental genes as well
as genes involved in oxidative stress adaptation, production of secondary metabolites
and iron homeostasis (Hortschansky et al.
2007, Thön et al. 2010).
It has long been an open question how the
CBC employs the HapC / HapE histone fold
motifs to gain sequence specificity for the
CCAAT box. In collaboration with the group of
Michael Groll (TU Munich) we determined the
crystal structure of the CBC:DNA complex.
HapC and HapE induce nucleosome-like
DNA bending by interacting with the sugarphosphate backbone, whereas HapB tightly
anchors the CBC to the CCAAT box by minor
groove sensing and widening (Fig. 6). Interestingly, the tertiary structure of subunit
72
HapB displays no similarity to any known
protein domains characterised today. It is
composed of an N-terminal helix followed
by a loop, a sensor helix S and an anchor A.
Whereas most transcription factors insert
into the major groove of the DNA, since it offers more contact sites with DNA bases, HapB
binds into the minor groove of the DNA and
widens it from 12 Å up to 19 Å. This structural deformation is not accomplished by the
sensor helix S, but by the anchor A, which
functions as a tentacle and inserts into the
CCAAT-box. Surface Plasmon Resonance
(SPR) measurements of structure-based truncated HapB mutants proved that the anchor
A is essential for high-affinity binding of the
CBC to the CCAAT-box.
Furthermore, the CBC:DNA structure visualised for the first time the position of the
HapE N-terminal helix (N) within the CBC
and relative to the DNA backbone. This domain was previously shown to be important
Figure 7
(A) An evolutionary conserved, bipartite motif present in cccA promoters
from 28 fungal species including A.
fumigatus. (B) Surface Plasmon Resonance analysis of in vitro formation of
the CBC/DNA-HapX ternary complex
on the conserved cccA promoter
motif from A. fumigatus. (C) Scheme
of the regulatory mechanism mediating both adaptation to iron starvation and iron resistance by the same
transcription factor complex, comprising the CBC and HapX.
for the interaction with HapX, a fourth CBC
subunit that regulates various iron-dependent pathways (e. g. heme and siderophore
biosynthesis) (Huber et al. 2012).
Aspergillus fumigatus HapX is a Janus
transcription factor required for adaptation to both iron starvation and iron
excess
Iron is an essential but, in excess, toxic nutrient. As a consequence, balance of physiological levels of iron is crucial for every organism. In A. fumigatus and other fungal
pathogens, the bZIP transcription factor
HapX mediates adaptation to iron limitation
and consequently virulence by repressing
iron-consumption and activating iron uptake
(Schrettl et al. 2010, Linde et al. 2012). Importantly, HapX activity depends on physical
interaction with the above described heterotrimeric CCAAT binding factor (CBC), which
is conserved in all eukaryotes. In collaboration with the group of Hubertus Haas (Inns-
bruck Medical University), we demonstrated
for the first time that HapX is not only crucial for adaptation to iron starvation but also
for iron resistance via activating vacuolar
iron storage, i.e. HapX is a Janus-type transcription factor acting as both an activator
and repressor depending on iron availability. Consistently, we identified evolutionarily
conserved protein domains within HapX that
are exclusively essential for adaptation to either limitation or excess of iron, and are presumably involved in iron sensing. We further
found that for promoter recognition, a HapX
homodimer and the CBC cooperatively bind
an evolutionarily conserved DNA motif in
the promoter of the cccA gene, encoding for
the vacuolar iron importer CccA (Fig. 7). As
the CBC can also act independently of HapX,
the latter reveals the mode of discrimination
between CBC and HapX/CBC target genes.
Collectively, our data uncovered a novel regulatory mechanism mediating both iron resistance and adaptation to iron starvation by
73
References
Burmester A, Shelest E, Glöckner G,
Heddergott C, Schindler S, Staib P,
Heidel A, Felder M, Petzold A, Szafranski K, Feuermann M, Pedruzzi I,
Priebe S, Groth M, Winkler R, Li W,
Kniemeyer O, Schroeckh V, Hertweck C, Hube B, White TC, Platzer M,
Guthke R, Heitman J, Wöstemeyer
J, Zipfel PF, Monod M, Brakhage AA
(2011) Comparative and functional
genomics provide insights into the
pathogenicity of dermatophytic
fungi. Genome Biol 12, R7.
Kroll K, Pähtz V, Kniemeyer O (2014)
Elucidating the fungal stress response by proteomics. J Proteomics
97, 151-163.
Bruns S, Seidler M, Albrecht D, Salvenmoser S, Remme N, Hertweck C,
Brakhage AA, Kniemeyer O, Müller
FM (2010) Functional genomic profiling of Aspergillus fumigatus biofilm
reveals enhanced production of the
mycotoxin gliotoxin. Proteomics 10,
3097-107.
Scherlach K, Nützmann HW, Schroeckh V, Dahse HM, Brakhage AA,
Hertweck C (2011) Cytotoxic pheofungins from an engineered fungus
impaired in posttranslational protein
modification. Angew Chem Int Ed Engl
50, 9843-9847.
Bacher P, Kniemeyer O, Schönbrunn
A, Sawitzki B, Assenmacher M, Rietschel E, Steinbach A, Cornely OA,
Brakhage AA, Thiel A, Scheffold A
(2013) Antigen-specific expansion
of human regulatory T cells as a
major tolerance mechanism against
mucosal fungi. Mucosal Immunol 7,
916-928.
Bacher P, Schink C, Teutschbein
J, Kniemeyer O, Assenmacher M,
Brakhage AA, Scheffold A (2013)
Antigen-reactive T cell enrichment
for direct, high-resolution analysis of
the human naive and memory Th cell
repertoire. J Immunol 190, 3967-3976.
König CC1, Scherlach K, Schroeckh
V, Horn F, Nietzsche S, Brakhage AA,
Hertweck C (2013) Bacterium induces
cryptic meroterpenoid pathway in
the pathogenic fungus Aspergillus
fumigatus. ChemBioChem 14, 938-942.
74
the same transcription factor complex with
activating and repressing functions depending on ambient iron availability.
5
6
Molecular biotechnology
Group Leaders: Vito Valiante,
Axel A. Brakhage
Molecular biology of natural
products
Group Leaders: Daniel Scharf,
Axel A. Brakhage
Gliotoxin (GT) is the most important epidithiodioxopiperazine (ETP)-type fungal
toxin. GT was the first ETP discovered and
has been investigated for years. This low
molecular mass secondary metabolite plays
a critical role in the pathobiology of Aspergillus fimigatus (Scharf et al. 2012). It modulates
the immune response and induces apoptosis
in cell culture models. The toxicity is due to
a disulfide bridge, which is the functional
motif within this metabolite. Because of the
extraordinary structure and activity of GT,
we have studied the biosynthesis of this molecule genetically, biochemically and also by
elucidation of the crystal structure of some
of the biosynthesis enzymes (in collaboration with Michael Groll, TU Munich). The
sulfur moieties are introduced into the diketopiperazine core by addition of glutathione
(GSH) and its sequential degradation to the
free thiol precursor. A flavin-adenine-dinucleotide (FAD)-dependent oxidoreductase,
termed GliT, finally oxidises an intermediate
to the disulfide containing GT, thereby conferring resistance of the fungus to its own
toxin. It is very conceivable that GliT-type enzymes catalyse the final biosynthesis step of
all fungal ETPs (Scharf et al. 2012, Scharf et
al. 2011, Scharf et al. 2010). Recently, we were
able to carry out most of the reactions in vitro
using purified enzymes. This will open up
the possibility to use these unusual enzymes
to engineer novel ETP analogs in vitro (Scharf
et al. 2013). Also, the crystal structure of the
oxidoreductase GliT was solved together with
the structures of two other oxidoreductases
which allowed proposing a unifying reaction
mechanism (in collaboration with Michael
Groll, TU Munich).
Signalling components in fungi
For growth in all habitats, fungi need to be
able to sense environmental stimuli and
transduce signals via phosphorylation cascades. Previously, we showed that cyclic
AMP signal transduction and the calcineurin pathway are involved in virulence of
A. fumigatus. Comparative genomic analyses
led to the identification of the member proteins of the different signalling pathways in
pathogenic fungi. Many of them were found
to be important for virulence in both plant
and human pathogens (Horn et al. 2012).
Among evolutionarily conserved pathways,
mitogen-activated protein kinases (MAPK)
cascades in all eukaryotes generally consist of three interlinked protein kinases.
A. fumigatus contains four putative MAPKs
named MpkA, MpkB, MpkC and SakA. MpkA
regulated cell wall integrity signalling is involved in regulation of a plethora of genes
ranging from those involved in cell wall
repair and synthesis, defence against oxidative stress, pigment and toxin biosynthesis
as well as siderophore production (Jain et al.
2011). MpkA appears to fine-tune the balance
between the stress responses and the energy
consumed in cellular processes required
for growth, development and secondary metabolite biosynthesis. Transcriptomics data
revealed that mutant strains of all MAPK
showed different expression of gene clusters.
For many of them, several MAPK pathways
contribute to their activation / repression,
revealing a cross-talk between the different
signalling pathways.
Another important signalling is the mTor
pathway. Tor is an evolutionary conserved
nutrient sensing protein kinase member of
the phosphatidylinositol 3-kinase (PI-3K)
superfamily that regulates cell growth and
differentiation in response to nutrients. A
deletion mutant for Tor in A. fumigatus appears not to be viable, revealing the importance of this gene for the fungus. An inducible Tor mutant, generated using the xylose
promoter, allowed us to characterise the role
of Tor in the adaptation to different stress
conditions. Proteome analysis of the Tor inducible mutant revealed that in A. fumigatus
this gene is responsible for major metabolic
activities, which include sugar metabolism,
amino acid biosynthesis, and polyamines
storage and consumption.
Hitting the caspofungin salvage pathway of human-pathogenic fungi with the
novel natural product humidimycin
The dramatic increase of fatal fungal infections, coupled with the emergence of antifungal drug resistant strains, makes it an
urgent task to search for new therapeutic
strategies against invasive mycoses. An innovative concept relies on the combination
of two drugs, which act on different targets
or even target a salvage pathway of one of
the drugs. The cell wall biosynthesis of the
two most prominent human-pathogenic
fungi, i.e. Candida albicans and Aspergillus
fumigatus, is inhibited by the clinically used
antifungal drug caspofungin (CAS). Here, by
screening 20,000 microbial extracts, we discovered a novel compound, designated humidimycin, produced by a strain of Streptomyces humidus, which strongly enhanced
the growth inhibitory effect of caspofungin
and also another clinically used antifungal,
i.e., itraconazole. The structural elucidation
of the new molecule was accomplished using
a combination of spectroscopic and chemical techniques, including 1D and 2D NMR,
HRMS and MS/MS sequencing. Transcriptome analyses revealed that CAS mainly
induced the expression of cell wall biogenesis genes of A. fumigatus. When both drugs
were combined, the number of CAS-induced
genes decreased, suggesting that humidimycin reduced the A. fumigatus stress response
against CAS. Phenotypic characterisation
of A. fumigatus mutants defective in different signal transduction pathways, measurements of the cell wall carbohydrate content
and western blot analyses of protein kinases
indicated that CAS affects the cell wall integrity pathway and the high osmolarity glycerol response (HOG) pathway, the latter acting
as a salvage pathway of CAS-triggered disturbance of cell wall integrity. When used in
combination, humidimycin silenced the HOG
pathway and thus increased the antifungal
activity of CAS. Additionally, humidimycin
alone or in combination with CAS did not
show any toxicity on human hepatocyte cell
lines. Taken together, our data demonstrate
that targeting compensatory signalling pathways provides an excellent basis to develop
novel anti-infective strategies.
The role of histone acetyltransferases
in regulation of natural product biosynthesis
In A. fumigatus the biosynthesis genes of
natural products are found in specific biosynthesis gene clusters. Under standard
laboratory conditions, many of these gene
clusters are not activated, making it difficult
to identify which secondary metabolites are
biosynthesised from a particular gene cluster. One approach to identify silent gene clusters involves the use of epigenetics methods
to manipulate the chromatin landscape. Previous studies in Aspergillus nidulans have
demonstrated that histone acetyl transferases (HATs) play an important role in secondary metabolism, but little is known so far in
A. fumigatus. There are approximately 46 putative HAT-encoding genes in A. fumigatus
and current work aims at creating a knockout library of these HATs. Further work will
help determine the function of HATs.
Synthetic biology tools for bioprospecting of natural products in eukaryotes
Most natural product biosynthesis pathways
are silent, many in intractable organisms,
and their products consequently unidentified. Viral 2A peptides have been shown to
promote cotranslational cleavage of eukaryotic polycistronic mRNA. Expression of
viral proteins often includes non-canonical
decoding events during translation. 2A peptides are able to drive such events, termed
References
Hortschansky P, Eisendle M, Al-Abdallah Q, Schmidt AD, Bergmann S,
Thön M, Kniemeyer O, Abt B, Seeber
B, Werner ER, Kato M, Brakhage AA,
Haas H (2007) Interaction of HapX
with the CCAAT-binding complex--a
novel mechanism of gene regulation
by iron. EMBO J 26, 3157-3168.
Thön M, Al Abdallah Q, Hortschansky
P, Scharf DH, Eisendle M, Haas H, Brakhage AA (2010) The CCAAT-binding
complex coordinates the oxidative
stress response in eukaryotes. Nucleic
Acids Res 38, 1098-1113.
Schrettl M, Beckmann N, Varga J,
Heinekamp T, Jacobsen ID, Jöchl
C, Moussa TA, Wang S, Gsaller F,
Blatzer M, Werner ER, Niermann WC,
Brakhage AA, Haas H (2010) HapXmediated adaption to iron starvation
is crucial for virulence of Aspergillus
fumigatus. PLoS Pathog 6, e1001124.
Linde J, Hortschansky P, Fazius E,
Brakhage AA, Guthke R, Haas H (2012)
Regulatory interactions for iron
homeostasis in Aspergillus fumigatus
inferred by a Systems Biology approach. BMC Syst Biol 6, 6.
Scharf DH, Chankhamjon P, Scherlach
K, Heinekamp T, Roth M, Brakhage
AA, Hertweck C (2012) Epidithiol
formation by an unprecedented
twin carbon-sulfur lyase in the gliotoxin pathway. Angew Chem Int Ed 51,
10064-10068.
Scharf DH, Chankhamjon P, Scherlach K, Heinekamp T, Willing K,
Brakhage AA, Hertweck C (2013)
Epidithiodiketopiperazine biosynthesis: a four-enzyme cascade converts
glutathione conjugates into transannular disulfide bridges. Angew Chem
Int Ed 52, 11092-11095.
Scharf DH, Remme N, Habel A,
Chankhamjon P, Scherlach K,
Heinekamp T, Hortschansky P,
Brakhage AA, Hertweck C (2011) A
dedicated glutathione S-transferase
mediates carbon-sulfur bond formation in gliotoxin biosynthesis. J Am
Chem Soc 133, 12322-12325.
75
Figure 8
Summary of heterologous production of natural product biosynthesis pathways using virus 2A
peptide.
The polycistronic mRNA is translated
by the ribosomes and the nascent
peptide chain cotranslationally
cleaved to the single biosynthesis
proteins.
References
Scharf DH, Remme N, Heinekamp T,
Hortschansky P, Brakhage AA, Hertweck C (2010) Transannular disulfide
formation in gliotoxin biosynthesis
and its role in self-resistance of the
human pathogen Aspergillus fumigatus. J Am Chem Soc 132, 10136-10141.
Horn F, Heinekamp T, Kniemeyer O,
Pollmächer J, Valiante V, Brakhage
AA (2012) Systems biology of fungal
infection. Front Microbiol 3, 108.
76
‘stop-carry on’ recoding. Nascent 2A peptides interact with the ribosomal exit tunnel
to promote an unusual stop codon-independent termination of translation at the final
Pro codon of 2A peptides. As synthetic tools
viral 2A peptides therefore provide an opportunity to express pathways as polycistronic
genes in eukaryotes. Obvious examples are
large non-ribosomal peptide synthetase
(NRPS) and polyketide synthase (PKS) proteins, which are often the key biosynthetic
enzymes of secondary metabolite pathways,
and are technically complicated to express
heterologously. The increased in vivo cloning efficiency, and the use of viral 2A peptide sequences to yield a large polycistronic
mRNA, has facilitated generation of large
constructs used for expression from a single
promoter of entire biosynthesis pathways.
As a proof-of-concept, we described how the
entire penicillin biosynthesis pathway from
Penicillium chrysogenum can be produced in
a heterologous host, allowing subsequent
characterisation, using a combination of (i)
improved yeast in vivo cloning technology,
(ii) generation of polycistronic mRNA for the
gene cluster under study and (iii) the use of
an amenable and easily manipulated fungal
host i.e. Aspergillus nidulans (Fig. 8) (Unkles
et al. 2014).
7
Zygomycetes – Pathogenicity and
Biodiversity
Group Leader: Kerstin Voigt
This project is described in more detail in
the section Jena Microbial Resource Collection (JMRC)
Group members
Visiting Scientists
Head
Prof. Dr. Axel Brakhage
Phone +49 3641 532-1001
Fax
+49 3641 532-0802
[email protected]
Dafna Ben-Yaacov
University of Tel Aviv, Israel
04/2013
Secretary
Cornelia Schwab (until 12/2013)
Daniela Wagner (since 12/2013)
Scientists
Dr. Falk Hillmann
Dr. Nora Köster-Eiserfunke
Dr. Thorsten Heinekamp
Dr. Olaf Kniemeyer
Dr. Thomas Krüger
Dr. Kerstin Hoffmann
Dr. Peter Hortschansky
Dr. Daniel Scharf
Dr. Volker Schroeckh
Dr. Maria Strassburger (since 07/2012)
Dr. Andreas Thywißen
Dr. Vito Valiante
Ph.D. Students
Clara Baldin
Juliane Fischer
Martin Föge
Benjamin Hanf (since 10/2013)
Christoph Heddergott (until 06/2012)
Sophia Keller
Claudia König
Antje Kröber
Kristin Kroll
Katrin Lapp
Ting Luo
Juliane Macheleidt
Derek Mattern
Tina Netzker
Vera Pähtz
Hea-Reung Park (since 09/2012)
Hella Schmidt (since 11/2012)
Hanno Schoeler (since 10/2013)
Volker Schwartze
Elena Shekhova (since 05/2013)
Iordana Shopova
Katja Tuppatsch
Jakob Weber
Jana Haueisen
Patrick Pann
Franziska Schmidt
Iordana Shopova
Lysett Wagner
Lars Wallstabe
Research Assistants
Sylke Fricke
Nancy Hannwacker (until 01/2012)
Maria Pötsch
Carmen Schult
Caroline Semm (until 07/2012)
Silke Steinbach
Christina Täumer
Dr. Grit Walther (since 06/2012)
Christiane Weigel
Dr. James Kinghorn
University of St. Andrews, Scotland
04/2013 – 07/2013
Susana Pamela Mejia
University of Antioquia, Medellín, Colombia
06/2012 – 07/2012
Dr. Shiela Unkles
University of St. Andrews, Scotland
04/2013 – 07/2013
External funding
ERA-NET PathoGenoMics 2
Apergillosis – Die Zellwand als Target zur
Verbesserung der antifungalen Therapie der
Aspergillose
Olaf Kniemeyer
ERA-NET – AspBIOmics
Biomarker zur Therapie von invasiver
Aspergillose
Olaf Kniemeyer
ERA-NET OXYstress
Humapathogene Pilze unter Sauerstoff-Stress:
Adaptation an Sauerstoffmangel und reaktive
Sauerstoffspecies und die Folgen für die Interaktion mit dem Wirt und die Therapie
Thorsten Heinekamp
Zwanzig20 – Partnerschaft für Innovation
InfectControl 2020 – Neue Antiinfektionsstrategien – Wissenschaft • Gesellschaft • Wirtschaft;
Strategievorhaben
Axel Brakhage
Integriertes Forschungs- und Behandlungszentrum (IFB) Sepsis und Sepsisfolgen (CSCC)
Project: Inprot – Identifizierung von C. albicans
und A. fumigatus Proteinantigenen für die
Pilzsepsisdiagnostik und Untersuchung der
A. fumigatus Stressantwort
Olaf Kniemeyer
Project: Aspergillus fumigatus – Analysis of the
interaction of the human pathogenic fungus
Aspergillus fumigatus with immune effector
cells by functional genomics
Axel Brakhage
Project: Interactions of the fungal pathogen
Aspergillus fumigatus with human macrophages
Axel Brakhage
Project: Functional analysis of major transcriptional regulators in pathogenic dermatophytes
Axel Brakhage
Project: Redox regulation, development and
hyphal growth in Aspergillus nidulans
Axel Brakhage, Peter Hortschansky
Project: Soil amoeba interactions with filamentous fungi as driving forces for pathogenicity
Falk Hillmann
SFB/TR FungiNet
Project Z02: Methoden zur Analyse der zeitlichen und räumlichen Änderung der Proteome
bei der Interaktion pilzlicher Pathogene mit
dem Wirt
Olaf Kniemeyer
SFB/TR FungiNet
Project Z01: Projektmanagement
Axel Brakhage
European Union
Framework Programm 7 European Re-integration Grants (ERG)
CellWallPath – Identification of new molecules
able to bypass the cell wall compensatory
pathways in the pathogenic fungus Aspergillus
fumigatus
Vito Valiante
European Union
Framework Programm 7 Marie Curie Initial
Training Network
ARIADNE – Signaling circuitry controlling fungal virulence: identification and characterization of conserved and specific fungal virulence
genes as common antifungal targets
Axel Brakhage, Thorsten Heinekamp
European Union
Framework Programm 7 Marie Curie Initial
Training Network
Quantfung – Quantitative Biology for Fungal
Secondary Metabolite Producers
Axel Brakhage
NanoConSens – Nanocontainer und nanostrukturierte Trägermaterialien für Sensorik und
Wirkstofftransport
Axel Brakhage, Christian Hertweck
MikroInter – Characterisation of receptors of
the human-pathogenic fungus Aspergillus
fumigatus – How does the fungus communicate
with the environment and the host
Axel Brakhage
77
Thüringer Aufbaubank
Geschäftsstelle InfectControl 2020
Axel Brakhage
Charité, Universitätsmedizin Berlin
Prof. Dr. Alexander Scheffold
German Israeli Foundation
The hypoxic response in the pathogenic mold
Aspergillus fumigatus and its relevance to disease
Olaf Kniemeyer
Leibniz-Gemeinschaft
Leibniz-Wettbewerb, Förderlinie 3: Vernetzung
Kryostress – Anpassungsmechanismen der
Zelle an Tiefstemperaturen (KAIT)
Olaf Kniemeyer
Scharf DH, Groll M, Habel A, Heinekamp T,
Hertweck C, Brakhage AA, Huber EM (2014)
Flavoenzyme-catalyzed formation of disulfide
bonds in natural products. Angew Chem Int Ed
53, 2221-2224.
Scharf DH, Chankhamjon P, Scherlach K,
Heinekamp T, Willing K, Brakhage AA, Hertweck C (2013) Epidithiodiketopiperazine biosynthesis. A four-enzyme cascade converts glutathione conjugates into transannular disulfide
bridges in fungal ETP toxins. Angew Chem Int Ed
52, 11092-11095.
Duke University, Durham, NC, USA
Prof. Dr. Rytas Vilgalys
Dr. Andrej Gryganskyi
Fundación MEDINA, Centro de Excelencia en
Investigación de Medicamentos Innovadores
en Andalucía, Armilla, Granada, Spain
Dr. Olga Genilloud
Friedrich Schiller University, Jena, Germany
Prof. Dr. Ulrich S. Schubert
Prof. Dr. Stefan Schuster
Konrad Grützmann
Prof. Dr. Jürgen Popp
Dr. Petra Rösch
Ute Müncheberg
PD Dr. Frank Peters
Dr. Akos Kovacs
Henkel AG & Co KGaA, Düsseldorf, Germany
Dr. Mirko Weide
Max-Planck-Institut für chemische
Ökologie Jena
Prof. Dr. Wilhelm Boland
Yamuna Sahadevan
Innsbruck Medical University,
Innsbruck, Austria
Brakhage AA (2013) Regulation of fungal secondary metabolism. Nat Rev Microbiol 11, 21-32.
Institut Pasteur, Frankreich, Paris
Prof. Dr. Jean-Paul Latgé
Bacher P, Kniemeyer O, Schönbrunn A, Sawitzki
B, Assenmacher M, Rietschel E, Steinbach A,
Cornely OA, Brakhage AA, Thiel A, Scheffold
A (2013) Antigen-specific expansion of human
regulatory T cells as a major tolerance mechanism against mucosal fungi. Mucosal Immunol
7, 916-928.
Institute of Animal and Invertebrate Genetics,
Czech Academy of Sciences
Dr. Katerina Fliegerová
König CC, Scherlach K, Schroeckh V, Horn F,
Nietzsche S, Brakhage AA, Hertweck C (2013)
Bacterium induces cryptic meroterpenoid
pathway in the pathogenic fungus Aspergillus
fumigatus. ChemBioChem 14, 938-942.
Montana State University, Bozeman, USA
Prof. Dr. Robert A.Cramer
Philipps University Marburg, Germany
Prof. Dr. Shu-Ming Li
Royal Botanic Gardens Kew, UK
Dr. Paul M. Kirk
Dr. Sandor Nietzsche
Collaborations
Albert Ludwigs University Freiburg, Germany
Prof. Dr. Michael Müller
University of Florida, Gainsville, FL, USA
Dr. Gerald L. Benny
BASF, Ludwigshafen, Germany
Dr. Lutz Petzke
Centraalbureau voor Schimmelcultures (CBS),
Utrecht, Netherland
Prof. Dr. Sybren de Hoog
Dr. Benjamin Stielow
Dr. Gerard Verkley
Centro Nacional de Microbiologia, Instituto
de Salud Carlos III, Servicio de Micologia,
Madrid, Spain
Dr. Emilia Mellado
78
University of California Los Angeles, USA
Prof. Dr. Scott F. Filler
University of Manchester, UK
Dr. Elaine Bignell
Dr. Michael Bromley
Dr. Paul Bowyer
University of Missouri-Kansas City, USA
Prof. Dr. Ted White
Technical University Munich, Germany
Prof. Dr. Michael Groll
Department of Cell and Molecular Biology
79
Department of Cell and Molecular Biology
Research in the Department of Cell and Molecular Biology is devoted to the flow of molecular information during host-pathogen
interactions. As model hosts we are using
human cell lines, mice and chick embryos
in ovo, and the pathogens of our main interest are Chlamydiales and pathogenic fungi.
Within this framework, we aim at the elucidation of how infections proceed in living organisms (imaging) and how infected organs
react on a molecular level (e.g. comparative genomics, transcriptomics and interactomics). Imaging is performed by means
of our latest generation positron emission
tomography-computed tomography (PET-CT)
instrument that provides co-registered images, ie. it combines the high spatial reso-
lution and anatomical detail of CT with the
molecular, quantifiable images obtained by
PET. Comparative genomics, transcriptomics
and interactomics involve massive parallel
sequencing.
To broaden information-solving abilities we
have set out to adopt and to develop highly
advanced micro- and nanosytems, which allow the simultaneous handling of multiple
samples within sets of different biomolecules
under nearly identical experimental conditions. At present we are focussing on multicolour hyperspectral imaging of biomolecules on solid body surfaces and in single
living infected cells.
Die Abteilung Zell- und Molekularbiologie
beschäftigt sich mit den molekularen Grundlagen von Wirt-Pathogen-Interaktionen. Als
Modellwirte untersuchen wir menschliche
Zellen, Mäuse und Hühnerembryonen im Ei.
Die Pathogene, die für uns eine zentrale Rolle spielen, sind Chlamydien und pathogene
Pilze.
Head:
Prof. Dr. Hans Peter Saluz
Im Rahmen der biologischen Projekte erforschen wir Infektionsabläufe in lebenden
Organismen (Imaging) und die Reaktion infizierter Organe auf molekularer Ebene (mittels komparative Genomics, Transcriptomics
und Interactomics). Imaging führen wir vor
allem mit einem modernen kombinierten
PET-CT (Positron Emission Tomography-Computed Tomography)-Instrument durch, das
die hohe räumliche Auflösung und anato-
80
mischen Details des CT mit molekular quantifizierbaren Bildern des PET kombiniert. Sowohl die Genom-, Transkriptom - als auch die
Interaktomanalysen involvieren Hochdurchsatzsequenzierung. Um die verschiedenen
wirts-spezifischen Antworten wirkungsvoll
erfassen zu können, entwickeln wir hochmoderne Mikro- und Nanosysteme. Mit ihnen
können jeweils mehrere tausend Proben aus
einer Reihe unterschiedlicher Biomoleküle
gleichzeitig und unter nahezu identischen
Versuchsbedingungen untersucht werden.
Zur Zeit beschäftigen wir uns mit Vielfarben-hyperspektralem Imaging von Biomolekülen auf Festkörperoberflächen und in einzelnen infizierten, lebenden Zellen.
Unsere Mikro- und Nanosysteme eignen
sich auch zur Anwendung in anderen Abtei-
HKI Research Report 2012 / 2013 Department of Cell and Molecular Biology
The micro- and nanosystems are also ideally
suited for the application in other departments of our institute, as well as within the
entire Beutenberg Campus in Jena, where research interests in different fields, such as
physics, chemistry, and biology meet. Combined with the automation of techniques,
these systems will also provide effective
tools for the rapid realisation of products
and instruments - an important aspect in the
context of the “BioRegio” Jena network.
To expand optimum access to the most advanced scientific know-how and technical
equipment we also cooperate with several
institutions and industries, locally and inter-
lungen unseres Institutes, sowie auf dem gesamten Beutenberg Campus in Jena, wo die
Forschungsinteressen aus so verschiedenen
Gebieten wie Physik, Chemie und Biologie
in einzigartiger Weise aufeinander treffen.
Außerdem liefern diese Systeme, kombiniert
mit der Möglichkeit zur Automatisierung,
eine Voraussetzung für die effiziente Realisierung von Produkten und Instrumenten,
für die „BioRegio“ Jena ein nicht unwesentlicher Aspekt.
Um eine optimale Erweiterung durch neueste wissenschaftliche Erkenntnisse und
technische Mittel zu haben, kooperieren
wir mit mehreren lokalen und internationalen Instituten und Firmen. Die Erfahrungen
und Kenntnisse, die wir aus unseren Arbeiten gewinnen, erlauben es uns, Studenten
nationally. Experience and knowledge gained
from our projects allow us to teach and confront students theoretically and practically
with modern aspects of basic and applied
research.
During the last two years, several bachelor,
master, diploma and doctoral students from
our department have graduated successfully. Within our technological framework, we
have had some real success related to rapid
heat block thermo cycling of small samples.
The ability to rapidly amplify nucleic acids
has been particularly important for pathogen detection in diagnostic applications, as
well as in life science research (e.g. library
theoretisch und praktisch mit modernsten
Aspekten der Grundlagenforschung und
angewandten Wissenschaft zu konfrontieren. So hatten wir in den letzten zwei Jahren
wiederum mehrere Bachelor- und Masterstudenten, Diplomanden und Doktoranden
in unserer Abteilung, die ihre Studien mit
Erfolg abschließen konnten.
Was unsere technischen Projekte anbetrifft,
konnten wir sehr gute Erfolge auf dem Gebiet der ultraschnellen PCR verzeichnen. Die
Möglichkeit, Nukleinsäuren sehr rasch und
präzise amplifizieren zu können, war vor
allem für den Nachweis von Pathogenen bei
diagnostischen Anwendungen, aber auch für
Forschungs- (z. B. für die Konstruktion von
Bibliotheken) und Industrieanwendungen
wichtig (z. B. für Pathogennachweise in ex-
Department of Cell and Molecular Biology HKI Research Report 2012 / 2013
81
constructions) and industrial applications
(e.g. pathogen detection in exported marine
organisms).
Additional developments concerning in vivo
ChIPSeq are currently providing novel and
unexpected information on regulatory protein-DNA interactions in promoter, intron,
and exon regions of the human genome. Finally, our home-made molecular hyper spectral imager (two dimensional) and our hyper
spectral microscope (one dimensional) coupled with multivariate data analysis provide
powerful new tools for understanding complex biological and biomedical samples and
infection studies in single cells.
portierten marinen Organismen). Andererseits ergaben die Interaktomanalysen neuartige und unerwartete Informationen über die
Interaktionen von regulatorischen Proteinen
mit DNA in allen Promoter-, Intron- und
Exonregionen des menschlichen Genoms.
Schließlich ermöglicht das selbstgebaute
hyperspektrale Imaging-Instrument (zweidimensional) und unser hyperspektrales
Mikroskop (eindimensional), kombiniert mit
multivariater Datenanalyse, neue Möglichkeiten zum Verständnis von biologischer
und biomedizinischer Materialien und Infektionsstudien in einzelnen Zellen.
82
Scientific Projects
1
The primary transcriptomes of
infectious and noninfectious chlamydial particles
Group Leader: Gerhard Schöfl
Chlamydiae are obligate intracellular Gramnegative bacteria parasitising a diverse array of eukaryotic hosts ranging from amoebas to humans. They are responsible for
a wide spectrum of medical conditions in
mammals and birds. All members of the family Chlamyidaceae share a unique biphasic life-cycle: Infectious elementary bodies
(EBs) attach to the host cell and are internalised by endocytosis. The DNA of EBs is
condensated by histone-like proteins, which
renders EBs small and dense, and effectively
suppresses most transcriptional activity.
Once enveloped by the host cell, EBs develop
into metabolically active, but non-infectuous
reticulate bodies (RBs). The RBs proliferate
by cell division, and, after several cycles of
reproduction, redifferentiate into EBs. The
host cell containing these EBs bursts and
new cells can be infected. EBs are slightly
larger than RBs, have a granular cytoplasm
and diffuse nucleic acid, which makes them
considerable less dense than the EBs.
The various species and strains within Chlamydiaceae are ecologically separated by differential host preferences, host specificities,
virulence, and tissue tropism. Investigations
into the molecular and genetic bases of these
differentiating factors have been hampered
by the fact that these bacteria cannot be cultivated outside of their host cells and their
targeted genetic manipulation is difficult.
Therefore, approaches such as comparative
genomics, microarray studies, and genomescale proteomics have been employed to
elucidate genes or genomic regions likely
responsible for differences in host range
and virulence. However, while these studies
reveal suggestive patterns of divergence between species they don’t convey information
about the relative importance of diverging
genes nor about the roles of regulatory RNAs
during different stages of the chlamydial life
cycle.
To address this question we initiated a project to monitor the primary and processed
transcriptomes of chlamydial EBs and RBs
at various stages of the chlamydial life cycle.
Our initial target species are the highly virulent host-generalist C. psittaci, the less virulent host-specialist C. abortus und and the
chlamydia-like Waddlia chondrophila as an
outgroup.
A basic requirement for investigating differential patterns of gene expression in EBs and
RBs was to develop a method for a clean mechanical separation of eukaryotic and prokaryotic material, as well as the separation of purification of the two life-cycle stages. Therefore, at a first stage, we established a protocol
for the purification of infectuous EBs and
metabolically active RBs utilising the differences in the density of these bodies. A separation of eukaryotic and prokaryotic material, as well as EBs and RBs was achieved by
ultracentrifugation through a discontinuous
gradient of Visipaque® 320, based on the estimated densities of EBs and RBs. The purity
of the EB and RB preparations was verified
by infection tests (Fig. 1A) and electron microscopy (Fig. 1B). Total-RNA extracts were
prepared from the EB and RB fractions (Fig.
1C) and subjected to a differential exonuclease treatment, which leads to a depletion of
processed transcripts in the treated samples.
A comparison of cDNA libraries generated
from untreated versus exonuclease-treated
samples will allow a global mapping of transcriptional start sites, the determination of
promoter regions and the definition of operon
and suboperon structures at whole-genome
83
Figure 1
(A): Representative visual fields of
infected HEp-2 cells with RB and EB
fractions obtained by the purification
protocol. Magnification 400x, bar
20 µm. (B) Electron micrographs of
RB and EB fractions. RBs and EBs are
tagged by white and black arrows
respectively. Bar 500 nm. (C) Gel-like
image of total RNA obtained from
the input material and the RB and EB
fractions. Prominent eukaryotic (28S,
18S) and prokaryotic (23S, 16S) rRNAs
were labelled.
level. It will also allow the global detection of
small regulatory RNAs and thus reveal the
whole complexity of gene regulation during
an infection time-course of Chlamydia.
2
Identification of antigens in calves
induced by Chlamydia psittaci infection
Group Leader: Hans Peter Saluz
(in cooperation with Konrad Sachse,
Friedrich-Loeffler-Institut, Federal Research
Institute for Animal Health, Jena
The obligate intracellular bacterium Chlamydia psittaci shows a unique life cycle consisting of infectious extracellular elementary bodies (EBs) and metabolically active
noninfectious intracellular reticulate bodies
(RBs). Because of the ability of C. psittaci to
enter host cells, the combined action of cellular and humoral immune response is often
84
needed to combat its infection. To study chlamydia-host interactions in calf we applied
modified in vivo-induced antigen technology
(IVIAT; Fig. 2) to C. psittaci (DC15 strain).
IVIAT is an in vivo high throughput immunoscreening technique capable of identifying
immunogenic antigens that are upregulated
or specifically expressed upon infection.
The aim of this study is the identification of
antigens to gain insights into the virulence of
these pathogenic organisms. Upon infection
of host cells, many chlamydial genes are expressed. We used IVIAT for the identification
of immunogenic bacterial proteins. Several
chlamydial antigenic determinants, being expressed during calf infection, have been identified and predicted to be involved in cell wall
synthesis/structures, transport, metabolism,
metal acquisition, virulence, regulatory and
hypothetical functions. Both humoral and
cell mediated immunity are important for the
protection against chlamydial infection and
Sera preparation
+
Y
Y
Y
Y
Y
Y Y
Y
Y
Generate Protein Expression Library
Y
Figure 2
Modified in vivo-induced antigen
technology (IVIAT).
…AGTATAGCA…
its clearance, thus IVIAT can point out the
antibody-mediated immune response.
3
Protein-protein interactions during
infection and cellular stress
Group Leader: Frank Hänel
(in cooperation with Shamci Monajembashi,
Leibniz Institute for Age Research – Fritz Lipmann Institute, Jena)
Association of Chlamydia psittaci inclusion membrane protein IncB with host
protein Snapin to interact with the host
microtubule network
All members of the family Chlamydiaceae are
obligate intracellular parasites that display
a biphasic developmental cycle. The elementary body (EB) form of the bacteria attaches and invades host-epithelial cells. The
nascent phagocytic compartment is rapidly
modified by Chlamydia-derived proteins to
generate a parasitophorous vacuole termed
an inclusion. Within the inclusion EBs differentiate into non-infectious but metabolically active reticulate bodies (RB), which proliferate within the expanding inclusion, giving rise to 1000 or more progeny per host cell.
The developmental cycle ends after 2 - 3 days
depending on the strain, when RBs transform back into EBs and are released into the
extracellular medium by cell lysis and/or
extrusion to initiate new rounds of infection.
During this unique biphasic developmental
cycle replicating bacteria acquire energy
and biosynthetic precursors from the infected cell. Furthermore, Chlamydiae modulate cellular functions such as apoptotic
programs and immune response (reviewed
by Bastidas et al. 2013). Studies on inhibitors of bacterial protein synthesis suggest
that modulation of the host cell functions
requires the activity of chlamydial proteins.
All Chlamydiaceae ssp. possess genes encod-
85
References
Bastidas RJ, Elwell CA, Engel JN,
Valdivia RH (2013) Chlamydial intracellular survival strategies. Cold Spring
Harb Perspect Med 3, a010265.
Peters J, Wilson DP, Myers G, Timms
P, Bavoil PM (2007) Type III secretion
à la Chlamydia. Trends Microbiol 15,
241-251.
Borth N, Litsche K, Franke C, Sachse
K, Saluz HP, Hänel F (2011) Functional
interaction between type III-secreted
protein IncA of Chlamydophila psittaci and human G3BP1. PLoS One 6,
e16692.
ing core components of a Type III Secretion
(TTS) apparatus, a protein transport system
used by Gram-negative bacteria to translocate proteins into the cytoplasm of the host
cell. Therefore, it is commonly accepted that
chlamydial effector proteins are targeted by
the TTS to the inclusion membrane and that
their interaction with host proteins cause
the modulation of host cell functions (Peters et al. 2007). Interactions of chlamydial
effector proteins with host proteins seem to
play a role at all stages of chlamydial developmental cycle – from adhesion and internalisation of EBs till their exit from the
host cell. In C. pneumoniae invasin protein
Pmp21 recruits the EGF receptor for host cell
entry. Other chlamydial proteins playing a
role in invasion are TARP and CT694. The
well conserved among all Chlamydia TARP
is bacterial nucleator of actin. An important
group of chlamydial infector proteins are
the Inc (inclusion) proteins which share a
common secondary structural feature of a
bilobed hydrophobic domain. While this hydrophobic domain enables the anchoring of
the proteins in the inclusion membrane the
cytoplasmic tail is responsible for interaction
with host proteins. IncD of C. trachomatis interacts with the lipid transfer proteins CERT
and CT229 from C. trachomatis binds to the
GTPase Rab4 – both interactions may affect
trafficking processes of the host cell. One of
the first discovered protein-protein interactions was the association of C. trachomatis
IncG with the mammalian 14-3-3 protein
which contributes to circumvention of host
cell apoptosis similar to the interaction of
TARP with the human adaptor protein SHC1.
Finally, C. trachomatis Inc protein CT228 recruits elements of the myosin phosphatase
pathway to regulate release mechanisms (reviewed by Bastidas et al. 2013).
acting host proteins that could indicate functions of this proteins. In a first attempt we
found the interaction between Type III-secreted protein IncA of C. psittaci and host protein G3BP1 in a yeast two-hybrid system. In
GST-pull down and co-immunoprecipitation
experiments both in vitro and in vivo interaction between full-length IncA and G3BP1
were shown. Using fluorescence microscopy, the localisation of G3BP1 near the inclusion membrane of C. psittaci-infected Hep2 cells was demonstrated. G3BP1 harbors a
phosphorylation-dependent RNase activity
which specifically cleaves the 3´-untranslated region of human c-myc mRNA. Notably, infection of Hep-2 cells with C. psittaci
and overexpression of IncA in HEK293 cells
led to a decrease in c-Myc protein concentration. This effect could be ascribed to the
interaction between IncA and G3BP1 since
overexpression of an IncA mutant construct
disabled to interact with G3BP1 failed to reduce c-Myc concentration. We hypothesise
that lowering the host cell c-Myc protein
concentration may be part of a strategy employed by C. psittaci to avoid apoptosis and
scale down host cell proliferation (Borth et
al. 2011). Furthermore we could show that
IncB in early inclusions of C. psittaci interacts und co-localises with components of the
dynein complex. It was known that within
the first few hours of infection, endocytosed
C. trachomatis EBs are transferred to a perinuclear location corresponding to the microtubule-organizing centre (MTOC) of the
host cell using the tubulin-dynein system.
Like C. trachomatis, C. psittaci also utilises
dynein motor proteins for optimal early development. The interplay of IncB of C. psittaci
with dynein components is the first allusion
to the nature of chlamydial proteins involved
in this transport process.
While the present knowledge on interactions
of chlamydial effector proteins with host
proteins almost entirely refers to the human
pathogens C. trachomatis and C. pneumoniae,
we focused on the IncA and IncB proteins
(Fig. 3) of the zoonotic agent C. psittaci using
yeast two-hybrid screens to search for inter-
86
Figure 3
IncB of C. psittaci localises to the
inclusion membrane.
HEp-2 cells were infected with C.
psittaci at a MOI of 3, and at 24 h post
infection, the cells were fixed and immunolabeled with anti-IncB (green),
counterstained with DAPI (blue,
nuclei), and viewed under a confocal laser-scanning microscope. IncB
shows a typical inclusion membrane
staining. Bar: 20 µm.
4
Interaction of the multifunctional
signal protein TopBP1 with the helicase RecQL4
Group Leader: Frank Hänel
(in cooperation with Peter Hortschansky,
Department of Molecular and Applied
Microbiology, HKI, and Shamci Monajembashi, Frank Grosse, Matthias Görlach and
Helmut Pospiech, Leibniz Institute for Age
Research – Fritz Lipmann Institute, Jena).
TopBP1 is a BRCT domain-rich protein that
is structurally and functionally conserved
throughout eukaryotic organisms. It is required for the initiation of DNA replication
and for DNA repair and damage signalling.
To further dissect its biological functions,
we explored TopBP1-interacting proteins by
yeast two-hybrid experiments. As TopBP1
binding partner we identified the helicase
RecQL4 and confirmed the physical interaction by GST pull-down assays, co-immuno-
precipitations and by surface plasmon resonance binding assays.
RecQ-like DNA helicases form a ubiquitous
protein family that plays a pivotal role in the
maintenance of genome stability from bacteria to man. Five RecQ-like DNA helicases
have been identified in the human cell, mutations of three of which are associated with
autosomal, recessive human disorders that
display various symptoms of genomic instability and premature ageing. Mutations
in the RecQL4 gene are associated with the
Rothmund–Thomson (RTS), RAPADILINO
and Baller-Gerold syndromes. Numerous
studies have indicated a role for RecQL4 helicase at the crossroads of DNA replication, recombination and DNA damage response.
The amino-terminus of RecQL4 possesses
limited homology to the essential yeast replication factor Sld2. In yeast, the binding of
Sld2 to Dpb11 (the yeast orthologue of TopBP1)
constitutes a critical regulatory step dur-
87
References
Ohlenschläger O, Kuhnert A, Schneider A, Haumann S, Bellstedt P, Keller
H, Saluz HP, Hortschansky P, Hänel
F, Grosse F, Görlach M, Pospiech H
(2012) The N-terminus of the human
RECQL4 helicase is a homeodomainlike DNA interaction motif. Nucleic
Acids Res 40, 8309-8324.
ing initiation of DNA replication, and Xenopus laevis RecQL4 (XRecQL4) has also been reported to bind to XCut5, the frog orthologue of
Dpb11. The amino-terminal ~55 amino acids
of human RecQL4 display the highest sequence homology to the N-terminus of Sld2,
and this region is conserved in metazoans.
This led us to speculate that this part of the
protein constitutes an autonomous subdomain of the RecQL4 N-terminal region. To
address this assumption, we assessed binding of the human Dbp11 homologue TopBP1
and of DNA to the N-terminal 54 residues of
RecQL4 (RecQL4_N54). In addition, we determined its solution structure via NMR spectroscopy. In essence, RecQL4_N54 represents
the minimal region for the interaction with
TopBP1, binds Y-shaped and double-stranded
(ds)DNA with no apparent sequence specificity and adopts a fold highly similar to homeodomains (Ohlenschläger et al. 2012).
5
Melanin dependent survival of
Apergillus fumigatus conidia in lung
epithelial cells
Group Leaders: Gerhard Schöfl,
Hans Peter Saluz
(in cooperation with the Leibniz Center
for Tropical Marine Ecology (ZMT), Bremen;
Leibniz Institute for Baltic Sea Research
Warnemünde; University Rostock; NTT,
and the Indonesian Ministry for Marine
Affairs and Fisheries)
In 2012 we launched a project within the
SPICE III framework that aims at the taxonomic and functional characterisation of the
microbial gut communities in economically
and alimentary relevant marine organisms
in Indonesian coastal areas under pollutant
and non-pollutant conditions. The microbiome includes bacterial, fungal, protistan,
and viral species, many of which may pose
threats to human and animal health, and/or
impact sustainability of food production. It is
estimated that about 98 % of the prokaryotic
species cannot be cultivated under laboratory conditions, and consequently are not
88
easily detectable. By employing a culture-independent metagenomic approach, we aim to
generate taxonomic profiles and snapshots of
metagenome gene content for faecal samples
collected in different coastal areas and under
different environmental conditions. At present, the main goals are to (i) establish the
bioinformatical infrastructure necessary to
handle and analyse metagenomic data generated by a next-generation sequencing platform, and (ii) to demonstrate, whether and
if yes, what kind of pathogenic microorganisms can be found in food fish derived from
Jakarta bay.
The most commonly used next-generation
sequencing platforms (454/Roche pyrosequencing and Illumina/Solexa) have now
been extensively applied to metagenome
samples. While the 454/Roche technology
produces average read-lengths of ~600 pb
(with a relatively high error rate), a typical run will yield only ~500 Mbp. Illumina
HiSeq2000 instruments on the other hand
will produce ~60 Gbp per channel with, however, drastically reduced read-lengths (only
up to 150 bp). The trade-off between information loss due to short read-lengths and information loss due to lower yield is alleviated by
the recent introduction of the Illumina MiSeq
technology. With read-lengths of 2x250 bp
(paired-end libraries) and a total output of
up to 15 Gbp, the most current MiSeq technology greatly surpasses 454 technology in
terms of yield at only a marginal loss of information due to read-length reduction. This
method has not yet been used extensively
for metagenomics, but first comparisons
with established methodologies highlight
its promise. Therefore we chose to evaluate
metagenomic MiSeq sequencing within the
framework of this SPICE III project.
Initially, a total of 24 faecal samples from
three species of local food fish, Epinephelus fuscoguttatus (brown-marbled grouper),
E. sexfasciatus (six-striped grouper), and
Atule mate (yellow-fin scad) were obtained
from Jakarta Bay (north-eastern Java, polluted) and Cilacap (south-western Java,
unpolluted). To generate a comprehensive
taxonomic profile, paired-end 16S rRNA
community sequencing for all samples was
performed on the Illumina MiSeq platform
using conserved bacterial/archeal primers
for the variable V4 region of the 16S gene. To
generate functional profiles, whole genome
metagenomic sequencing was performed on
total DNA extracted from 12 E. fuscoguttatus
samples.
For the 16S amplicon sequencing, all 24
samples were multiplexed on a single MiSeq
channel, yielding approx. 15.2 m 250 bp
paired-end reads, of which 8.9 m could be
mapped to one of 530 operational taxonomic
units (OTUs) inferred using the uparse OTU
clustering method. Total DNA fragments
from 12 samples were sequenced on 2 MiSeq
channels, yielding a total of 20.6 m and 20.9
m paired-end reads, respectively. The development of a classification pipeline optimised
for whole-genome DNA derived MiSeq reads
is currently ongoing.
Preliminary analyses of the taxonomic composition of the gut microbiomes suggests
some habitat effects, possibly attributable to
pollution levels, as well as strong betweenspecific variability, with, e.g. E. fuscoguttatus microbiomes being dominated by gamma-proteobacteria, while the dominating
taxa in E. sexfasciatus and Atule mate were
members of the classes beta-proteobacteria
and clostridia, and alpha-proteobacteria and
beta-proteobacteria, respectively.
With the establishment of an analysis pipeline a larger-scale comparison of the compositions of microbial communities harvested
from fish and shellfish exposed to the relatively polluted conditions of Jakarta Bay with
control samples from less polluted areas will
be conducted to elucidate the influence of environmental conditions on intestinal microbiomes. A special interest will be qualitative
and quantitative differences in the pathogenic or potentially pathogenic gut microbiota.
6
Melanin dependent survival of
Apergillus fumigatus conidia in lung
epithelial cells
Group Leaders: Hans Peter Saluz
(in cooperation with Axel A. Brakhage,
Department of Molecular and Applied
Microbiology, HKI)
Host cell death is a critical component of
innate immunity and often determines the
progression and outcome of infections. The
opportunistic human pathogen Aspergillus
fumigatus can manipulate the immune system either by inducing or inhibiting host cell
apoptosis dependent on its distinct morphological form. Previous studies (Volling et al.
2011) showed that conidia of Aspergillus ssp.
inhibit apoptosis of macrophages induced via
the intrinsic (staurosporine) and extrinsic
(Fas ligand) pathway. Hence, mitochondrial
cytochrome c release and caspase activation
were prevented. It was further found that the
anti-apoptotic effect depends on both host
cell de novo protein synthesis and phagocytosis of conidia by macrophages. For the
first time, it could be shown that the fungal
dihydroxynaphthalene (DHN) melanin is
a crucial component to inhibit macrophage
apoptosis which may contribute to dissemination of the fungus. Apoptosis inhibition
even works with the isolated DHN melanin
and also with the corresponding synthetic
melanin.
These findings have been further investigated these findings in apoptosis-induced
human lung A549 epithelial cells. Aspergillus
fumigatus enters the human body through
inhalation of airborne conidia, thus making close contact with lung epithelial cells,
which only possess low phagocytic activity.
These cells are in particular interesting to
address the question whether there is some
form of persistence of A. fumigatus conidia in
the human host. Therefore, by using uracilauxotrophic mutant strains, one has been
able to investigate the interaction of the lung
epithelial cells and A. fumigatus conidia in
detail for long periods. Interestingly, un-
89
like professional phagocytes, the study has
shown that the presence of conidial DHN
melanin enhances the uptake of A. fumigatus
conidia by epithelial cells when compared
with non-pigmented pksP mutant conidia.
Furthermore, conidia of A. fumigatus are able
to survive within epithelial cells. This is due
to the presence of DHN melanin in the cell
wall of conidia, because melanised wild-type
conidia showed a higher survival rate inside
epithelial cells and led to inhibition of acidification of phagolysosomes. Both effects cannot be observed for white (non-melanised)
conidia of the pksP mutant strain. Moreover,
in contrast to pksP mutant conidia, melanised wild-type conidia are able to inhibit
both the intrinsic and extrinsic apoptotic
pathway in the lung epithelial cells even
for longer periods. The anti-apoptotic effect
is not restricted to conidia, because both conidia-derived melanin ghosts (cell-free DHN
melanin) and a different type of melanin, dihydroxyphenylalanine (DOPA) melanin, act
anti-apoptotically. Taken together, these data
indicate the possibility of melanin-dependent persistence of conidia in lung epithelial
cells.
7
Positron emission tomography/
computed tomography (PET/CT) for
depicting infection, bone metabolism and inflammation: reduction,
refinement, and replacement of
animal experiments
(in cooperation with Bernhard Hube,
Department of Microbial Pathogenicity
Mechanisms, HKI; Marc Thilo Figge, Research Group Applied Systems Biology, HKI;
Konrad Sachse, Friedrich-Loeffler-Institut,
Federal Research Institute for Animal Health,
Jena, and Thomas Kamradt and Ingo Irmler,
Institute for Immunology, University Hospital Jena)
After establishing molecular imaging techniques by combining positron emission tomography (PET) with computed tomography
90
(CT) we started investigating the pathogenicity of microbial infections, bone growth and
inflammations using established and new
animal model systems. Because PET/CT is a
non-invasive imaging method, stress on laboratory animals is heavily reduced (refinement). A reduction of animal experiments by
using PET/CT is achieved because the same
animal can be subjected to repeated measurements and moreover, it can act as its own
control. Finally, by introducing the embryonated chicken egg as an alternative animal
model, we were able to replace rodents with
bird embryos, especially well suited for in
vivo experiments in the context of infections
with C. psittaci, the original hosts of which
are birds.
8
Automated segmentation and object classification of CT images: Application to in vivo molecular imaging of avian embryos
Although chick embryogenesis has been
studied extensively, there has been growing
interest in the investigation of skeletogenesis. In addition to improved poultry health
and minimised economic loss, a better understanding of skeletal abnormalities can also
have implications for human medicine. True
in vivo studies require non-invasive imaging techniques such as high-resolution µCT.
However, the manual analysis of acquired
images is both time consuming and subjective. For these reasons we have developed a
system for automated image segmentation
that entails object-based image analysis followed by the classification of the extracted
image objects. For image segmentation, a
rule set was developed using Definiens image analysis software. The classification
engine was implemented using the WEKA
machine learning tool. Our system reduces
analysis time and observer bias while maintaining high accuracy (Fig. 4). Applying the
system to the quantification of long bone
growth has allowed us to present the first
Figure 4
Graphical representation of the complete automated image segmentation
procedure. (From: Heidrich et al. 2013)
true in ovo data for bone length growth recorded in the same chick embryos. The procedures developed represent an innovative
approach for the automated segmentation,
classification, quantification, and visualization of µCT images (Fig. 5). µCT offers the possibility of performing longitudinal studies
and thereby provides unique insights into
the morphogenesis and embryogenesis of
live chick embryos.
9
Insights into bone metabolism of
avian embryos in ovo via 3d and 4d
18
F-fluoride Positron Emission Tomography
Group Leader: Hans Peter Saluz (in cooperation with Thomas Opfermann, Institute of
Nuclear Medicine, University Hospital Jena)
Although the chick embryo is a well-known
economical and widely applied in vivo model
system for e.g., bone development studies,
it is surprising that no studies concerning
the application of 18F-fluoride microPET to
bone metabolism were reported so far. Reasons for this lack might be motion artifacts
and the lack of convenient tracer injection
tools and sites. We therefore resolved the
above problems by a combination of embryo
in ovo anesthesia, microPET imaging, followed by special computational processing.
By these means we developed a convenient
way of visualising three- and four- dimensional features of bone metabolism in living
chick embryos. Thereby the application of
18
F-fluoride microPET facilitates repeat measurements, highly reproducible and motionartifact-free skeletal imaging, and provides
quantitative measurements of in ovo metabolic activities in the bones of developing
chicks. During micro-PET measurement, a
radio tracer was injected intravascularly using a custom-made catheter system, allowing us to additionally investigate early time
References
Heidrich A, Schmidt J, Zimmermann J,
Saluz HP(2013) Automated Segmentation and Object Classification of CT
Images: Application to In Vivo Molecular Imaging of Avian Embryos. Int J
Biomed Imaging, 508474.
91
Figure 5
Segmentation and classification of
micro CT images of Group 1 chick
embryos from d 13 to d19 . The bone
names and their corresponding colors
are presented. Image artifacts are
circled in green. (From: Heidrich et
al. 2013)
points in tracer kinetics and uptake. Our
results clearly show that bone metabolism
in living chick embryos can be reproducibly studied and quantified in ovo, even for
multiple tracer injections over a longer time
period. The use of dynamic 18F-fluoride microPET imaging made it possible to visualise and analyse even small bone structures
with excellent quality. Moreover, as our data
are comparable to data from corresponding
rodent experiments, the use of embryonated
chicken eggs is a convenient and economical
alternative to other animal models.
10
PET/CT image analysis in experimental arthritis
Group Leader: Hans Peter Saluz (in cooperation with Ingo Irmler and Thomas Kamradt,
Institute for Immunology, University Hospital Jena, and Marc Thilo Figge, Research
Group Applied Systems Biology, HKI)
92
Rheumatoid arthritis (RA) is one of the most
common autoimmune diseases and mainly
characterised by joint inflammation, bone erosion and deformation. Up to now there exists
no cure and the causes of RA are also not
resolved in detail. Early detection of the disease can improve the treatment success and
is important to evaluate the effectiveness of
new drugs. The gold standard for exploring
experimental arthritis in murine models is
histopathological investigation, meaning
that animals have to be sacrificed to assess
joint inflammation and bone destruction. In
contrast, imaging with positron emission
tomography-computed tomography (PET/CT)
is minimal invasive and therefore a promising approach to investigate arthritic progresses in murine models. This multimodal
imaging technique offers the opportunity to
explore experimental arthritis in vivo and in
longitudinal studies. Besides this the number of animals needed for preclinical studies
can be reduced in a large amount. In this
Figure 6
PET/CT imaging in experimental
arthritis and high-resolution surface reconstruction.
(A) 18F-fluoride PET and CT image
fusion at day 28 after G6PI immunization. Inflamed joints show a high
tracer uptake. (B) High-resolution
surface reconstruction of the zoomed
region in A. (C) Highlighting of bone
destruction. Blue colour denotes
smooth regions, green colour denotes
rough and eroded regions of metatarsal bones.
subproject the usability of the PET tracer 18Ffluorodeoxyglucose (18F-FDG) for quantification of joint inflammation in a model of glucose-6-phosphate isomerase (G6PI)-induced
murine arthritis could already been shown.
The current research covers the application
of 18F-fluoride as a tracer for bone metabolism as well as a more extensive quantification of bone erosion in the paws based on CT
images. CT offers a higher spatial resolution
than PET imaging and therefore may allow
better discrimination of different stages in
arthritic progress, but also earlier detection of the disease based on symptoms like
bone erosion and malformation. New computer aided approaches for (semi-)automated
image analysis are under development in
a close cooperation with the HKI research
group Applied Systems Biology. Two different
concepts are pursued within these studies.
The first one is based on measurements of
cortical bone thickness, as arthritic mice
show a loss of cortical bone substance dur-
ing progress of the disease. Additionally,
the second approach uses high-resolution
surface reconstruction of metatarsal bones
to quantify bone erosion based on surface
roughness (Fig. 6).
The combination of functional imaging via
PET, giving insights into bone metabolism,
and high-resolution anatomical imaging via
CT may give the opportunity to improve diagnostics and also treatment testing in murine
models, towards RA in humans.
References
Irmler IM, Opfermann T, Gebhardt P,
Gajda M, Brauer R, Saluz HP, Kamradt
T (2010) In vivo molecular imaging of
experimental joint inflammation by
combined (18)F-FDG positron emission tomography and computed
tomography. Arthritis Res Ther 12, R203.
93
Group members
Dr. Kusuma Jitsaeng
Ubon Ratchathani University, Thailand
10/2012–11/2012
Head
Prof. Dr. Hans Peter Saluz
Phone +49 3641 532-1200
Fax
+49 3641 532-0805
[email protected]
Dr. Brent Sørensen
Max Planck Institute for Chemical Ecology,
Jena, Germany
04/2013–06/2013
Secretary
Svetlana Bauer
Dr. Ingo Irmler
Since 03/2012
Scientists
Dr. Anne Bleicher (until 11/2013)
Dr. Frank Hänel
Dr. Peter Gebhardt
Dr. Hans Krügel (until 10/2012 )
Dr. Jens Müller (since 11/2013)
Dr. Gerhard Schöfl
Dr. Brent Sørensen
Dr. Alexander Tretiakov
External funding
Thüringer Aufbaubank
Integriertes Multiplex-Genotyping-System basierend auf einem hyperspektralen Vielfarben-Scanner zur schnellen qualitativen und quantitativen
Sequenzanalyse von SNPs und DNA-Polymorphismen auf Microarrayoberflächen
Hans Peter Saluz
Ph.D. Students
Shayista Amin
Thomas Beder (since 11/2013)
Selina Böcker
Alexander Heidrich (until 06/2013)
Bianca Hoffmann
Julia Kästner
Anja Kuhnert (until 02/2012)
Katrin Litsche (until 03/2012)
Sara Mohebbi
Philipp Sehnert
Arndt Steube
Anja Voigt
Lydia Würbach (until 04/2012)
Verbundprojekt Zoonotische Chlamydien –
Wechselwirkung von zoonotischen Chlamydien
mit ihren Wirtszellen
Hans Peter Saluz, Frank Hänel
Resistom – Antibiotika-Resistom in der Lebensmittelkette
Hans Krügel
Jochen Bick (until 05/2013)
Thomas Beder (10/2012–10/2013)
Anna Dettling
Sabine Kupis
Martin Schneider (since 10/2012)
Philipp Sehnert (until 07/2012)
Martin Thamm (until 03/2013)
Ding Wang
Christina Wichmann
PET-CT II – In-vivo-Quantifizierung von Inflammation und Destruktion in murinen Arthritismodellen
mittels PET/CT als Beitrag zur Reduktion der Zahl
der in Forschung und Entwicklung verwendeten
Versuchstiere
Hans Peter Saluz
SPICE III – Wissenschaftlich-technische Zusammenarbeit mit Indonesien: MABICO - Einfluss von
Meeresverschmutzung auf Biodiversität und den
Lebensunterhalt von Küstenbewohnern; Mikrobielle Biodiversität
Hans Peter Saluz
Research Assistants
Claudia Franke
Grit Mrotzek
Vera Mittenzwei-Klujewa (until 09/2012)
Yvonne Hupfer (until 03/2012)
Project: Comparative genomics of host /
chlamydiales interactions
Hans Peter Saluz
Trainees
Karthika Annamalai (07/2012–05/2013)
Sarah Behr
Alexander Korn (02/2013–03/2013)
Juliane Kießling
Sabine Kupis
Anna Lisa Mitter
Abdul Muslihudeen
Hanna Noack (until 04/2012)
Dominique Prätzsch
Mareike Scheven
Sebastian Schneider (until 03/2012)
Project: Pathogen/host communication: dual RNA
expression dynamics to investigate differential
patterns of virulence
Hans Peter Saluz
Klimina KM, Kjasova DCh, Poluektova EU, Krügel
H, Leuschner Y, Saluz HP, Danilenko VN (2013)
Identification and characterization of toxin-antitoxin systems in strains of Lactobacillus rhamnosus
isolated from humans. Anaerobe 22, 82-89.
Bleicher A, Schöfl G, Rodicio MD, Saluz HP (2013)
The plasmidome of a Salmonella enterica serovar
Derby isolated from pork meat. Plasmid 69, 202-210.
Groot AT, Staudacher H, Barthel A, Inglis O, Schöfl
G, Santangelo RG, Gebauer-Jung S, Vogel H, Emerson J, Schal C, Heckel DG, Gould F (2013) One
quantitative trait locus for intra- and interspecific
ariations in a sex pheromone. Mol Ecol 22, 10651080.
Rassmann A, Martin U, Saluz HP, Peter S, Munder
T, Henke A (2013) Identification of gene expression
profiles in HeLa cells and HepG2 cells infected with
Coxsackievirus B3. J Virol Methods 187, 190-194.
Collaborations
Food GmbH Jena, Germany
Dr. Michael Grün
Friedrich-Loeffler-Institut, Federal Research
Institute for Animal Health, Jena
Dr. Konrad Sachse
Dr. Thomas Opfermann
Gesellschaft zur Förderung von Medizin-, Bio- und
Umwelttechnologien e.V. (GMBU), Jena, Germany
Dr. Emanuel Gutmann
Dipl.-Ing. Florian Erfurth
Gondol Research Institute for Mariculture,
Indonesia
Dr. Haryanti
Leibniz Center for Marine Tropical Ecology (ZMT),
Bremen, Germany
Dr. Claudia Schultz
Dr. Sebastian Ferse
Leibniz Institute for Age Research, Fritz-Lipmann-Institut, Jena, Germany
Prof. Frank Große
Max Planck Institute for Chemical Ecology, Jena,
Germany
Dr. Ales Svatos
Research Ministry of Marine Affairs and Fisheries,
Jakarta, Indonesia
Dr. Ketut Sugama
Universtiy of Applied Sciences, Jena, Germany
Prof. Dr. Thomas Munder
Prof. Dr. Harry Palm
Visiting Scientists
Dr. Haryanti
Gondol Research Institute for Mariculture,
Indonesia
11/2012–12/2012
11/2013
94
Heidrich A, Schmidt J, Zimmermann J, Saluz HP
(2013) Automated segmentation and object classification of CT images: application to in vivo
molecular imagin of avian embryos. Int J Biomed
Imaging 2013, 508474.
Research Group Applied Systems Biology
95
Research Group Applied Systems Biology
The focus of the research group Applied
Systems Biology (ASB) is on the quantitative analysis and mathematical modelling of
microscopy image data. Image-based Systems Biology represents an essential connective link in joint studies of experiment
and theory and complements bioinformatics
analysis of “omics”-data by addressing the
dynamical, functional and morphological
aspects of biological processes. Image-based
Systems Biology includes the following three
main aspects: (i) acquisition and automated
analysis of image data for high-content and
high-throughput screening, (ii) quantitative
description of biological processes by appropriate characteristic measures, (iii) con-
struction of image-derived spatiotemporal
models and predictive computer simulations.
Applying the Image-based Systems Biology
approach is a unique feature of the research
group ASB at the HKI which collaborates
with experimentalists at the HKI and beyond
on both of the institute’s research priorities:
natural product research and infection biology of human-pathogenic fungi. Microscopy
data are provided by the Research Group
Fungal Septomics, the Department Microbial
Pathogenicity Mechanisms and the Bio Pilot
Plant. Horn. External collaborations exist
with Prof. Dr. J. Stein (Lymphocyte Trafficking and Activation) at the Theodor Kocher
Head:
Prof. Dr. Marc Thilo Figge
Der Schwerpunkt der Forschungsgruppe
Angewandte Systembiologie (ASB) liegt auf
der quantitativen Analyse und mathematischen Modellierung von Mikroskopie-Bilddaten. Die Bildbasierte Systembiologie ist
ein wesentliches Bindeglied in gemeinsamen Studien von Experiment und Theorie
und die Berücksichtigung von dynamischen,
funktionellen und morphologischen Aspekten biologischer Prozesse ist ein zur bioinformatorischen Analyse von „omics“-Daten komplementärer Ansatz. Bildbasierte
Systembiologie umfasst die folgenden drei
Hauptaspekte: (i) Erstellung von Bilddaten
und deren automatisierte Analyse für ein
high-content- und high-troughput-screening,
(ii) quantitative Beschreibung biologischer
Prozesse durch geeignete charakteristische
Maße, (iii) Generierung von Bild-basierten
96
Raum-Zeit-Modellen und prädiktiven Computersimulationen.
Die Anwendung der Bildbasierten Systembiologie ist ein Alleinstellungsmerkmal der
Forschungsgruppe ASB am HKI. Die theoretischen Studien werden in Zusammenarbeit
mit Experimentatoren am HKI und darüber
hinaus durchgeführt und umfassen beide
Forschungsschwerpunkte des Instituts: die
Naturstoff-Forschung und die Infektionsbiologie human-pathogener Pilze. Mikroskopie-Daten werden erstellt in der Gruppe Fungal Septomics, in der Abteilung Mikrobielle
Pathogenitätsmechanismen und im Biotechnikum. Externe Kooperationen sind etabliert
mit Prof. Dr. J. Stein (Transport und Aktivierung von Lymphozyten) am Theodor-Kocher-Institut in Bern, Prof. Dr. A. Hauser (Im-
HKI Research Report 2012 / 2013 Research Group Applied Systems Biology
Institute in Bern, Prof. Dr. A. Hauser (Immunodynamics) at the Leibniz Institute German Rheumatism Research Centre in Berlin
and Prof. Dr. M. Gunzer (Experimental Immunology and Imaging) at the University
Duisburg-Essen.
In the research group ASB, Image-based
Systems Biology is realized with regard to
all three aforementioned aspects: (i) Fully
automated image analysis is performed by
the postdoctoral researchers Dr. Franziska
Mech and Dr. Carl Svensson as well as the
PhD student Susanne Brandes. The implementation ranges from the use of the commercial software Definiens Developer XD®
mundynamik) am Leibniz-Institut Deutsches
Rheuma-Forschungszentrum in Berlin und
Prof. Dr. M. Gunzer (Experimentelle Immunologie und Bildgebung) an der Universität
Duisburg-Essen.
In der Forschungsgruppe ASB wird die
Bild-basierte Systembiologie in Bezug auf
alle drei zuvor genannten Aspekte realisiert: (i) Die vollautomatische Analyse von
Bilddaten wird von den Postdoktoranden Dr.
Franziska Mech und Dr. Carl Svensson sowie
von der Doktorandin Susanne Brandes bewerkstelligt. Die Implementierung der Algorithmen reicht von der Programmierung im
Rahmen der kommerziellen Software Definiens Developer XD® und Imaris® zu selbst geschriebenen Codes in den Sprachen C++ und
Python mit modernen Methoden aus dem
and Imaris® to self-written code in C++ and
Python languages using state-of-the-art machine learning methods. (ii) Mathematical
analysis of the image-derived raw data to
quantify biological processes is realized by
the PhD student Zeinab Mokhtari. (iii) Integration of the quantified spatio-temporal information into models – to generate testable
predictions and hypotheses by computer
simulations – is realized by the PhD students
Johannes Pollmächer and Teresa Lehnert
through applying ordinary differential equations, state-based models or agent-based
models – depending on the properties of the
biological process under consideration.
Bereich des maschinellen Lernens. (ii) Die
Anwendung von mathematischen Konzepten
zur Herleitung charakteristischer Größen für
die Quantifizierung von biologischen Prozessen, die in den Rohdaten aus der Bildanalyse enthalten sind, wird von der Doktorandin
Zeinab Mokhtari realisiert. (iii) Die Integration der quantitativ erfassten raum-zeitlichen
Information in Modelle, die zur Generierung
von überprüfbaren Voraussagen und Hypothesen auf dem Computer simuliert werden
können, wird von den Doktoranden Johannes
Pollmächer und Teresa Lehnert realisiert.
Abhängig von den Eigenschaften des betrachteten biologischen Prozesses reichen
die Modellierungsansätze von gewöhnlichen
Differentialgleichungen über Zustand-basierte Modelle bis hin zu Agenten-basierten Modellen.
Research Group Applied Systems Biology HKI Research Report 2012 / 2013
97
In joint experimental and theoretical studies,
we investigate phagocytosis assays for alveolar macrophages versus Aspergillus fumigatus or Lichtheimia, as well as invasion assays
for Candida albicans on oral epithelial cells,
and a whole blood model to quantify innate
effector mechanisms and immune evasion
by C. albicans in human blood. In addition,
we develop image processing algorithms for
recognition and sorting of Actinobacteria in
picoliter-droplets in real time. The development of tracking algorithms goes hand in
hand with the identification of mathematical
measures for the characterization and classification of cell tracks from image sequences.
In gemeinsamen experimentellen und theoretischen Studien untersuchen wir Phagozytose-Assays für Alveolarmakrophagen und
Aspergillus fumigatus oder Lichtheimia, sowie
einen Invasionsassay für Candida albicans
auf oralen Epithelzellen und ein Vollblut-Modell zur Quantifizierung von angeborenen Effektor-Mechanismen und der Immunevasion
von C. albicans im menschlichen Blut. Darüber hinaus entwickeln wir Bildverarbeitungsalgorithmen zur Erkennung und Sortierung
von Actinobacteria in Pikoliter-Tröpfchen in
Echtzeit. Die Entwicklung von Tracking-Algorithmen geht Hand in Hand mit der Identifizierung mathematischer Maße, mit denen
Zell-Tracks aus Bildsequenzen charakterisiert und klassifiziert werden können.
98
Scientific Projects
Figure 1
(A) Overview of segmentation and
subsequent classification of C. albicans cells by the automated image
analysis. (a) Original image. (b) Application of the maximum intensity
transformation. (c) Recognition of the
region of interest (white outline) as
a C. albicans cell. (d) The filamentous
cell was classified into a yeast cell (red
outline) and its two hyphae (green
outline). (e) Hyphae were further
partitioned into invaded (blue) and
non-invaded (pink) segments. (B)
Original and analysed images for six
time points. Hyphae and yeast segmentation is shown in comparison
with the original images for each
time point.
1
Interaction between host cells and
human pathogenic fungi: Quantification of microscopic image data for
different confrontation assays
Group Leaders: Franziska Mech,
Marc Thilo Figge
In collaboration with the Department Molecular and Applied Microbiology (A. Brakhage),
automated analysis of microscopic image
data was previously established for phagocytosis assays of murine alveolar macrophages
and Aspergillus fumigatus conidia (Mech et
al. 2011). This ongoing collaboration is now
focused on the quantitative image analysis
of phagocytosis assay with Lichtheimia species. Futhermore, in cooperation with the Department Microbial Pathogenicity Mechanisms (B. Hube), the automated analysis of
image data was applied to invasion assays for
Candida albicans and human epithelial cells.
This required an adaptation of the exist-
ing algorithm for spherical image objects
to recognize the morphologically changed
yeast cells forming hyphae and invading the
epithelium. A selection of typical image data
is presented in Figure 1. Based on the automated image analysis, the kinetics of hyphal
growth and of epithelial invasion were quantitatively described by mathematical models
consisting of coupled differential equations.
This analysis revealed that the initiation of
hyphae formation represents an ultimate
commitment to invasive growth, which suggests that the yeast to hypha transition in
vivo must be under exquisitely tight negative
regulation to avoid the transition from commensal to pathogen and hyphal invasion of
the epithelium (Mech et al. 2012, Mech et al.
2013).
References
Coelho FM, Natale D, Soriano SF,
Hons M, Swoger J, Mayer J, Danuser
R, Scandella E, Pieczyk M, Zerwes HG,
Junt T, Sailer AW, Ludewig B, Sharpe
J, Figge MT, Stein J (2013) Naïve B
cell trafficking is shaped by local
chemokine availability and LFA-1
independent stromal interactions.
Blood 121, 4101-4109.
Figge MT, Reichert AS, MeyerHermann M, Osiewacz HD (2012)
Deceleration of fusion–fission cycles
improves mitochondrial quality control during aging. PLoS Computational
Biology 8, e1002576.
Figge MT, Osiewacz HD, Reichert AS
(2013) Quality control of mitochondria
during aging: Is there a good and a
bad side of mitochondrial dynamics?
Bioessays 35, 314-322.
99
Figure 2
Schematic representation of the
state-based model.
Circular symbols depict different
states of the model, i.e. the green
circle represents extracellular alive
C. albicans (CAE), the red circle indicates extracellular killed C. albicans
(CKE), the black circles symbolise
resistant C. albicans that are alive (CAR)
or killed (CKR), orange circles represent states of monocytes (Mi,j) with
i alive and j killed C. albicans and the
blue circles depict different states
of granulocytes (Gi,j). The arrows
between states represent transitions
studied in the virtual whole-blood
infection model.
References
Guthke R, Linde J, Mech F, Figge MT
(2012) Systems biology of microbial
infection. Frontiers in Microbiology
3, 328.
Horn F, Heinekamp T, Kniemeyer O,
Pollmächer J, Valiante V, Brakhage
AA (2012) Systems biology of fungal
infection. Frontiers in Microbiology
3, 108.
Mech F, Thywißen A, Guthke R,
Brakhage AA, Figge MT (2011)
Automated image analysis of the
host-pathogen interaction between
phagocytes and Aspergillus fumigatus.
PLoS ONE 6, e19591.
Mech F, Figge MT (2012) Image-based
systems biology: A quantitative
approach to elucidate the kinetics of
fungal morphologies and virulence.
Proceedings of the German Conference
on Bioinformatics 2012, HighlightPaper.
100
2
Mathematical modelling: From
strategies of the immune response
to intracellular infection processes
Group Leaders: Johannes Pollmächer,
Teresa Lehnert, Marc Thilo Figge
Modelling of the immune response to
human-pathogenic fungi
Computer simulations are a valuable tool
to investigate strategies of the immune response against fungal infections within
mathematical models (Guthke et al. 2012). In
the research group ASB, agent-based models
are developed that describe each biological object within a system as an individual
agent, which moves in space and time and
interacts with other agents according to defined rules. This approach allows to integrate
the information obtained from image data
analysis, e.g. on the migration behaviour
of cells, into computer simulations (Horn et
al. 2012). This was realized in a compara-
tive study of virtual phagocytosis assays for
neutrophils versus A. fumigatus conidia to
investigate the phagocytosis efficiency as a
function of the migration behaviour of neutrophils (Tokarski et al. 2012).
We currently develop a software package for
agent-based modelling that will allow testing
of hypotheses about the immune response in
complex and dynamic structures, such as
alveoli in the presence of respiration. Such
structures are not accessible for investigation under physiological conditions and represent an ideal field for application of virtual
infection models. Furthermore, in collaboration with the Research Group Fungal Septomics (O. Kurzai) a virtual infection model
for infection assays of whole blood with C. albicans is developed. Using the state-based
model depicted in Figure 2, in combination
with a Metropolis Monte Carlo algorithm to
quantitatively determine the optimal model
parameters, we investigate innate effector
Figure 3
Schematic representation of antibody feedback during a germinal
centre reaction.
Circulating high-affinity antibodies
(red) that are dynamically binding to
antigen presented on follicular dendritic cells reduce its availability for
B lymphocytes and by that increase
the selection pressure for retrieval of
antibodies with even higher binding
affinities.
mechanisms and immune evasion by C. albicans in human blood.
the product and the regulator of the germinal
centre reaction.
Modelling antibody affinity maturation
in germinal centres
Studies on the antibody affinity maturation
in germinal centres pose a prime example
for the fruitful exchange between experimental and theoretical research. In such a
joint study with scientists from Geneva (CH),
Birmingham (UK) and Braunschweig (G), we
recently proved that germinal centres can
communicate with each other through the
direct exchange of high-affinity antibodies
(Zhang et al. 2013). Computer simulations
of agent-based models did not only predict
an increased selection pressure and thus
the production of even higher affinity antibodies, but also the termination of a germinal centre reaction, if no further improvement of the antibody affinity was possible.
Thus, as is schematically shown in Figure
3, antibodies play the central role of being
Modelling of mitochondrial network dynamics – a view of aging as intracellular
infection process
In cooperation with experimentalists from
Goethe University Frankfurt, the spread of
molecular damage in the mitochondrial network of a cell was investigated by a statebased model. The loss of mitochondrial
functionality contributes to the formation of
degenerative processes, which are thought
to be responsible for neurodegenerative
diseases and to be a cause of aging. The dynamics of the mitochondrial network plays
a crucial role for the bio-energetic quality
control. However, the model developed in the
research group ASB reveals that mitochondrial dynamics is a double-edged sword: too
frequent fusion and fission processes can
highly increase the probability of molecular
damage spread and degeneration of the intra-
References
Mech F, Wilson D, Lehnert T, Hube B,
Figge MT (2013) Epithelial invasion
outcompetes hypha development
during Candida albicans infection as
revealed by an image-based systems
biology approach. Cytometry Part A,
DOI: 10.1002/cyto.a.22418.
Mokhtari Z, Mech F, Zitzmann C,
Hasenberg M, Gunzer M, Figge MT
(2013) Automated characterization
and parameter-free classification of
cell tracks based on local migration
behavior. PLoS One 8: e80808.
Tokarski C, Hummert S, Mech F, Figge
MT, Germerodt S, Schroeter A, Schuster S (2012) Agent-based modelling
approach of immune defense against
spores of opportunistic human
pathogenic fungi. Frontiers in Microbiology 3, 129.
101
Figure 4
Three phases for triggered
imaging and sorting.
Droplet detection by the photodiode
and subsequent triggered imaging
occurs in phase I. During phase II,
image retrieval and analysis is executed. Based on the outcome of
the image analysis, a sorting pulse is
generated and transmitted through
the electrodes in phase III, changing
the directionality of the droplets.
References
Zang E, Brandes S, Tovar M, Martin K,
Mech F, Horbert P, Henkel T, Figge MT,
Roth M (2013) Real-time image processing for label-free enrichment of
Actinobacteria cultivated in picolitre
droplets. Lab on a Chip 13, 3707-3713.
Zhang Y, Meyer-Hermann M, Geroge
L, Figge MT, Khan M, Goodall M,
Young SP, Reynolds A, Falciani F, Waisman A, Notley CA, Ehrenstein MR,
Kosco-Vilbois M, Toellner KM (2013)
Germinal center B cells govern their
own fate via antibody feedback. The
Journal of Experimental Medicine 210,
457-464.
102
cellular mitochondrial network, ultimately
resulting into aging of cells (Figge et al.
2012, Figge et al. 2013).
3
Image processing for recognition
and sorting of Actinobacteria in
pL-droplets in real time
Group Leaders: Susanne Brandes,
Marc Thilo Figge
The subject of this cooperation with the Bio
Pilot Plant (U. Horn, M. Roth) is the development of an automated image analysis procedure for microfluidic application. The aims
are (i) to detect the growth of actinobacteria
in microfluidic droplets with pico-liter (pL)
volumes and (ii) to sort the droplets depending on the content of actinobacteria in real
time. This was successfully achieved by a
setup that is depicted in Figure 4 and yielded
the high-throughput cultivation of more than
half a million of cultures per hour and subsequent sorting of unlabelled droplets in a
real-time analysis of triggered images with a
frequency above 100 Hz (Zang and Brandes
et al. 2013). The technique has a high potential for screening of unknown or difficult to
cultivate actinobacteria and thus for the discovery of novel natural products.
4
Characterisation and classification
of cell migration from image sequences
Group Leaders: Zeinab Mokhtari,
Susanne Brandes, Marc Thilo Figge
The characterisation and classification of cell
migration generally requires the analysis of
individual cell tracks relative to their surroundings, because useful knowledge about
the migration behaviour can be lost in the
process of averaging over cell populations
Group members
Head
Prof. Dr. Marc Thilo Figge
Phone +49 3641 532-1416
Fax
+49 3641 532-2416
[email protected]
Scientists
Dr. Carl Svensson
Anna Medyukhina (since 11/2013)
Dr. Zoltán Cseresnyés (since 10/2013)
Dr. Franziska Mech (until 09/2013)
Ph.D. Students
Susanne Brandes
Bianca Hoffmann
Kaswara Kraibooj (since 09/2013)
Teresa Lehnert
Zeinab Mokhtari
Johannes Pollmächer
Sandra Timme (since 11/2013)
Sandra Timme (until 11/2013)
Bertram Vogel
Carolin Zitzmann (until 12/2013)
Research Assistants
Sören Totzauer (since 07/2013)
Vladimir Shelest
External funding
Project: Development of a multi-scale simulator
to model host-pathogen interactions
Marc Thilo Figge
(Coelho et al. 2013). The research group ASB
develops algorithms for the automated cell
tracking as well as methods for the systematic and quantitative characterization of cell
tracks and the classification of cell populations (Mokhtari et al. 2013). The aim is to arrive at a comprehensive tool for the extraction and analysis of cell tracks in image data.
JIMI – ein Netzwerk für intravitale Mikroskopie:
Gezielte Projektbetreuung von der Mikroskopie
in der Zelle bis zum Zelltracking in lebenden
Organismen
Marc Thilo Figge
SFB/TR FungiNet
Project B04: Bilddatenanalyse und Agenten-basierte Modellierung der Wechselwirkung zwischen
Immunzellen und humanpathogenen Pilzen in
Raum und Zeit
Marc Thilo Figge
Mech F, Wilson D, Lehnert T, Hube B, Figge MT
(2014) Epithelial invasion outcompetes hypha
development during Candida albicans infection
as revealed by an image-based systems biology
approach. Cytometry Part A 85, 126-139.
Mokhtari Z, Mech F, Zitzmann C, Hasenberg M,
Gunzer M and Figge MT (2013) Automated characterization and parameter-free classification of
cell tracks based on local migration behavior. PLoS
ONE 8, e80808.
Zang E*, Brandes S*, Tovar M, Martin K, Mech F,
Horbert P, Henkel T, Figge MT, Roth M (2013) Realtime image processing for label-free enrichment
of Actinobacteria cultivated in picolitre droplets.
Lab Chip 13, 3707-3713. *Authors contributed
equally.
103
Coelho FM, Natale D, Soriano SF, Hons M, Swoger
J, Mayer J, Danuser R, Scandella E, Pieczyk M,
Zerwes HG, Junt T, Sailer AW, Ludewig B, Sharpe J,
Figge MT, Stein J (2013) Naïve B cell trafficking is
shaped by local chemokine availability and LFA1-independent stromal interactions. Blood 121,
4101-4109.
Zhang Y, Meyer-Hermann M, Geroge L, Figge MT,
Khan M, Goodall M, Young SP, Reynolds A, Falciani
F, Waisman A, Notley CA, Ehrenstein MR, KoscoVilbois M, Toellner KM (2013) Germinal center B
cells govern their own fate via antibody feedback.
J Exp Med 210, 457-464.
Collaborations
Buchmann Institute for Molecular Life Sciences,
Frankfurt, Germany
Prof. Dr. Andreas Reichert
Frankfurt Institute for Advanced Studies (FIAS),
Frankfurt, Germany
Dr. Jörg Lücke
Friedrich Schiller University, Jena, Germany
German Rheumatism Research Centre (DRFZ),
Berlin, Germany
Dr. Raluca Niesner
Prof. Dr. Anja Hauser
Goethe University Frankfurt, Germany
Prof. Dr. Heinz Osiewacz
Helmholtz Centre for Infection Research, Braunschweig, Germany
Prof. Dr. Michael Meyer-Hermann
University of Birmingham, UK
Dr. Kai Tollner
University of Duisburg-Essen, Germany
Prof. Dr. Matthias Gunzer
Theodor Kocher Institut, Bern, Switzerland
Prof. Dr. Jens Stein
104
Research Group Fungal Septomics
105
Research Group Fungal Septomics
Fungal Septomics investigates the infection
biology of invasive fungal infections (IFI),
focusing on the interaction between fungal
pathogens and the human host. The group
is located at the Center for Innovation Competence Septomics and forms an independent associated research group at the HKI.
Septomics is a faculty-spanning centre of
the Friedrich-Schiller-University and scientifically interconnected with the Department
of Anesthesiology and Intensive Care Medicine, the Institute for Medical Microbiology
and the HKI. Within Septomics, the fields of
pathogen and host biology in sepsis are approached complementary and linked to clinical practice.
Numbers of IFI have constantly increased
over the last decades and a steadily growing number of patients must be considered
at risk for IFI. The human pathogenic yeast
Candida albicans and the filamentous fungus Aspergillus fumigatus are by far the most
important causes of life-threatening mycoses in Europe, whereas Cryptococcus neoformans plays a major role in Africa. Sepsis
- an uncontrolled immune reaction to infection affecting the whole body - can be both
a consequence of and a risk factor for IFI.
Within Fungal Septomics, we work with infection models for a range of primary human
immune cells, including neutrophilic granulocytes, monocytes, monocyte derived den-
Die Forschungsgruppe Fungal Septomics
untersucht die Infektionsbiologie invasiver
Pilzinfektionen und legt dabei den Schwerpunkt auf die Interaktion zwischen den
pathogenen Pilzen und dem menschlichen
Wirt. Die Forschungsgruppe gehört zum Zentrum für Innovationskompetenz (ZIK) Septomics und bildet eine unabhängige assoziierte Forschungsgruppe am HKI. Septomics
ist ein fakultätsübergreifendes Zentrum der
Friedrich-Schiller-Universität und wird wissenschaftlich getragen von der Klinik für
Anästhesiologie und Intensivmedizin, dem
Institut für Medizinische Mikrobiologie und
dem HKI.
Head:
Prof. Dr. Oliver Kurzai
Im Septomics Forschungszentrum wird die
Biologie von Erregern und Wirt im Rahmen
der Sepsis in komplementärer Weise bearbeitet und mit der klinischen Praxis dieser
Infektion verknüpft. Die Zahl invasiver Pilz-
106
infektionen hat in den letzten Jahrzehnten
konstant zugenommen und eine ständig
wachsende Zahl von Patienten besitzt ein hohes Risiko für invasive Pilzinfektionen. Die
humanpathogene Hefe Candida albicans und
der Schimmelpilz Aspergillus fumigatus sind
mit Abstand die wichtigsten Erreger lebensbedrohlicher Mykosen in Europa. In Afrika
ist zusätzlich insbesondere Cryptococcus
neoformans relevant.
Sepsis – eine unkontrollierte Immunreaktion
auf eine invasive Infektion, die den gesamten Körper betrifft – kann sowohl eine Folge als auch ein Risikofaktor invasiver Pilzinfektionen sein. In der Arbeitsgruppe Fungal Septomics haben wir Infektionsmodelle
für ein breites Spektrum primärer humaner
Immunzellen, darunter neutrophile Granulozyten, Monozyten, Monozyten-generierte
dendritische Zellen und natürliche Killerzel-
HKI Research Report 2012 / 2013 Research Group Fungal Septomics
dritic cells and natural killer (NK-) cells.
For this purpose, a large cohort of healthy
blood donors as well as several protocols for
isolation and cultivation of primary human
immune cells have been established. Our
work addresses aspects of fungal virulence
biology that determine the human host response to the pathogen. A major example is
filamentation of C. albicans which is closely
related to tissue invasion and innate immune
activation. A whole blood infection model is
used to analyse host-pathogen interaction in
a complex environment that closely mimics
early stages of in vivo interaction. In addition, Fungal Septomics is analysing interindividual differences in human susceptibil-
ity to fungal pathogens. For this we look at
genetic diversity and correlate genetic polymorphisms with functional consequences in
immune responses to fungal pathogens. A
major effort is the coordination of the AspIRS
international multi-centre study on the genetic risk for invasive aspergillosis in stem
cell transplant recipients.
len etabliert. Um diese Modelle bearbeiten
zu können, wurde eine große Kohorte von
gesunden Blutspendern rekrutiert und verschiedene Protokolle für die Isolierung und
Kultivierung primärer humaner Immunzellen etabliert. Unsere Arbeit adressiert verschiedene Aspekte der Pathobiologie von Pilzen, die einen Einfluß auf die Interaktion mit
dem menschlichen Immunsystem haben. Ein
wichtiges Beispiel hierfür ist die Filamentierung von C. albicans, die eine zentrale Rolle
bei der Gewebsinvasion und der Aktivierung
des angeborenen Immunsystems spielt. Wir
verwenden ein Vollblutinfektionsmodell, um
die pathogene Interaktion in einem komplexen Umfeld, das eng mit der in vivo Situation
verwandt ist, zu untersuchen.
invasive Pilzinfektionen. Zu diesem Zweck
untersuchen wir die genetische Diversität
bei Patienten und korrelieren genetische
Polymorphismen mit funktionellen Aspekten der Immunantwort auf humanpathogene Pilze. Ein wesentliches Standbein dieses
Arbeitsfelds ist die Koordinierung der internationalen Multicenterstudie AspIRS (Aspergillosis Intrinsic Risk Stratification), die das
genetische Risiko für invasive Aspergillose
bei stammzelltransplantierten Patienten adressiert.
Zusätzlich bearbeitet Fungal Septomics die
genetischen Grundlagen für interindividuelle Unterschiede in der Suszeptibilität für
The resulting data will lead to a better understanding of molecular mechanisms in IFI.
Furthermore they build a basis for translational projects that should result in the development of diagnostic tests which will help
to improve clinical management of patients
at risk for or affected by IFI.
Die erzielten Ergebnisse sollen neben einem
verbesserten Verständnis der Pathophysiologie invasiver Pilzinfektionen auch die Entwicklung neuer diagnostischer Verfahren im
klinischen Management von Risikopatienten
ermöglichen.
Research Group Fungal Septomics HKI Research Report 2012 / 2013
107
Scientific Projects
1
Adaptation of fungal pathogens
to host niches
Virulence of C. albicans is directly related to
the ability of this yeast to respond to changes
in environmental conditions with a morphological switch between the yeast form and
filamentous growth. In our group we work
on several aspects of filamentation in C. albicans. To determine the effects of filamentation on the interaction with human host
cells, we identified a group of genes that are
differentially regulated under all conditions
which favour filamentous growth, termed
core filamentation response. By correlationbased network modelling of the transcriptional response to different defined external
stimuli for morphogenesis (cooperation with
the Research Group Systems Biology / Bioinformatics, R. Guthke) we identified a set of
eight genes with highly correlated expression patterns (Fig. 1; Martin et al. 2013). The
validity of network modelling was confirmed
using datasets of advanced complexity that
describe the transcriptional response of C. albicans during epithelial invasion as well as
comparing our results with other previously
published transcriptome studies. As filamentation is mainly triggered by environmental
cues we have studied the role of CO2 as an environmental signal for C. albicans in cooperation with the group of Fritz Mühlschlegel,
University of Kent (GB). High levels of CO2
are linked to regulation of filamentation in
C. albicans. On a molecular level CO2 adaptation involves activation of the multi-sensor
adenylyl cyclase (AC), and additionally an
AC-independent CO2 sensing pathway. The
central transcriptional regulator of the latter,
Rca1p, was recently identified by Fritz Mühlschlegel in collaboration with Fungal Septomics. Unravelling of this alternate cascade,
which is conserved in yeasts, is currently in
progress. For this we make use of the genomic resources available for the closely re-
108
lated model yeast Saccharomyces cerevisiae,
including completed knock-out libraries,
possibility of synthetic-lethal screens, highthroughput transformation and multiple
reporter systems (Cottier et al. 2012, 2013).
Integrated knowledge on the initial triggers
for filamentation and the resulting changes
in gene expression is currently used in ongoing projects to extent our knowledge on
the impact of this central virulence trait on
pathogen – host interaction.
2
Innate immune recognition
of fungal pathogens
The contact between specialised immune
effector cells and invading pathogens is of
central importance in invasive infections.
Activation of innate immunity can result
both in the successful elimination of invading pathogens and in systemic hyperinflammation and sepsis. Therefore a major focus
in the group are studies on the activation of
innate immune cells by fungal pathogens.
The preferred model organism is C. albicans.
Whenever possible, comparative analyses
with the second major fungal pathogen in
Europe, A. fumigatus are performed in cooperation with the Department Molecular
and Applied Microbiology (A. Brakhage)
and Jürgen Löffler, University of Würzburg.
Furthermore, we have recently established
infection models for C. neoformans, thus enabling us to perform comparative analyses
for the major invasive fungal pathogens. A
major focus is put on polymorphonuclear
neutrophils (PMN), which play a central role
in antifungal immunity. In addition, the role
of monocytes, dendritic cells and NK cells is
studied.
PMN activation is mainly triggered by filamentous forms of C. albicans and results in
targeted migration and uptake of the fun-
Figure 1
Defining a core filamentation response (CFR) in Candida albicans.
Candida albicans wild type was
grown for a total of 12h at 37°C and
in M199 medium with a pH8. (A) This
environment triggered the transition
from unicellular yeasts to long and
branching filaments as shown by
microscopy. It was therefore possible
to distinguish between germ tube
formation and hyphal elongation.
(B) From these cells, total RNA was
isolated and used for whole- genome
expression analysis with DNA- microarrays. Out of these data, the transcriptional landscape was defined by
correlationbased networks. (C) Independent from the external stimulus,
distinct networks of upregulated
genes which were specific for germ
tube formation or hyphal elongation
could be identified. Combination
of these two networks allowed the
modelling of a core filamentation
network which was restricted to the
eight genes which were upregulated during all stages of filamentous
growth in C. albicans.
gus. We have established live cell imaging
systems for primary human PMN to further analyse these activation patterns. For
analysis of video microcopy we established
a close cooperation with the Research Group
Applied Systems Biology (M. T. Figge) who
develop automated analysis tools for live cell
imaging. In addition, comparative studies
for PMN activation by C. albicans and C. glabrata are performed in cooperation with the
Department Microbial Pathogenicity Mechanisms (B. Hube). NK cells are innate lymphocytes with potent cytotoxic activity. In cooperation with the group of Jürgen Löffler
activity of NK cells has been demonstrated
against the fungal pathogen A. fumigatus.
Other groups have shown activity against
C. neoformans, but little was known about
their role in invasive candidiasis. Primary
human NK cells were isolated from buffy
coats, primed with a cytokine cocktail and
used for confrontation assays with C. albicans. Interaction was monitored and quantified using live cell imaging, confocal microscopy, flow cytometry and Luminex/ELISA.
Cytokine-primed primary human NK cells
recognised C. albicans, resulting in degranulation and secretion of GM-CSF, IFN-g and
TNF-a. Activation of NK cells is triggered
by actin-dependent phagocytosis of C. albicans. Although antifungal activity could be
detected and attributed to secreted perforin,
NK cells were unable to inhibit filamentation
of C. albicans. Human PMN potently counteracted the pro-inflammatory reaction of NK
cells by preventing a direct contact between
109
Figure 2
NK cells completely engulf C. albicans.
To confirm internalisation of C. albicans by human NKC we performed differential staining of extracellular and intracellular fungal cells. Extracellular C. albicans
cells were stained with Calcofluor-White before permeabilisation (blue). After
permeabilisation, both intra- and extracellular C. albicans were stained using a
specific antibody (red). Immune cells were identified as NKC by CD56-positivity
(orange) and staining of the actin cytoskeleton (green) further confirmed an
embedding of the fungal cells within the NKC. Pictures are representative for
cells from three independent experiments using cells from different donors.
NK cells and the fungal pathogen. Activation
of human PMN was enhanced in the presence of NK cells. Our results clearly assign a
function to NK cells in C. albicans infection,
which involves both direct pro-inflammatory
activation and modulation of PMN activity.
For the first time, phagocytosis of a pathogen
is shown to contribute to NK cells activation.
3
In/ex vivo infection models
All in vivo studies are performed in cooperation with the Research Group Microbial Immunology (I. Jacobsen) using the expertise
of the HKI infection models group. To back
up in vitro data of the infection model with
NK cells and C. albicans a mouse model was
established by generating NK cell deficient
mice using an anti-NK1.1 antibody. Compared to wild type mice NK cell deficient
mice showed prolonged survival and lower
pro-inflammatory cytokine levels in blood
110
upon infection with a lethal dose of C. albicans. These data indicate a pro-inflammatory
role of NK cells in mice during C. albicans
infection. In cooperation with the Research
Groups Microbial Biochemistry and Physiology (M. Brock) and Microbial Immunology,
we have used the Septomics infrastructure to
establish in vivo imaging in murine models
of systemic candidiasis. We developed a novel
real-time non-invasive bioluminescence imaging technique which allows monitoring of
infection progression and determination of
fungal burden in vivo in mice. In addition to
murine infection models, we work with a human whole blood model to address immune
activation in response to fungal pathogens in
a situation close to in vivo. While many parameters of host-pathogen interaction are accessible to direct experimental quantification
in the whole-blood infection assay, others are
not (Fig. 3). To overcome these limitations,
in cooperation with the Research Group Applied Systems Biology we generated a virtual
Figure 3
C. albicans predominantly associates with PMN.
Representative blood smears of C.
albicans-infected blood after 30
min (A), 60 min (B) and 240 min
(C) demonstrate association of C.
albicans with PMN and continuous
filamentation of extracellular fungi.
infection model that allowed detailed and
quantitative predictions on the dynamics
of host-pathogen interaction. Experimental
time-resolved data were simulated using a
state-based modelling approach combined
with the Monte Carlo method of simulated annealing to obtain quantitative predictions on
a priori unknown transition rates and to identify the main axis of antifungal immunity.
Results clearly demonstrated a predominant
role of PMN, mediated by phagocytosis and
intracellular killing as well as the release of
antifungal effector molecules upon activation, resulting in extracellular fungicidial
activity. Both mechanisms together account
for almost 98 % of C. albicans killing. Independent of filamentation and not linked to
exhaustion of inactivation of innate immune
cells a fraction of C. albicans cells escaped
phagocytosis and remained extracellular
which may account for the high proportion
of dissemination in C. albicans bloodstream
infection (Hünniger, Lehnert et al. 2014).
Furthermore, in cooperation with Niels Riedemann (InflaRx GmbH, Jena) and the Department Infection Biology (P. Zipfel) we have
used the human whole blood model to analyse the impact of C5a inhibition on the immune response against C. albicans. We have
found that early PMN activation and effector function against C. albicans clearly rely
on C5a – a highly potent pro-inflammatory
product, which is generated right upon activation of the complement cascade.
4
Genetic risk factors for fungal
infection
Fungal Septomics coordinates the AspIRS
(Aspergillosis – intrinsic risk stratification)
study which is the first systematic approach
to identify genetic markers associated with
invasive aspergillosis (IA). For this purpose,
genome-wide screening for single nucleotide
polymorphisms (SNPs) in allogeneic stem cell
111
transplant (SCT) recipients, a well-defined
high-risk cohort of patients, is performed in
a multi-center approach, including 16 leading clinical partners from Europe and the
US. Within AspIRS an unprecedented cohort
of >500 cases of IA and >900 control SCT patients has been collected. The resulting data
will (i) increase our understanding of the genetic background for IA, (ii) allow the design
of diagnostic tools to estimate the intrinsic
risk of individual patients and (iii) open new
ways to analyse pathogenesis and immune
response in IA.
In addition to genome-wide analyses, we have
used AspIRS samples for confirmation of prior
work from our cooperation partner A. Carvalho (Perugia, Italy), who has studied the long
pentraxin 3 (PTX3) in IA. PTX3 has a nonredundant role in antifungal immunity as it
promotes the control of A. fumigatus infection in vivo. The contribution of SNPs in PTX3
in the development of IA is unknown. We
screened an initial cohort of 229 recipients of
hematopoietic stem cell transplants and their
donors for PTX3 SNPs modifying the risk for
developing IA. The analysis was validated
in a large multi-center study involving 107
cases of IA and 223 matched controls. The
functional consequences of PTX3 SNPs were
investigated in vitro and in lung specimens
from SCT recipients. A donor homozygous
haplotype (h2/h2) in PTX3 increased the risk
of infection in the discovery (adjusted hazard
ratio, 3.15; P=0.002) and validation (adjusted
odds ratio, 2.14; P=0.01) studies and was associated with defective expression of PTX3
in transplant recipients. Functionally, PTX3
deficiency in h2/h2 PMN, presumably due
to impaired mRNA stability, led to impaired
phagocytosis and clearance of the fungus. Genetic deficiency of PTX3 affects the antifungal
capacity of PMN and contributes to risk of IA
in SCT recipients (Cunha et al. 2014).
112
Group members
DFG KU1540/3-1
Project: CO2 Adaptation in Candida glabrata und
ihre Rolle in der Pathogen-Wirt-Interaktion
Oliver Kurzai
Head
Prof. Dr. Oliver Kurzai
Phone +49 3641 532-1347
Fax
+49 3641 532-2347
[email protected]
SFB/TR 124 FungiNet
Project: Intrinsic Modulation of Neutrophil antifungal Activity against Candida albicans
Oliver Kurzai
Secretary
Silke Pfohl
Administrative Manager
Dr. Katrin Haupt
Scientists
Dr. Michael Böhringer
Dr. Daniela Hellwig (since 02/2013)
Dr. Kerstin Hünniger
Dr. Ronny Martin
Dr. Jessica Voigt (since 05/2013)
Dr. Michael Weber (since 06/2013)
Dr. Hanna Windecker (until 01/2013)
InflaRx GmbH
Prof. Dr. Niels Riedemann
Prof. Dr. Ren-Feng Guo
Prof. Dr. Dagmar Barz
PD Dr. Ralf Claus
Prof. Dr. Michael Kiehntopf
Fritz-Lipmann-Institute, Jena
PD Dr. Matthias Platzer
Voigt J, Hünniger K, Bouzani M, Jacobsen I, Barz
D, Hube B, Löffler J, Kurzai O (2014) Phagocytosis
of Candida albicans by human NK Cells results in a
proinflammatory response J Inf Dis 209, 616-626.
Martin R, Albrecht-Eckardt D, Brunke S, Hube B,
Hünniger K, Kurzai O (2013) A core filamentation
response network in Candida albicans is restricted
to eight genes. PLoS One 8, e58613.
Laura Bauer (since 10/2013)
Fabian Essig (03/2013–10/2013)
Antje Häder
Johanna Huyke (since 02/2013)
Toni Kaulfuß (until 03/2013)
Judith Piegsa (04/2013–08/2013)
Ann-Katrin Schmidt (04/2013–08/2013)
Steffi Spielberg (10/2013)
Cunha C, Aversa F, Lacerda JF, Busca A, Kurzai O,
Grube M, Löffler J, Maertens JA, Bell AS, Inforzato
A, Barbati E, Almeida B, Santos e Sousa P, Barbui A,
Potenza L, Caira M, Rodrigues F, Salvatori G, Pagano L, Luppi M, Mantovani A, Velardi A, Romani
L, Carvalho A. (2014) Genetic PTX3 deficiency and
aspergillosis in stem-cell transplantation. N Engl J
Med (accepted for publication)
Research Assistents
Cindy Reichmann
Collaborations
Trainees
Josephine Dietrich (07/2013–09/2013)
External funding
Zentren für Innovationskompetenz – Unternehmen Region
Zentrum für Innovationskompetenz Septomics –
Nachwuchsgruppe Fungal Septomics
Oliver Kurzai
Project: VisIPLI – Visualisierung der Immunzellaktivierung durch Sepsispathogene durch
Life-Cell Imaging
Oliver Kurzai
Project: The interaction of Candida glabrata with
human neutrophils
Oliver Kurzai
AspIRS Study Group (Medizinische Klinik und
Poliklinik II, University Hospital Würzburg, Microarray Facility, University Hospital Tübingen,
Bundesinstitut für Arneimittelforschung, Bonn,
University Hospital Dresden, Jena, Regensburg,
Köln, Mannheim, Allgemeines Krankenhaus Wien,
Karolinska Institute, Stockholm, Trinity College &
Saint James Hospital, Dublin, University of Leuven,
Universitätsklinikum Jena, Abteilung Hämatologie
und Internistische Onkologie, University Hospital
of Wales, Cardiff, Medizinische Universität Innsbruck, University of Perugia, University Hospital of
Leiden, Fred Hutchinson Cancer Research Center,
Seattle)
Julius Maximilians University, Würzburg, Germany
Prof. Dr. Jürgen Löffler
Prof. Dr. Hermann Einsele
PD Dr.Johannes Schumacher
University of Kent, Canterbury, UK
Prof. Dr. Fritz Mühlschlegel
Radboud University Nijmegen Medical Center,
Nijmegen, Netherlands
Prof. Dr. Mihai G. Netea
University of Perugia
Dr. Agostinho Carvalho
Prof. Dr. Luigina Romani
113
114
Research Group Microbial Immunology
115
Research Group Microbial Immunology
The independent Research Group Microbial
Immunology, established in August 2013,
derives from a research group that was formerly integrated in the Department Microbial Pathogenicity Mechanisms. The group
investigates pathogenesis and host-pathogen
interactions of human pathogenic fungi with
a focus on Candida albicans. We aim to elucidate which mechanisms contribute to the
outcome of infection by using microbiology,
cell biology, biochemistry, infection biology
and immunology approaches in combination
with complex infection models.
We are especially interested in how Candida
albicans interacts with the host’s immune
system in disseminated infections and which
attributes contribute to either protection or
disease. In this context, projects address the
role of fungal morphology in the development
of disease and fungal factors that modulate the host immune response. Recently, we
began a project that extends our work to the
mechanisms that facilitate translocation of
C. albicans from the intestinal tract, which
is an essential step in the development of
disseminated candidiasis. Further projects
address the pathogenesis of infection with
less common fungal pathogens, Aspergillus
terreus and Lichtheimia species.
In 2012 to 2013, our research was supported
Die unabhängige Forschungsgruppe Mikrobielle Immunologie ist im August 2013 aus
einer Arbeitsgruppe innerhalb der Abteilung Mikrobielle Pathogenitätsmechanismen hervorgegangen. Die Forschungsgruppe
untersucht die Pathogenese und Wirt-Erreger-Interaktionen humanpathogener Pilze,
insbesondere Candida albicans. Um Mechanismen zu identifizieren und zu charakterisieren, die den Infektionsverlauf maßgeblich
bestimmen, setzen wir mikrobiologische,
zellbiologische, infektionsbiologische und
immunologische Methoden ein. Unsere besondere Expertise liegt dabei im Bereich
komplexer Infektionsmodelle.
Head:
Ilse D. Jacobsen, PhD
Der Forschungsschwerpunkt der Gruppe
Mikrobielle Immunologie liegt in der Un-
116
tersuchung der Interaktion von C. albicans
mit dem Immunsystem des Wirtes während
disseminierter Infektionen, da diese Interaktionen sowohl zur Protektion als auch zum
Fortschreiten der Erkrankung beitragen
können. Dabei interessieren uns insbesondere die Rolle der Pilzmorphologie bei der
Entstehung von Erkrankungen und Pilzfaktoren, die die Immunantwort des Wirtes modulieren können. Neben disseminierten Infektionen adressieren wir dabei seit kurzem
auch die Translokation von C. albicans aus
dem Darm, die den initialen Schritt in der
Entstehung der disseminierten Candidiose
darstellt. Weitere Projekte beschäftigen sich
mit der Pathogenese seltener Pilzinfektionen, ausgelöst durch Aspergillus terreus und
Lichtheimia-Arten.
HKI Research Report 2012 / 2013 Research Group Microbial Immunology
by the Jena School for Microbial Communication (JSMC), the Deutsche Forschungsgemeinschaft (DFG), the Studienstiftung des
Deutschen Volkes and the Federal Ministry
of Education and Research (BMBF). Projects
are integrated into the Center for Sepsis
Control and Care (CSCC; joined project with
MPM) and the DFG Collaborative Research
and their human host: Networks of interaction – FungiNet”.
Unsere Forschung wurde in den Jahren 2012
und 2013 finanziell von der Jena School for
Microbial Communication (JSMC), der Deutschen Forschungsgemeinschaft (DFG), der
Studienstiftung des Deutschen Volkes und
dem Bundesministeriums für Bildung und
Forschung (BMBF) unterstützt. Wir sind mit
unseren Projekten an dem Center for Sepsis
Control and Care (CSCC, gemeinsames Projekt mit der Abteilung MPM) und dem DFG
Sonderforschungsbereich / Transregio
124
“Pathogenic fungi and their human host: Networks of interaction – FungiNet” beteiligt.
Research Group Microbial Immunology HKI Research Report 2012 / 2013
117
Scientific Projects
1
The role of morphology for fungal
pathogenicity
Group Leader: Ilse D. Jacobsen
The switch between the spherical yeast and
the filamentous hyphal growth form of Candida albicans is a two-step process including
the initial formation of germ tubes, subsequent elongation and maintenance of hyphal
growth. A complex network of regulatory
factors controls the morphological state.
1.1 The role of filamentation in the
pathogenesis of candidiasis
The ability of C. albicans to switch between
yeast and hyphal growth forms is believed to
be crucial for virulence during systemic infections and sepsis. In fact, all growth forms,
yeast, pseudohyphae and true hyphae are
found during systemic infection. In general,
mutants locked either in the yeast or hyphal
phase are often strongly attenuated in virulence. Although it is clear that hyphal formation is essential for tissue invasion in vitro,
the role of filamentation in the pathogenesis
of systemic candidiasis remains unknown.
However, we found that the filament-deficient mutant eed1∆ is able to cause mortality
in the absence of hyphae. This recent finding
challenges the long-standing hypothesis that
morphogenesis is essential for disseminated
candidiasis. We aim to clarify the role of hyphal formation for pathogenesis of disseminated C. albicans infection. Therefore, conditional knock-out or overexpression mutants
of EED1 and UME6, regulatory factors supporting the maintenance of hyphal growth,
and NRG1, a negative regulator of yeast-tohypha transition, have been constructed using a tetracycline-regulable expression system (Fig. 1). With the help of these strains,
the effect of morphology on virulence and
immune response is studied in different infection models. Further, in vitro studies with
immune cells, endothelial and epithelial
118
cells are performed to understand the role
of morphology in the interaction with distinct host cell types and the contribution of
morphology to direct vs. immune-mediated
tissue damage. This combination of in vitro
and in vivo approaches will allow us to dissect the different mechanisms that contribute to the development of disease and will
provide insight into the role of filamentation
in pathogenesis.
1.2 Eed1 – a regulator of hyphal
maintenance
The C. albicans EED1 gene plays a crucial role
in hyphal extension and maintenance of filamentous growth. Mutants lacking the EED1
gene (eed1∆) do not form true hyphae but
only pseudohyphae before switching back
into an elongated yeast cell-like growth form
(Martin et al. 2011). We observed that this
phenotype depends on the inoculum size:
eed1∆ cells seeded at moderate cell densities
formed shorter filaments than cells seeded at
low cell densities. Using filamentation studies
we were able to show that this effect is due
to the quorum sensing molecule farnesol to
which eed1∆ mutant cells are significantly
more sensitive than wild type cells. Since it
has been described that farnesol especially
acts on the cAMP/PKA signalling pathway
to inhibit filamentation, the association between EED1 and cAMP signalling is currently under investigation by testing the
effect of db-cAMP on cells and constructing
overexpression or deletion strains of several
factors within this signalling pathway in an
eed1∆ mutant background. To provide further insights into the role of Eed1p within
the cellular machinery, His-tagged protein
versions have been constructed to analyse
the protein localisation. Eed1 C-terminal
peptides were produced using a heterologous
E. coli expression system which are tested
in ongoing experiments for interaction with
DNA and proteins. In collaboration with Vito
Valiante (Department MAM) another expres-
Figure 1
Morphology of C. albicans wild
type and regulable mutant strains
in vivo.
Calcofluor white stained C. albicans
cells from murine liver tissue 24 h
after intraperitoneal infection. – Dox:
Absence of doxycycline, the respective target genes are expressed.
+ Dox: Doxycyclin added to the
drinking water, gene expression
is repressed. Repression of Nrg1, a
transcriptional repressor of hyphal
formation, results in hyperfilamentous growth while the presence of
doxycyclin allows NRG1 expression
resulting in yeast growth. Doxycyclin
represses expression of the positive
regulators EED1 or UME6, thus cells
grow dominantly in yeast or pseudohyphal-like form. In the absence of
doxycyclin EED1 and UME6, respectively, are expressed, resulting in
production of true hyphae.
sion system involving the yeast Saccharomyces cerevisiae is used to generate full length
Eed1 protein for further studies. These experiments will provide valuable information
about the possible role of EED1 in C. albicans
tolerance to farnesol and contribute to the elucidation of its molecular function in morphogenesis.
The projects are supported by the DFG
and Studienstiftung des Deutschen Volkes
(joined project with MPM).
2
Molecular mechanisms of Candida
sepsis
Group Leader: Betty Hebecker
During systemic infections, C. albicans can
infect almost all organs; however, the host
response and the ability to clear C. albicans
cells is organ-specific. While C. albicans is
gradually cleared from liver and spleen, infection progresses in the kidneys due to a
delayed, but prolonged influx of neutrophils
and is accompanied by a strong cytokine response. The mechanisms underlying these
organ-specific differential host responses
are not known. We hypothesised that distinct fungal genes elicit an organ-specific
host response, which is either protective or
non-protective, without directly affecting
host cell damage.
To identify C. albicans factors or activities
which contribute to protective or non-protective host responses, we use large scale
host response screening (Fig. 2). Therefore,
screening protocols for several host cell
lines including hepatocytes, renal, oral and
intestinal epithelial cells, and macrophages
were established. So far, 1180 defined fungal
mutants were screened for their potential to
damage renal, oral and intestinal epithelial
cells and to induce the expression of Il-6
119
Figure 2
Molecular mechanisms of Candida
sepsis: Project outline.
(1) Organ-specific outcome of
systemic infection with C. albicans.
Approaches to identify fungal factors involved in the organ-specific
host responses and outcome: (2)
Large-scale host response screening
of C. albicans mutants. Mutants with
significantly altered induction of
cytokines without changes in growth
rates, filamentation and cytotoxicity
are selected for further analysis. (3)
Identification of C. albicans genes
that are differentially expressed in
different organs. C. albicans RNA is
isolated from infected organs using
an optimized protocol to perform
microarray analysis.
and Il-8 in these host cell lines. From this
mutant collection 37 knock out mutants
could be identified which showed a significantly altered induction of cytokines without changes in growth rates, filamentation
and cytotoxicity. These candidate genes can
be categorized in three classes: (a) mutants
with known reduced virulence potential,
which demonstrate the suitability of this
approach, (b) mutants causing reduced cytokine induction in all analysed cell lines and
(c) mutants which have a host cell type specific phenotype indicating organ specific
factors. These genes and their role during
systemic C. albicans infection will be further
investigated in detail.
Another approach to identify niche-specific
fungal genes is in vivo transcriptional profiling of C. albicans from different organs. In
vivo transcriptional profiling is technically
challenging due to low amounts of fungal
RNA in the organs. Therefore, we established
120
an efficient RNA isolation protocol, which includes centrifugation and washing steps to
separate host and fungal RNA. Preliminary
tests using this isolation protocol applied to
mouse organs spiked with C. albicans cells
allowed the reproducible isolation of good
quality RNA in sufficient amounts to perform microarray analysis. We could show
that this optimised RNA isolation protocol
has no impact on gene expression by analysing the expression of heat shock genes before
and after spiking. Analysis of organ samples
from infected mice is currently under way.
The combination of these two approaches will
allow us to detect genes which are both infection-associated and niche-specific and which
contribute to organ-specific pathogenesis.
The project is performed in close collaboration with the Department MPM and supported by the CSCC (Project D1.15: MomCanSep).
A . te rreus 5 d p. inf.
Figure 3
Persistence of A. terreus conidia in
immunocompetent mice.
Histological sections of murine lung,
5 days after infection with A. terreus
SBUG844, PAS-stained. Left panel: arrow indicates immune cell infiltrates;
right panel: arrows indicate A. terreus
conidia stained in pink; scale bars
represent 100µm (left panel) and
10µm (right panel).
3
Pathogenesis of aspergillosis
caused by Aspergillus terreus
Aspergillus terreus is an emerging life-threatening agent of invasive aspergillosis. In collaboration with Matthias Brock (Research
Group Microbial Biochemistry and Physiology) and financially supported by the DFG,
two murine infection models were developed
to study A. terreus mediated disease. Leukopenic mice showed a similar outcome of infection in comparison to A. fumigatus, though
a 102 to 103 fold higher infectious dose was
required for 100 % lethality. In surviving
animals conidia were found to persist in pulmonary tissue. Contrasting an A. fumigatus
infection, a fatty liver degeneration appeared
in all animals infected with A. terreus, suggesting production of secondary toxic metabolites. Persistence and subclinical liver damage are unique features of A. terreus invasive
aspergillosis in mice (Slesiona et al. 2012a).
The murine data implied that the initial disease establishment might be fundamentally
different between A. terreus and A. fumigatus. Therefore, we compared the interaction
of A. terreus and A. fumigatus conidia with
alveolar macrophages, one of the initial
phagocytes facing inhaled conidia in the
lung. A. terreus conidia were phagocytosed
more rapidly than A. fumigatus conidia, possibly due to a higher exposure of β-1,3-glucan
and galactomannan on the conidial surface,
as confirmed by reduced phagocytosis rates
when blocking dectin-1 and the mannose-receptor. Once phagocytosed A. terreus did not
inhibit acidification of phagolysosomes, but
remained viable, as inert conidia both in vitro
and in immunocompetent mice, contrasting
A. fumigatus (Fig. 3).
The inability of A. terreus to germinate and
pierce macrophages resulted in significantly
121
A
B
lower cytotoxicity compared to A. fumigatus.
Blocking phagolysosome acidification by
bafilomycin, a v-ATPase blocker, increased
A. terreus germination rates and cytotoxicity. In summary A. terreus and A. fumigatus have evolved significantly different strategies to survive the attack of host immune
cells. While A. fumigatus prevents phagocytosis and phagolysosome acidification and
escapes from macrophages by germination,
A. terreus is rapidly phagocytosed, but conidia show long-term persistence in macrophages even in immunocompetent hosts (Slesiona et al. 2012b).
mycosis in Europe, virulence and pathogenesis of this genus has not been addressed in
detail yet. Only three of the five species of
the genus Lichtheimia have been found to
be involved in mucormycosis. Using an alternative infection model based on chicken
embryos to screen the virulence potential of
the different Lichtheimia species we showed
that the three clinically relevant Lichtheimia
species, L. ramosa, L. corymbifera and L. ornata, have a higher virulence potential than
the species that have only been isolated from
the environment. Furthermore, we observed
differences in virulence between strains of
the same species, independent of their origin
(environmental vs. clinical) or phylogenetic
position within the species (Schwartze et al.
2012).
Figure 4
Lung pathology after infection
with L. corymbifera.
(A) HE-stained overview of an infected murine lung showing alterations
typical for mucormycosis. (B) PASstained area indicated by the box in
(A). Arrows indicate fungal hyphae
(stained in pink) within the thrombus. H: hemorrhage, T: thrombosis.
References
Martin R, Moran GP, Jacobsen ID,
Heyken A, Domey J, Sullivan DJ,
Kurzai O, Hube B (2011) The Candida
albicans-Specific Gene EED1 Encodes
a Key Regulator of Hyphal Extension.
PLoS One 6, e18394.
Slesiona S, Ibrahim-Granet O, Olias P,
Brock M, Jacobsen ID (2012) Murine
infection models for Aspergillus terreus pulmonary aspergillosis reveal
long-term persistence of conidia and
liver degeneration. J Infect Dis 205,
1268-1277.
Slesiona S, Gressler M, Mihlan M,
Zaehle C, Schaller M, Barz D, Hube B,
Jacobsen ID, Brock M (2012) Persistence versus Escape: Aspergillus terreus
and Aspergillus fumigatus employ
different strategies during interactions with macrophages. PLoS ONE
7, e31223.
Schwartze VU, Hoffmann K, Nyilasi I,
Papp T, Vágvölgyi C, de Hoog S, Voigt
K, Jacobsen ID (2012) Lichtheimia Species Exhibit Differences in Virulence
Potential. PLoS One 7, e40908.
122
4
Pathogenesis of Lichtheimia
infections
Although Lichtheimia species represent the
second to third most common cause of mucor-
We selected a L. corymbifera strain of typical
virulence in the chicken embryo model to establish murine pulmonary infection models
(Fig. 4). Clinical disease developed only in
Group members
Head
PD Ilse D. Jacobsen, PhD
Phone +49 3641 532-1223
Fax
+49 3641 532-2223
[email protected]
Scientists
Dr. Betty Hebecker (together with MPM)
Ph.D. Students
Christine Dunker (since 06/2013)
Tony Pawlik (since 04/2013, together with MPM)
Melanie Polke
Marcel Sprenger (since 10/2013)
Research Assistents
Birgit Weber
Visiting Scientists
03/2013-05/2013
Josephine Fischer
Laboratory of Molecular Stem Cell Biology,
Westfälische Wilhelms-Universität Münster
11/2013
External funding
immunocompromised animals. Acute disease was characterized by respiratory symptoms and extensive necrosis and hemorrhage
in the lung. Histology revealed abundant
mycelia, angioinvasion and thrombosis. Dissemination from the lung to other internal
organs, especially the kidneys, was observed
in all animals that became moribund ≥ 6 days
post infection. Similar to the lung, abundant
mycelia, angioinvasion and thrombosis characterised alterations in the various internal
organs. Thus, the pathological alterations in
the murine model accurately resemble disease in human patients. We are currently employing this model to analyse whether fungal
strains differ in virulence and to determine
the immune response to infection.
This project is performed in close collaboration with Kerstin Voigt, JMRC.
Project: Mechanisms of Candida albicans colonization and translocation
Ilse Jacobsen
MorphPath – The Role of morphogenesis in the
pathogenesis of systemic candidiasis
Ilse Jacobsen
SFB/TR FungiNet
Project C05: Mechanismen der gastrointestinalen
Kolonisierung und der Translokation von Candida
albicans durch die Darmschleimhaut
Ilse Jacobsen
Lüttich A, Brunke S, Hube B, Jacobsen ID (2013)
Serial passaging of Candida albicans in systemic
murine infection suggests that the wild type strain
SC5314 is well adapted to the murine kidney. PLoS
One 8, e64482.
Schwartze VU, Hoffmann K, Nyilasi I, Papp T, Vágvölgyi C, de Hoog S, Voigt K, Jacobsen ID (2012)
Lichtheimia species exhibit differences in virulence
potential. PLoS One 7, e40908.
Slesiona S, Ibrahim-Granet O, Olias P, Brock M,
Jacobsen ID (2012) Murine infection models for
Aspergillus terreus pulmonary Aspergillosis reveal
long-term persistence of conidia and liver degeneration. J Infect Dis 205, 1268-1277.
123
Slesiona S, Gressler M, Mihlan M, Zaehle C, Schaller
M, Barz D, Hube B, Jacobsen ID, Brock M (2012)
Persistence versus escape: Aspergillus terreus and
Aspergillus fumigatus employ different strategies
during interactions with macrophages. PLoS One
7, e31223.
Jacobsen ID, Wilson D, Wächtler B, Brunke S,
Naglik JR, Hube B (2012) Candida albicans dimorphism as a therapeutic target. Expert Rev Anti Infect
Ther 10, 85-93.
Collaborations
Max von Pettenkoffer-Institut, LudwigMaximilians-Universität, Munich, Germany
Prof. Dr. Frank Ebel
Laboratory of Molecular Stem Cell Biology,
Medizinische Fakultät, Westfälische WilhelmsUniversität, Münster, Germany
Prof. Dr. Frank Rosenbauer
Leibniz Institute for Age Research,
Fritz-Lipmann-Institute, Jena, Germany
Prof. Dr. Falk Weih
VIB Department of Molecular Microbiology,
K. U. Leuven, Laboratory of Molecular Cell Biology,
Belgium
Prof. Patrick van Dijck
ZIK Septomics, Research Group Host Septomics,
Jena, Germany
124
013 Research Group Microbial Immunology
Research Group
Systems Biology and Bioinformatics
125
Research Group Systems Biology and Bioinformatics
The Research Group Systems Biology and
Bioinformatics (SBI) is dedicated to the integrated computational analysis of genome,
transcriptome, proteome and metabolome
as well as microbiological and clinical data.
Data analysis comprises the management of
heterogeneous large-scale experimental data
in a data warehouse, pre-processing, discovery of the main features and patterns by data
mining techniques and – finally – prediction of experimentally testable hypotheses.
Hypotheses of interest concern molecular
interactions between host and pathogen, or
between transcription factors and their target genes within the host or the pathogen or
the fungus that produces an natural product. The hypotheses have been embedded in
predictive mathematical and computational
models that represent both prior knowledge
available from experts or extracted from biomolecular databases or by text mining in research papers and the pattern found by the
computational analysis of experimentally
measured data. We developed and applied
novel bioinformatic tools that identify the
structure and parameter values of network
models of gene regulation and signalling. In
addition, we developed and applied bioinformatic tools of genome sequence analysis to
predict protein coding genes, transcription
factor binding sites and secondary metabolite synthesis gene clusters.
Fungi are known to produce pharmaceutically important natural products (secondary
metabolites, SMs) synthesised by polyketide
Die Forschungsgruppe Systembiologie und
Bioinformatik (SBI) befasst sich mit der
bioinformatischen Analyse von genomweiten Hochdurchsatzdaten, wie Genom-, Transkriptom- und Proteom-Daten in Verbindung
mit mikrobiologischen und klinischen Daten.
Die Datenanalyse umfasst das Management
von umfangreichen heterogenen experimentellen Daten in einem „Data Warehouse“,
die Vorverarbeitung, die Entdeckung von
Mustern in den Daten, sowie schließlich die
Vorhersage von experimentell prüfbaren Hypothesen. Interessante Hypothesen betreffen
vor allem die Wirt-Pathogen-Interaktion,
aber auch molekulare Wechselwirkungen,
darunter Transkriptionsfaktor-Zielgen-Beziehungen innerhalb der Wirt- oder der
Erregerzelle oder in einem Pilz, der einen
Wirkstoff produziert. Die Hypothesen sind
Head:
Prof. Dr. Reinhard Guthke
126
in mathematischen Modellen formuliert, die
einerseits das Vorwissen und andererseits
die entdeckten Muster in den experimentell
gemessenen Daten widerspiegeln. Wir haben
neue bioinformatische Werkzeuge entwickelt
und angewandt, die der Identifikation der
Struktur und Parameterwerte von Netzwerkmodellen der Genregulation und von Signalwegen dienen. Ferner haben wir bioinformatischen Werkzeuge für die Genomsequenzanalyse entwickelt, die der Vorhersage von
Genen, Transkriptionsfaktor-Bindestellen und
von Genclustern der SekundärmetabolitSynthese dienen. Pilze sind bekannt als
Produzenten von pharmazeutisch wichtigen Naturstoffen (Sekundärmetabolite, SM),
die mittels Polyketidsynthetasen (PKS) und
Nicht-ribosomalen Peptidsynthetasen synthetisiert werden. Zahlreiche „kryptische“
HKI Research Report 2012 / 2013 Research Group Systems Biology and Bioinformatics
synthases (PKS) and nonribosomal peptide
synthetases (NRPS). Analyses of sequenced
fungal genomes have revealed numerous examples of ‘cryptic’ biosynthetic gene clusters,
which can potentially be responsible for the
production of the novel SMs. Our work is devoted to the genome-driven detection of new
complex natural products in selected fungi
and bacteria.
Next Generation Sequencing techniques
paved the way for advanced genomic and
transcriptomic studies, in particular for infection research. We demonstrated how to
model the host-pathogen interaction by simultaneous monitoring of gene expression
profiles from both the pathogen and the host
using RNA-seq technique. In addition, using
Biosynthese-Gencluster wurden mittels
bioinformatischer Untersuchungen sequenzierter Pilzgenomen gefunden. Unsere Forschung zielt auf die Genom-getriebene Entdeckung neuer komplexer Naturstoffe in
ausgewählten Pilzen und Bakterien.
Die großen Fortschritte bei der Etablierung
neuer Seqenziertechniken (sogenanntes
Next Generation Sequencing, z. B. RNA-seq)
haben neue Wege für Genom- und Transkriptom-Studien, die besonders wertvoll für
die Infektionsforschung sind, eröffnet. Wir
konnten zeigen, wie die Wirt-Pathogen-Interaktion mittels eines Netzwerkmodells
beschrieben werden kann auf der Grundlage
von simultan (mittels RNA-seq) gemessenen
Genexressionsprofilen. Außerdem konnten
wir mittels RNA-seq-Datenanalyse Pilzgeno-
RNA-seq analysis, we established and applied
a pipeline for structural fungal genome annotation and improved the genome annotation
e.g. for A. fumigatus and C. glabrata. Thus,
we found that the transcriptional potential of
A. fumigatus was underestimated in the past.
The research was done within collaborative
networks, such as the graduate schools JSMC
and ILRS. The research within the DFG’s
Priority Programme SPP1160 ‘Colonisation
and Infection by Human-Pathogenic Fungi’
and the newly established Collaborative Research Centre/Transregio 124 “Pathogenic
fungi and their human host: Networks of
interaction – FungiNet” were focused on infections by fungal pathogens, in particular
A. fumigatus und C. albicans.
me neu annotieren bzw. für Aspergillus fumigatus and Candida glabrata die strukturelle
Genomannotation verbessern.
Diese Forschungen wurden in interdisziplinärer Zusammenarbeit in Forschungsverbünden wie den Graduiertenschulen JSMC and
ILRS durchgeführt. Im DFG-Schwerpunktprogramm SPP1160 „Colonisation and Infection by Human-Pathogenic Fungi“ und im
erst kürzlich etablierten Sonderforschungsbereich / Transregio 124 “Pathogenic fungi
and their human host: Networks of interaction – FungiNet” wurden die Forschungen auf
die Infektion mit human-pathogenen Pilzen,
insbesondere A. fumigatus und C. albicans
konzentriert.
Research Group Systems Biology and Bioinformatics HKI Research Report 2012 / 2013
127
Scientific Projects
1
Systems Biology of Fungal Infections
Project Leader: Jörg Linde
Systems biology of fungal infection comprises all levels of interactions between the
pathogen and the host. Here, we aim at describing and analysing the confrontation of
the host with fungal pathogens as well as
the confrontation of fungal cells with host
immune cells of antifungal drugs. We intend
to model and to understand the interactions
of the host, in particular the immune system
of humans, with components of pathogens.
Our analysis and models are mainly based
on transcriptome data but do also integrate
other data as well as current literature
knowledge. With help of global transcriptome sequencing, we identified chlamydospore specific markers in Candida albicans
and Candida dubliniensis. With the help of
microarray expression data we studied the
interaction between the soil-derived bacterium Streptomyces rapamycinicus and the
human pathogenic fungus Aspergillus fumigatus. Here we found that this interaction
leads to the activation of an otherwise silent
polyketide synthase gene cluster.
For sepsis, the quick identification of the
pathogen is crucial to increase the patient’s
chances of survival. Currently we are using
supervised learning algorithms to analyse
the transcriptomic response of human cells
to reliably and quickly determine whether
the patient is infected by bacteria or fungi.
Networks of host-pathogen interactions
During the last decade, the group SBI has
facilitated transcriptome data to predict
mathematical network models which predict
regulatory interactions in infection-related
conditions.
128
In collaboration with H. Haas (Medical
University Innsbruck) and the Department
MAM, we reconstructed gene regulatory
networks that model the regulation of iron
homoeostasis in A. fumigatus (Linde et
al. 2012). The network inference was performed by analysis of gene expression profiles as well as soft integration of already
known regulatory interactions. The inferred
model predicts new interactions between
transcription factors and target genes, in
particular the activation of the gene expression of hapX by the transcriptional regulator
SrbA. This interaction was experimentally
verified by northern blots by the collaboration partners.
In cooperation with the Department MPM,
we predicted regulatory networks of C. albicans under nitrogen starvation as well as
after feeding pre-starved cell with proteins
(BSA) or arginine. We learnt that C. albicans
adopts a particular nitrogen response network, defined by sets of specific gene-gene
connections, for each of the environmental
condition.
In collaboration with the Department MAM,
we predicted regulatory networks of A. fumigatus when treated with the antifungal
drug Caspofungin. Emergence and spreading of antifungal drug resistance is a growing clinical concern. The RNA-seq based expression data suggests that mpkA and sakA
signalling plays an important role for the
drug resistance.
In order to focus the network modelling on
human cells confronted with fungal pathogens we collaborate with the Research Group
Fungal Septomics. Here, we predicted a gene
regulatory network of MAPK- and NFkBsignalling pathway of epithelial cells infected by C. albicans (Fig. 1). In the future, we
will augment this study and analyse these
Candida albicans
97%
PTGS2
26%
100%
87%
NRAS
RIPK1
99%
90%
DUSP16
46%
DUSP6
91%
94%
63%
99%
DUSP1
90%
87%
65%
100%
70%
80%
DDIT3
GDF15
99%
86%
FLNB
GADD45B
88%
91%
90%
99%
58%
GADD45A
100%
86%
BMP2
MAP3K11
58%
98%
100%
FOS
12%
66%
100%
DUSP5
50%
76%
JUN
100%
49%
NFKB1/IA
67%
97%
96%
72%
20%
9%
CXCL2
EDN1
35%
TRIM25
TICAM1 100%
38%
66%
74%
41%
69%
HBEGF
60%
CCL20
56%
64%
80%
Figure 1
Network model focused on MAPKand NFkB Pathways.
The simulation of the network describes the response of intestinal
epithelial cells to C. albicans infection.
24 gene nodes (grey boxes indicate
transcript decay or autoregulation).
Inferred connections are coloured
green if prior knowledge was integrated and black without prior
knowledge. Edge percentage labels
give the frequency of occurrence in
the robustness test procedure (from
Schulze, 2013).
HSPA1A
80%
networks of the human immune system confronted with different fungal pathogens.
In principal, a molecular model of infection has to comprise interacting molecular
networks of both the host and the pathogen. Gene expression analysis by Next Generation Sequencing offers the possibility to
monitor the transcriptomic behaviour of host
and pathogen simultaneously (Dual RNAseq) and thus, provide data to infer the interaction network of both. A pioneering study
for such an integrated systems analysis was
done in collaboration with K. Kuchler and coworkers (Medical University of Vienna) and
the Department MPM. With the help of our
modelling approach we were able to predict
a small-scale host-pathogen interaction network (Fig. 2).
Improving structural gene prediction of
pathogenic fungi by RNA-seq
The correct prediction of the localisation of
genes in genomes is the most important basis
for transcriptomics as well as small-scale experiments, such as knockouts. With the help
of RNA-seq data, we are able to identify expressed regions in the genome; which have so
far not been indicated as genes. Thus, we can
improve the structural genome annotation.
In collaborative work with the Department
MAM the transcriptome of the wild type and
the mpkA mutant of A. fumigatus was analysed using paired-end RNA-seq data and data
from four different microarray platforms in
comparison with proteome data (Müller et al.
2012). The mutant was investigated to study
the regulatory role of the mitogen activated
protein kinase MpkA, which has an important function in passing on external cell wall
signals. It was demonstrated that by deactivating this kinase, many genes in the pri-
129
Infec&on SOD5 ZPF36 DDR48 HAP3 ALS3 Figure 2
Gene regulatory network inferred
from dual RNA-seq data from murin
dendritic cells infected with C. albicans. (from Tierney, Linde et al. 2012)
References
Linde J, Hortschansky P, Fazius E,
Brakhage AA, Guthke R, Haas H (2012)
Regulatory interactions for iron
homeostasis in Aspergillus fumigatus
inferred by a Systems Biology approach. BMC Syst Biol 6, 6.
Müller S, Baldin C, Groth M, Guthke R,
Kniemeyer O, Brakhage AA, Valiante V
(2012) Comparison of transcriptome
technologies in the pathogenic
fungus Aspergillus fumigatus reveals
novel insights into the genome and
MpkA dependent gene expression.
BMC Genomics 13, 519.
Wolf T, Shelest V, Shelest E (2013):
Motif-based method for genomewide prediction of eukaryotic gene
clusters. 2nd International Workshop
on Pattern Recognition in Proteomics,
Structural Biology and Bioinformatics. In: ICIAP 2013 Workshops, Lecture
Notes in Computer Science (LNCS) 8158,
Petrosino A, Maddalena L, Pala P (eds),
pp. 389–398, Springer-Verlag, Berlin,
Heidelberg.
130
CITED2 PTX3 MTA2 FRE10 LIF C. albicans M. musculus mary and secondary metabolism are consequently regulated. In 2013, we implemented
a pipeline for structural genome annotation
which can be used for re-annotation as well
as completely new annotation. This pipeline
integrates eleven different tools for structural genome annotation. It has been used for
re-annotation of A. fumigatus, C. glabrata as
well as de novo annotation of the basal fungal
pathogens Lichtheimia corymbifera and Lichtheimia ramosa.
2
RGS1 Genome mining, transcription factors and secondary metabolites in
fungi
Project Leader: Ekaterina Shelest
For prediction of SM genes’ functionality it is
essential to know which genes belong to the
cluster besides the backbone enzymes. To
this end, we have developed a novel non-sim-
ilarity based method for the prediction of SM
gene clusters in fungal genomes. The motif
density method (MDM) is based on the statistical over-representation of binding sites
for cluster-specific regulators (Wolf et al.
2013). Importantly, MDM not only allows to
reliably predict the SM clusters but also provides information about the potential regulators of the cluster by description of their
binding sites.
The methods of genome mining and comparative genomics allow to predict the approximate product structure based on the genes’
similarity and/or their domain structure.
However, in fungi the amino acid selectivity
can be hardly deduced from the adenylation
domain sequence and remains a challenging
problem. By methods of comparative analysis we showed that the signature for the
amino acid selectivity can be found for aromatic amino acids, but not for aliphatic ones;
moreover, in PKS-NRPS hybrids the amino
acid selectivity is additionally influenced by
the condensation (C) domain (Boettger et al.
2012). We could also infer a non-canonical
function of C-terminal C domains in truncated PKS–NRPS homologues. Moreover, sequence and phylogenetic analyses revealed
an evolutionary imprint of the PKS–NRPS
domains, which reflect the evolutionary history of the entire megasynthase.
Transcription factors (TFs) are in the other
focus of our research. Families of TFs are not
distributed evenly across the fungal kingdom. Combining the information about the
expansions and losses of the TF families in
different genomes with the specific biological traits of the species, we obtain a valuable
source of insights into the evolutionary history and functional importance of particular
features, in which the TFs are involved. This
is of special value when we discuss such features as virulence of pathogenic fungi, or
specific traits of symbionts, adaptation to a
particular niche, etc.
3
Gene regulatory network inference
Project Leader: Reinhard Guthke
Systems biology aims to model living systems to uncover the interaction of molecules,
cells, tissues, organs and organisms by dynamic networks. The research of the SBI
group has a longstanding tradition in the
field of gene expression data-based network
modelling of gene regulation and signalling.
In 2012 and 2013, we published three novel
tools for network modelling.
First, Vlaic et al. (2012) presented the ExTILAR approach that combines the advantages
of the regression-based inference algorithm
TILAR (Transcription factor binding sites
Integrating Least Angle Regression) with
the advantages of dynamic network modelling based on ordinary differential equations (ODE). TILAR is able to infer large scale
networks, whereas ODE-based models allow
process simulation of e.g. in silico knockdown experiments. Initially, the novel tool
ExTILAR was applied for dynamic modelling
of the TF network regulating the adaptation
of murine hepatocytes to changed nutrition.
Second, NetGenerator 2.0, a new version of
the in-house ODE-based network inference
tool was developed, assessed and published
(Weber et al. 2013a). The tool has many new
features, such as to process experimental
data of multiple experiments, to model the
influence of multiple external signals and to
softly integrate prior knowledge. It was applied for various tasks, in particular to model
the gene regulation of the fungi A. fumigatus
and C. albicans (see above: Networks of hostpathogen interactions). For the first time,
we studied the influence of four different
stimuli on synovial fibroblast of rheumatoid
arthritis patients. In addition, we modelled
the dynamic influence of microRNA on the
differentiation of human mesenchymal stem
cells (Weber et al. 2013b).
Third, GRN2SBLM is the name of the third
tool developed by the SBI group (Vlaic et al.
2013). It is dedicated to the automated encoding and annotation of inferred gene regulatory networks complying with SBML, the
international standard for the storage, exchange and use of quantitative models by the
systems biology markup language.
By constructing correlation-based difference
networks of gene regulation and signalling,
we identified those signalling pathways that
are most vigorously changed by impaired
glycolysis in the roundworm Caenorhabditis
elegans (Priebe et al. 2013). Thus, we found
genes and pathways that are potentially involved in the extension of life span of this
model organism in ageing research.
References
Boettger D, Bergmann H, Kuehn B,
Shelest E, Hertweck C (2012) Evolutionary imprint of catalytic domains
in fungal PKS-NRPS hybrids. ChemBioChem 13, 2363-2373.
Vlaic S, Schmidt-Heck W, Matz-Soja
M, Marbach E, Linde J, Meyer-Baese
A, Zellmer S, Guthke R, Gebhardt R
(2012) The extended TILAR approach:
a novel tool for dynamic modeling
of the transcription factor network
regulating the adaption to in vitro
cultivation of murine hepatocytes.
BMC Syst Biol 6, 147.
Weber M, Henkel SG, Vlaic S, Guthke
R, van Zoelen EJ, Driesch D (2013) Inference of dynamical gene-regulatory
networks based on time-resolved
multi-stimuli multi-experiment data
applying NetGenerator V2.0. BMC Syst
Biol 7, 1.
Weber M, Sotoca AM, Kupfer P,
Guthke R, van Zoelen EJ (2013)
Dynamic modelling of microRNA
regulation during mesenchymal stem
cell differentiation. BMC Syst Biol 7, 124.
Vlaic S, Hoffmann B, Kupfer P, Weber
M, Drager A (2013) GRN2SBML: automated encoding and annotation of
inferred gene regulatory networks
complying with SBML. Bioinformatics
29, 2216-2217.
Priebe S, Menzel U, Zarse K, Groth M,
Platzer M, Ristow M, Guthke R (2013)
Extension of life span by impaired
glucose metabolism in Caenorhabditis elegans is accompanied by structural rearrangements of the transcriptomic network. PLoS One 8, e77776.
Tierney L, Linde J, Müller S, Brunke S,
Molina JC, Hube B, Schock U, Guthke
R, Kuchler K (2012) An interspecies
regulatory network inferred from
simultaneous RNA-seq of Candida
albicans invading innate immune
cells. Front Microbiol 3, 85.
131
Group members
External funding
Head
Prof. Dr. Reinhard Guthke
Phone +49 3641 532-1083
Fax
+49 3641 532-0803
[email protected]
GERONTOSYS II: Rolle von oxidativen Stress in der
Alterung und therapeutische Implikationen
Reinhard Guthke
Jenaer Centrum für die Systembiologie des Alterns
– JenAge: Systembiologie von mildem Stress beim
gesunden Altern ein Multi-Spezies-Ansatz
Reinhard Guthke
Scientists
Dr. Ekaterina Shelest
Dr. Uwe Menzel
Wolfgang Schmidt-Heck
Dr. Jörg Linde
Eugen Fazius (until 10/2012)
Dr. Fabian Horn (since 09/2012)
Dr. Sebastian Müller (06/2012–10/2012)
Jonas Schaer (since 02/2012)
Philipp Kämpfer (since 10/2012)
Janine Freitag (since 11/2012)
ERASysBio – LINCONET
Project: Modellierung des genregulatorischen
Netzwerks für die Linien-spezifische Differenzierung in menschlichen mesenchymalen Stammzellen: Identifikation von potenziellen Zielmolekülen für die anabole Therapie von Gewebserkrankungen
Reinhard Guthke
Ph.D. Students
Robert Altwasser
Fabian Horn (until 08/2012)
Peter Kupfer
Sebastian Müller (until 05/2012)
Steffen Priebe (until 06/2013)
Sylvie Schulze (since 12/2013)
Sebastian Vlaic
Michael Weber
Thoms Wolf (since 04/2012)
Andreas Dix (since 06/2012)
Müller S, Baldin C, Groth M, Guthke R, Kniemeyer
O, Brakhage AA, Valiante V (2012) Comparison of
transcriptome technologies in the pathogenic
fungus Aspergillus fumigatus reveals novel insights
in genome structure and MpkA-dependent gene
expression. BMC Genomics 13, 519.
Vlaic S, Schmidt-Heck W, Matz-Soja M, Marbach
E, Linde J, Meyer-Baese A, Zellmer S, Guthke R,
Gebhardt R (2012) The Extended TILAR Approach:
A novel tool for dynamic modeling of the transcription factor network regulating the adaption
to in vitro cultivation of murine hepatocytes. BMC
Syst Biol 6, 147.
Collaborations
Kompetenznetz Die Virtuelle Leber
Reinhard Guthke
Jonathan Adam
Martin Bens
Thomas Brockmöller
Andreas Dix
Janine Freitag
Bianca Hoffmann
Phillip Kämpfer
Sylvie Schulze
Thomas Wolf
Maximilian Fichtner
Project: Network Model Fungi – Systems biology
of human pathogenic fungi: Modeling transcriptional networks of virulence
Reinhard Guthke
Project: Transcriptome data analysis and modeling
of Aspergillus fumigatus – phagocytes interaction
Reinhard Guthke
SFB/TR FungiNet
Project B03: Prädiktive Modellierung von WirtPathogen Interaktionen durch Rekonstruktion
genregulatorischer Netzwerke
Reinhard Guthke, Ekaterina Shelest
Research Assistants
Sylvie Schulze
Carolin Zitzmann (until 08/2012)
Vladimir Shelest
SFB/TR FungiNet
Project INF: Integrierte Datenbank für Experimentaldaten (Data Warehouse OmniFung)
Reinhard Guthke
Trainees
Sylvie Schulze (since 10/2012)
Christian Kreisel (since 10/2012)
European Union
Framework Programme 7: Marie Curie Action
International Research Staff Exchange Scheme
KRAB-ZNF – KRAB zinc finger gene biology in
evolution and disease
Reinhard Guthke
Visiting Scientists
Himanshu Manchanda
since 02/2012
Laetitia Muszkieta
Institut Pasteur, Laboratoire des Aspergillus, Paris
05/2013
José Muñoz
Universidad del Rosario, Bogotá,
Medellin, Colombia
06/2012
Linde J, Hortschansky P, Fazius E, Brakhage AA,
Guthke R, Haas H (2012) Regulatory interactions
for iron homoeostatis in Aspergillus fumigatus
inferred by a Systems Biology approach. BMC Syst
Biol 6, 6.
BioControl Jena GmbH, Jena, Germany
Sebastian Henkel
Dominik Driesch
Florida State University, USA
Prof. Dr. Anke Meyer Baese
Innsbruck Medical University, Innsbruck, Austria
Medical University of Vienna, Austria
Prof. Dr. Karl Kuchler
Radboud University Nijmegen, Nijmegen,
The Netherlands
Prof. Dr. Joop van Zoelen
Prof. Dr. Raimund Kinne
Prof. Dr. Thomas Kamradt
Prof. Dr. Utz Settmacher
University of Birmingham, U.K.
Prof. Dr. Francesco Falciani
University of Leipzig, Germany
Prof. Dr. Rolf Gebhard
Prof. Dr. Hans-Jürgen Thiesen
Tongji University, Shanghai, China
Prof. Dr. Cao Zhiwei
Technical University München, Germany
Prof. Dr. Andreas Kremling
Weber M, Henkel SG, Vlaic S, Guthke R, van
Zoelen EJ, Driesch D (2013) Inference of dynamical
gene-regulatory networks based on time-resolved
multi-stimuli multi-experiment data applying the
NetGenerator algorithm V 2.0. BMC Syst Biol 7, 1.
Vlaic S, Hoffmann B, Kupfer P, Weber M, Dräger
A (2013) GRN2SBML: Automated encoding and
annotation of inferred gene regulatory networks
complying with SBML. Bioinformatics 29, 2216-2217.
132
Junior Research Group
Secondary Metabolism of Predatory Bacteria
133
Secondary Metabolism of Predatory Bacteria
Considering the quick spread of bacterial resistance against antibiotics and the increasing emergence of highly invasive pathogens,
the discovery of new antiinfective drugs is
urgently needed. Natural products are structurally highly diverse compounds that have
been selected during evolution for their affinity to biological targets. Therefore, natural products are ideal candidates for drug
discovery. Genomic analyses illuminated
the huge proficiency of microorganisms for
natural product biosynthesis. Moreover, it
became clear that the potential of these organisms for the production of antibiotics is
far from being exhausted (Nett 2014). Bacteria and fungi that have as yet been ignored
in drug discovery programs are of particular
interest in this context, as they provide fresh
opportunities for the identification of bioactive molecules.
The research program of the Junior Research
Group Secondary Metabolism of Predatory
Bacteria (SRB) is centred on the exploitation
of such neglected microbial resources, focusing mainly on two bacterial groups:
•Predatory bacteria, such as Cupriavidus
necator or Herpetosiphon aurantiacus, can
feed on other microbes under conditions
when nutrients become scarce. Available genome sequences show that preda-
Aufgrund der raschen Ausbreitung von Antibiotika-resistenten Bakterien und dem zunehmenden Auftreten von hochinvasiven
Krankheitserregern, besteht ein dringender
Bedarf neue antimikrobielle Wirkstoffe zu
identifizieren. Naturstoffe umfassen strukturell höchst unterschiedliche Verbindungen,
die im Verlauf der Evolution auf ihre Affinität zu biologischen Zielstrukturen selektiert
worden sind. Aus diesem Grund sind Naturstoffe ideale Kandidaten bei der Suche nach
neuen Wirkstoffen. Genomische Analysen
haben das enorme Leistungsniveau von Mikroorganismen für die Biosynthese von Naturstoffen offengelegt. Zudem stellte sich heraus, dass das Potential dieser Organismen
zur Produktion von Antibiotika bei weitem
nicht ausgeschöpft wurde (Nett 2014). Von
Head:
Dr. Markus Nett
134
besonderem Interesse sind dabei Bakterien
und Pilze, die bislang in der Wirkstoffsuche
ignoriert wurden, da sie neue Chancen für
die Findung bioaktiver Moleküle bieten.
Das Forschungsprogramm der Nachwuchsgruppe Sekundärmetabolismus räuberischer
Bakterien (SRB) hat die Nutzbarmachung
dieser brachliegenden mikrobiellen Ressourcen zum Ziel. Im Fokus des Interesses stehen
dabei zwei bakterielle Gruppen:
•Räuberische Bakterien, wie Cupriavidus
necator oder Herpetosiphon aurantiacus,
können im Falle eines Nahrungsmangels
Jagd auf andere Mikroorganismen machen.
Verfügbare Genomsequenzen zeigen, dass
viele räuberische Bakterien spezifische
HKI Research Report 2012 / 2013 Junior Research Group Secondary Metabolism of Predatory Bacteria
tory bacteria possess specific biosynthetic
pathways that are not found in other organisms. Furthermore, there is growing
evidence that many of the secreted natural
products contribute to the predatory activity, e.g., in the form of signalling molecules
or antibiotics, which promote the killing of
the prey. From a pharmaceutical perspective, predatory bacteria are hence a promising source for finding new medicinal
drugs.
lanacearum, demonstrated that the expression of several biosynthesis genes responds to virulence-associated regulators,
assuming a role of natural products in the
infection process (Schneider et al. 2009).
In the context of drug discovery, secondary metabolites from plant pathogenic bacteria can be valuable lead structures for the
development of antibiotics or anticancer
agents, as exemplified by micacocidin and
rhizoxin.
References
Nett M (2014) Genome mining –
concept and strategies for natural
product discovery. Prog Chem Org
Nat Prod, in press.
•Plant pathogenic bacteria are, with few
exceptions, barely characterised with regard to their chemistry. Recent studies,
e. g., in the tomato pathogen Ralstonia so-
Stoffwechselwege für die Biosynthese von
Naturstoffen besitzen, die sich nicht in
anderen Produzentengruppen finden. Zudem gibt es vermehrt Hinweise, dass die
sezernierten Naturstoffe zur räuberischen
Lebensweise beitragen, z. B. in Form von
Signalstoffen oder antimikrobiellen Verbindungen, die das Töten der Beute begünstigen. Aus pharmazeutischer Sicht
sind räuberische Bakterien damit eine vielversprechende Quelle für die Entdeckung
neuer Wirkstoffe.
arum, haben gezeigt, dass die Expression
zahlreicher Biosynthesegene durch Virulenz-assoziierte Regulatoren kontrolliert
wird, was eine Funktion von Naturstoffen
im Rahmen des Infektionsprozesses nahe
legt (Schneider et al. 2009). Für die Nutzung von pflanzenpathogenen Bakterien
als Wirkstoffquelle spricht, dass diese Produzentengruppe bereits einige sehr vielversprechende Leitstrukturen für Antibiotika und Zytostatika geliefert hat. Beispiele
sind das Micacocidin und das Rhizoxin.
•Pflanzenpathogene Bakterien sind von
wenigen Ausnahmen abgesehen bislang
nur unzureichend chemisch charakterisiert. Neuere Untersuchungen, u.a. in dem
Tomaten-Schaderreger Ralstonia solanace-
Junior Research Group Secondary Metabolism of Predatory Bacteria HKI Research Report 2012 / 2013
135
Scientific Projects
1
Natural products from the predatory bacterium Cupriavidus necator
and related Cupriavidus species
Anecdotal evidence suggests a correlation between the predatory activity of Cupriavidus
necator and natural product biosynthesis.
In the presence of prey organisms, C. necator secretes a copper-binding peptide, which
might be used to deliver toxic amounts of the
transition metal to the prey cells. Moreover,
the same peptide was also proposed to play
an important role in the interaction with the
actinomycete Agromyces ramosus. The latter
is itself a microbial predator and spreads via
the formation of mycelia. Once a mycelial
contact is established with potential prey,
the actinomycete starts to secrete lytic enzymes in order to feed on these organisms.
Interestingly, A amosus was also found to attack C. necator, but in this particular case,
the assaulted cells struck back. The counterattack coincided with the release of the aforementioned copper-binding molecule and led
to a complete eradication of the A. ramosus
mycelium. Notwithstanding its biological
significance, the identity of the copper-binding peptide was not resolved. The objective
of the junior research group is thus to find
this potential antibiotic and to determine its
structure.
Using a genome mining strategy, a gene
cluster was identified in the genome of the
C. necator strain H16 that could likely code
for the biosynthesis of the molecule sought.
The candidate locus contained the genes for
the nonribosomal assembly of a peptide and
it also included genes involved in the formation of bidentate ligand groups, which would
be required for the coordination of metal
ions. To retrieve the metabolic product of the
cluster, one of the core biosynthesis genes
was disrupted through insertional mutagenesis. Comparative profiling of mutant and
136
wildtype strain revealed several peaks that
were completely absent in the mutant (Fig.
1). The corresponding compounds were isolated and the planar structure of the main
metabolite, cupriachelin, was elucidated by
NMR spectroscopy. The stereochemistry of
this lipopeptide was solved following chemical derivatisation. A model for the biosynthesis of cupriachelin was subsequently deduced from bioinformatic analyses and the
biochemical characterisation of adenylation
domain specificity (Kreutzer et al. 2012a).
Since the predatory activity of C. necator
cannot be detected in established assays, the
group also developed a new predation test
system. The latter relies on the co-cultivation
of predatory bacteria with prey organisms
producing distinctive pigments and the subsequent enumeration of the individual populations at defined time points. Using the new
assay, the predatory activity of C. necator
could be confirmed. However, cupriachelindeficient mutants of C. necator H16 were not
affected in their capability to feed on prey
bacteria, indicating that cupriachelin is not
essential for predation. This result was further corroborated by comparative genomics,
which revealed that the cupriachelin biosynthesis genes are restricted to C. necator
strains originating from freshwater environments. Subsequent studies showed a preference of cupriachelin for the chelation of ferric iron. The resulting complex is prone to
photolytic degradation, which comes along
with the release of ferrous iron. This finding
suggests an ecological role for cupriachelin
in supplying ferrous iron to planktonic communities in exchange against organic carbon
(Kreutzer et al. 2012a).
In a follow-up study, a cupriachelin-like
gene cluster was discovered in the genome
of the bacterium Cupriavidus taiwanensis
LMG19424. The structure of the encoded lipo-
Figure 1
Structure of cupriachelin (A). Metabolic profiles of the C. necator H16
wildtype (profile a) and a cupriachelin-deficient mutant strain (profile b).
The metabolites 1-3 represent cupriachelin congeners, which possess
differing acyl chains (B).
peptide could be predicted with sufficient accuracy from the molecular assembly line to
enable an NMR-assisted isolation. This study
hence represents the first example of a genome mining attempt that relied on the readout
of distinctive NMR resonances (Kreutzer et
al. 2012b). Similar to cupriachelin, the isolated taiwachelin was shown to undergo a
photoreductive dissociation, when an aqueous solution of its ferric iron complex was
exposed to sunlight.
2
Natural products from the predatory bacterium Herpetosiphon aurantiacus
The genome of the predatory bacterium Herpetosiphon aurantiacus harbours a plethora
of genes that are predicted to be involved in
natural product assembly. Although a total of
20 biosynthetic gene clusters had been identified in a preceding study (Kiss et al. 2011),
their metabolic products remained elusive,
most likely due to cultivation issues of this
filamentous microorganism. To retrieve the
natural products of H. aurantiacus, we joined
forces with Martin Roth (Bio Pilot Plant) to
optimise the fermentation conditions. A systematic evaluation of different cultivation parameters in combination with comparative
NMR analyses led to the discovery of a new
diterpene featuring an octahydro-1H-indenyl
skeleton. The isolated compound, herpetopanone, bears structural resemblance to the
plant-derived cadinane-type sesquiterpenes,
which are completely unprecedented in bacteria. Motivated by this finding, we continued to alter the herpetopanone production
medium and to analyse the metabolic effects.
In this way, two further natural products
could be identified, namely the nucleoside
antibiotic futalosine and the nonribosomal
peptide synthetase-derived niccicin. While
the former had been previously described
as the intermediate of an alternative mena-
137
Figure 2
Structurally diverse natural products
from the predatory bacterium Herpetosiphon aurantiacus.
References
Schneider P, Jacobs JM, Neres J, Aldrich CC, Allen C, Nett M, Hoffmeister
D (2009) The global virulence
regulators VsrAD and PhcA control
secondary metabolism in the plant
pathogen Ralstonia solanacearum.
ChemBioChem 10, 2730-2732.
Kreutzer MF, Kage H, Nett M (2012a)
Structure and biosynthetic assembly
of cupriachelin, a photoreactive
siderophore from the bioplastic
producer Cupriavidus necator H16.
J Am Chem Soc 134, 5415-5422.
Kreutzer MF, Nett M (2012b) Genomics-driven discovery of taiwachelin, a
lipopeptide siderophore from Cupriavidus taiwanensis. Org Biomol Chem
10, 9338-9343.
138
quinone pathway, niccicin represents a new
compound (Fig. 2). Efforts are now underway
to determine the absolute stereochemistry of
herpetopanone and niccicin, as well as their
biological effects. In addition, we could verify that H. aurantiacus possesses pathways
to specific biosynthetic building blocks, such
as the non-proteinogenic amino acid 4-hydroxyphenylglycine, which is known as a
constituent of glycopeptide antibiotics. In
collaboration with the Research Group Systems Biology and Bioinformatics (Reinhard
Guthke), we confirmed that the respective
4-hydroxyphenylglycine pathway in H. aurantiacus was not acquired by horizontal
gene transfer. According to a phylogenetic
reconstruction, it arose by intraspecies gene
duplication and concomitant, mutation-induced neofunctionalization (Kastner et al.
2012).
3
Natural Products from the plant
pathogenic bacterium Ralstonia
solanacearum
Although Mycoplasma pneumoniae is actually considered the leading cause of community-acquired pneumonia, the treatment of
its infections is still restricted to few antibiotics. In recent years, a dramatic resistance development was observed for this bacterium, underscoring the desperate need for
new effective drugs. Micacocidin is a thiazoline-containing natural product that exerts
strong and selective inhibitory effects in the
nanomolar range against M. pneumoniae. In
the previous reporting period, we discovered
the micacocidin biosynthesis gene cluster
in the plant pathogenic bacterium Ralstonia
solanacearum and, together with the group
of Hans Peter Saluz (Department CMB), a
siderophore function was demonstrated
for this molecule in cellular uptake studies
(Kreutzer et al. 2011). Subsequent testing re-
Figure 3
Modular biosynthesis enzymes for
the production of micacocidin.
Domain architecture of the micacocidin (Mic) assembly line. Domains
that likely account for the structural
differences to yersiniabactin (Ybt)
are highlighted in blue (A). Chemical
structures of micacocidin and yersiniabactin (B).
vealed that the antibiotic effect of micacocidin is not linked to its metal-chelating properties. An iron(III)-loaded complex exhibited
the same level of activity as the free ligand.
Aiming at the engineering of new analogs
for structure-activity relationship studies,
we then set out to unravel the biosynthesis
of micacocidin.
The chemical structure of micacocidin is
reminiscent of the natural product yersiniabactin, which is also reflected in a largely conserved enzymatic assembly line (Fig. 3). Notwithstanding the close relatedness of the two
molecules, the minor variations apparently
have important consequences in terms of antibiotic activity, as yersiniabactin is a much
weaker antimycoplasma drug than micacocidin. The molecular basis for the subtle,
yet biologically significant, structural differences were not clear. In particular, the origin of the distinctive pentylphenol moiety in
micacocidin was puzzling, and it was thus
interrogated by a combination of stable isotope feeding experiments, reconstitution of
biosynthetic steps, and targeted inactivation
of catalytic domains. These analyses pointed
to an iterative type I polyketide synthase
(iPKS) as the key enzyme in the construction
of the aromatic residue. The iPKS was shown
to generate a linear tetraketide intermediate
from acyl carrier protein-tethered hexanoic
acid by three consecutive, decarboxylative
Claisen condensations with malonyl-coenzyme A. The final conversion into the pentylphenol building block, however, depends on
a ketoreductase domain within the enzyme
(Fig. 4), as demonstrated by heterologous
expression in E. coli and subsequent sitedirected mutagenesis experiments (Kage et
al. 2013). It should be noted that the characterized iPKS from R. solanacearum is totally
unprecedented in bacteria. Not only is the
iteratively acting enzyme integrated into a
multimodular assembly line, but it seems
mechanistically also related to partially re-
References
Kiss H, Nett M, Domin N, Martin K,
Maresca JA, Copeland A, Lapidus A,
Lucas S, Berry KW, Glavina Del Rio T,
Dalin E, Tice H, Pitluck S, Richardson P,
Bruce D, Goodwin L, Han C, Detter JC,
Schmutz J, Brettin T, Land M, Hauser
L, Kyrpides NC, Ivanova N, Goker M,
Woyke T, Klenk HP, Bryant DA (2011)
Complete genome sequence of the
filamentous gliding predatory bacterium Herpetosiphon aurantiacus type
strain (114-95(T)). Stand Genomic Sci
5, 356-370.
Kastner S, Müller S, Natesan L, König
GM, Guthke R, Nett M (2012) 4-Hydroxyphenylglycine biosynthesis in
Herpetosiphon aurantiacus: a case of
gene duplication and catalytic divergence. Arch Microbiol 194, 557-566.
139
Figure 4
Initial steps in micacocidin assembly leading to the formation of the
6-pentylsalicylate starter unit (A).
A mutation in the KR domain of the
iPKS results in a premature offload
of a shunt product due to lactonisation (B).
References
Kreutzer MF, Kage H, Gebhardt P,
Wackler B, Saluz HP, Hoffmeister
D, Nett M (2011) Biosynthesis of a
complex yersiniabactin-like natural
product via the mic locus in phytopathogen Ralstonia solanacearum.
Appl Environ Microbiol 77, 6117-6124.
Kage H, Kreutzer MF, Wackler B,
Hoffmeister D, Nett M (2013) An
iterative type I polyketide synthase
initiates the biosynthesis of the
antimycoplasma agent micacocidin.
Chem Biol 20, 764-771.
Kreutzer MF, Kage H, Herrmann
J, Pauly J, Hermenau R, Müller
R, Hoffmeister D, Nett M (2014)
Precursor-directed biosynthesis of
micacocidin derivatives with activity
against Mycoplasma pneumoniae. Org
Biomol Chem 12, 113-118.
140
ducing polyketide synthases from fungi,
which utilise programmed keto reduction
to direct the biosynthesis toward a defined,
aromatic product. The possibility to independently reconstitute the function of this enzyme and to heterologously produce 6-pentylsalicylate may ultimately prove useful in
the field of combinatorial biosynthesis.
We now plan to introduce clickable groups
into micacocidin by directed feeding and
genetic engineering, respectively, to set the
stage for target fishing studies.
Together with the group of Dirk Hoffmeister
(PB), we then probed the substrate specificity of the hexanoic acid-loading enzyme. A
biochemical analysis revealed an extended
plasticity, which was exploited for the generation of new micacocidin derivatives by
precursor-directed biosynthesis (Kreutzer et
al. 2014). The engineered compounds were
profiled against M. peumoniae in collaboration with the group of Rolf Müller (Helmholtz Institute for Pharmaceutical Research
Saarbrücken), yielding valuable insights
with regard to the structural changes that
are tolerable in terms of antibiotic activity.
Group members
Head
Dr. Markus Nett
Phone +49 3641 532-1297
Fax
+49 3641 532-2297
[email protected]
Kage H, Kreutzer MF, Wackler B, Hoffmeister
D, Nett M (2013) An iterative type I polyketide
synthase initiates the biosynthesis of the antimycoplasma agent micacocidin. Chem Biol 20,
764-771.
Scientists
Dr. Hirokazu Kage
Dr. Soledad Vela Gurovic (until 03/2012)
Ph.D. Students
Nicole Domin (until 06/2013)
Martin Kreutzer (unitl 11/2013)
Xinli Pan (since 10/2013)
Sebastian Schieferdecker
Ivana Seccareccia
Kreutzer MF, Kage H, Nett M (2012) Structure
and biosynthetic assembly of cupriachelin, a
photoreactive siderophore from the bioplastic
producer Cupriavidus necator H16. J Am Chem
Soc 134, 5415-5422.
Juliane Korp (since 07/2013)
Colette Kurth (since 09/2013)
Tom Lauterbach (since 10/2013)
Bastien Schnell (02/2013–07/2013)
Research Assistants
Julia Greßler (until 09/2012)
Trainees
Ron Hermenau (03/2013–03/2013)
Juliane Korp (04/2012–07/2012)
Colette Kurth (04/2013–05/2013)
Franziska Müller von Klingspor
(09/2013–09/2013)
Sebastian Peil (07/2013–08/2013)
Martin Peschel (03/2013–03/2013)
Christiane Ring (08/2013–08/2013)
Marko Rodewald (09/2013–10/2013)
Sophie Seng (02/2013–03/2013)
Collaborations
University of Potsdam, Germany
Prof. Dr. Elke Dittmann
Scripps Institution of Oceanography and
the Skaggs School of Pharmacy and
Pharmaceutical Sciences, University of
California at San Diego, USA
Prof. Dr. Bradley S. Moore
Helmholtz-Institut für Pharmazeutische
Forschung Saarland (HIPS),
Helmholtz-Zentrum für Infektionsforschung
(HZI), Institute of Pharmaceutical Biology,
University of Saarland, Germany
Prof. Dr. Rolf Müller
PD Dr. Michaela Schmidtke
External funding
DiNaMid – Genom-basierte Findung neuer
antimikrobieller Naturstoffe in mikrofluidischen
Chips; Identifizierung neuer Stoffwechselwege
und Naturstoff-Isolierung
Markus Nett, Martin Roth
Project: Molecular basis fror predatory interactions of Cupriavidus necator wich ist prey
bacteria
Markus Nett
141
142
Cross-sectional Unit Bio Pilot Plant
143
Cross-sectional Unit Bio Pilot Plant
The research of the HKI Bio Pilot Plant aims
at the development and optimisation of biotechnological processes of natural product
producers, including strain development
and optimisation. The Bio Pilot Plant offers
opportunities for the cultivation of a wide
spectrum of microorganisms (bacteria, lower
and higher fungi, genetically modified organisms, safety group 1 and 2) from flask
cultures via laboratory bioreactor to pilot
scale fermenter of 2.5 m³ that are unique in
the German academic area. All necessary
purification processes and the appropriate
technical equipment for well-performed purifications of natural products, proteins and
biopolymers are available. The development
of analytical methods is mainly focused on
process-related parameters and the analysis
of synthesised products. We are experienced
in the cultivation of microorganisms in the
chemostat, under anaerobic fermentation
conditions, and at high cell densities. The
establishment of a microfluidic platform for
ultra-high-throughput screening of antimicrobial natural products and enzymes is an
example for the development of innovative
technologies. The Bio Pilot Plant closely collaborates with the departments and junior
research groups of the HKI, institutes of the
Friedrich Schiller University, and the Ernst
Abbe University of Applied Sciences.
Die Forschungsarbeiten des Biotechnikum
sind auf die Entwicklung und Optimierung
bio
technologischer Prozesse von Naturstoff-Produzenten einschließlich der Stamm
optimierung fokussiert. Das Biotechnikum
bietet im deutschsprachigen, akademischen
Bereich einzigartige Möglichkeiten, ein weites Spektrum von Mikroorganismen (Bakterien, niedere und höhere Pilze, gentechnisch
veränderte Organismen der Risikogruppen
1 und 2) vom Schüttelkolben bis zum Pilotmaßstab von 2,5 m³ Arbeitsvolumen zu
kultivieren. Für die dabei gebildeten Naturstoffe, Proteine und Biopolymere stehen
alle erforderlichen Aufreinigungsverfahren
und die dazu benötigten technischen Aus
rüstungen zur Verfügung, um diese bis zur
Reinsubstanz zu isolieren. Die Entwicklung
Head:
Dr. Uwe Horn
144
analytischer Methoden konzentriert sich besonders auf prozessbegleitende Parameter
und auf den Nachweis der synthetisierten
Produkte. Außerdem haben wir umfangreiche Erfahrungen bei der Kultivierung von
Mikroorganismen im Chemostaten, unter
anaeroben Fermentationsbedingungen, sowie der Hochzelldichtefermentation. Die Etablierung einer mikrofluidischen Plattform
für die Ultrahochdurchsatz-Suche nach antimikrobiellen Naturstoffen und Enzymen ist
ein Beispiel für die Entwicklung innovativer
Technologien. Das Biotechnikum arbeitet intensiv mit den Forschungsabteilungen und
Nachwuchsgruppen des HKI, mit Instituten der Friedrich-Schiller-Universität Jena
sowie der Ernst-Abbe-Fachhochschule Jena
zusammen.
HKI Research Report 2012 / 2013 Cross-sectional Unit Bio Pilot Plant
Scientific Projects
aminoglycosides β-‐lactams 1940 glycopepAdes 1950 1960 1970 muAlins 1980 1990 2000 innovaAon gap sulpha drugs macrolides quinolones, streptogramins 2010 lipopepAdes oxazolidinones chloramphenicol, tetracyclines Figure 1
Timeline for the discovery of antibiotic substance classes (after Fischbach and Walsh, 2009)
1
Picoliter droplet-based search
for natural products – the future of
biotechnological screening
Group Leaders: Emerson Zang, Martin Roth
Antibiotic screening: the past
Nature proved to be a rich source of active
compounds in the past. Starting with the
discovery and commercialisation of penicillin in 1928, an impressive variety of antimicrobial substances was unveiled from soil
and marine habitats until the early 1960s.
However, after this golden era of antibiotic
discovery, researchers increasingly struggled to find new substances as a result of
permanent rediscovery of already known
producers (Fig. 1). Simultaneously, first evidence of antibiotic-resistant pathogens was
reported, aggravating the demand for new
substances in the decades to follow. With
the dawn of molecular biological methods for
target-identification in pathogens, the focus
of screening efforts shifted from phenotypical screening to target-based screening approaches. At the same time, natural product
libraries were replaced by combinatorial
libraries of synthetic compounds. Despite
extensive screening programmes, the yield
of such techniques was disappointing. The
diversity and biological relevance of chemical libraries was found to be inferior to the
natural compound portfolio. Moreover, hits
that were active in vitro often suffered from
unspecificity or insufficient trans-membrane
mobility.
Antibiotic screening: the future
To overcome the crisis in antibiotic research,
leading experts postulate the return to phenotypic screening of natural products – the
hitherto most successful approach in antibiotic research. However, new cultivation and
screening methods are required for significantly increased sample throughput and to
provide new means of de-replication.
Cross-sectional Unit Bio Pilot Plant HKI Research Report 2012 / 2013
145
Figure 2
Screening for antibiotic compounds with emulsionbased microfluidics.
(1) Droplet generation from spore suspension by flow
focusing. (2) Droplet incubation for propagation of
Actinobacteria. (3) Dosing of reporter cells. (4) Droplet
incubation for propagation of reporter cells. (5) Droplet
interrogation and sorting.
In 2003, droplet-based microfluidics was
used at the HKI for the first time to cultivate soil-borne bacteria by Martin et al. On
the basis of this seminal work, a nanoliterdroplet-based screening concept was developed, relying on in-droplet isolation, cultivation and antibiotic activity assessment of
Actinobacteria derived from soil samples.
The assay was implemented using so-called
“micro-segmented” flow, where droplets are
continuously kept in serial trains, separated
by carrier oil. It could be shown that cultivation of Actinobateria and other organisms on
that platform is feasible, as well as the detection of miniminal inhibitory concentrations
for various antibiotics. Yet, micro-segmented
flow suffered from frequent unintended fusion of droplets, so that complex assay regimes as the intended antibiotic screening
were not realizable.
As a consequence, we focused on emulsionbased microfluidics in 2013 (Fig. 2), a droplet-
146
Figure 3
Microcultures of green fluorescent
Streptomyces lividans in picoliter
droplets.
based technique that has several advantages
over previously applied micro-segmented
flow: Droplets are stabilised by surfactant
molecules in the carrier oil, so that unintended droplet fusion is avoided. The increased
droplet robustness furthermore allows bulk
incubation of droplets, which can be exploited for a massive increase in throughput, easily achieving more than 106 samples per day.
To make emulsion-based microfluidics available at the Bio Pilot Plant, compliant microfluidic chips were designed in-house and
manufactured externally at LionixBV (NL).
Apart from droplet generation by flow focusing, the chips allow dosing and sorting of
re-injected droplets by integrated electrodes,
which are needed to specifically destabilise the surfactant-loaded droplet interface.
These operations also require electronic periphery (function generators, voltage amplifiers), and its commissioning was successfully accomplished in 2013.
Figure 4
Triggered imaging of droplets.
ADC: A/D-converter, ST: Schmitttrigger
pL-droplets as high-throughput
cultivation platform
Having this platform at hand, emulsionbased growth of Actinobacteria was first investigated. To this end, spore suspensions
of diverse actinobacterial species were employed as aqueous phase for droplet generation, and the resulting droplet populations
were incubated off-chip in handcrafted vials.
After one day of incubation, in-droplet germination of spores was noticed and development of small filamentous structures was
observed. With prolonged incubation, the filaments grew to dense microcultures (Fig. 3)
and the occupied droplets showed shrinkage
as a result of metabolic activity concomitant
with an osmotic mismatch to non-occupied
droplets. In extreme cases, the droplet interface was deformed by growing microcultures. However, filament penetration of the
droplet interface was rarely observed, so that
the droplet integrity as an isolated cultivation vessel was maintained. Nevertheless,
the unequally distributed shrinkage of occupied droplets led to increased polydispersity
of the droplet population, impeding subsequent unit operations as dosing and sorting.
Image-based droplet sorting
To avoid above-mentioned polydispersity
during prolonged incubation, image-based
droplet sorting (IDS) for growth-dependent
fractionation of droplets was developed (Fig.
4): Spore-loaded droplets are incubated until
germination of spores and formation of microcultures occurs. The droplets are reinjected into a microfluidic chip and separately imaged using a brightness-dependent
trigger signal. Each image is subjected to an
online “Difference of Gaussians” analysis developed by the Research Group Applied Systems Biology, enabling detection of droplet
boundaries and enclosed filamentous structures within milliseconds. Depending on the
presence or absence of filamentous structures, droplets are sorted into either one of
147
no/little
mycelium
droplet
generation
incubation
reinjection
image-based
droplet sorting
dense
mycelium
antimicrobial
activity testing
Figure 5
Screening with image-based droplet
sorting for droplet population maintenance and enrichment of slowgrowing actinobacteria.
two efferent channels. Using this approach,
growth-dependent droplet sorting at 100 Hz
was demonstrated. The technique will not
only enable reduction of droplet population
polydispersities. More importantly, IDS will
allow fractionation of unknown Actinobacteria according to their speed of growth, finally achieving enrichment of slow-growing
and rare species, being producers of hitherto
undiscovered compounds with high probability (Fig. 5).
In summary, emulsion-based microfluidics
was successfully established as a cultivation platform for Actinobacteria, enabling
a culture throughput of far more than 106
samples per day. By the implementation of
image-based droplet sorting, a powerful tool
for droplet maintenance and enrichment of
slow-growing Actinobacteria was developed
(Zang et al. 2013). Yet, the proof-of-concept
for antibiotic screening in pL-droplet-emulsions must still be provided, but important
148
steps to master this demanding challenge
have been taken.
2
Stress response in Escherichia coli
bioprocesses related to rare amino
acids
Group Leaders: Michael Biermann, Uwe Horn
Escherichia coli is a commonly used host in
industrial applications for the production
of pharmaceutical proteins. Because of its
high volumetric productivity the fed-batch
fermentation under high cell density conditions is one of the most widely used process
strategies for the expression of such proteins. Although the fermentation processes
are very well-developed today, they demand
very high standards on the used host-vector-systems and bioreactors. Particularly in
industrial large-scale bioreactors inhomogeneities may occur causing substrate gra-
dients and local limitations of dissolved
oxygen. The resulting stress situations can
lead to instabilities of the process and can
have negative effects on the protein yield and
quality. Concerning the protein quality, a
phenomenon studied intensely in the recent
years is the formation and misincorporation
of non-canonical amino acids such as norvaline and norleucine into recombinant proteins. This can result in modified variants
of the final product. According to the guidelines of regulatory health authorities such as
the US Food and Drug Administration (FDA)
and the European Medicines Agency (EMA),
all of these modified variants need intensive
analytical characterisation and are therefore
highly undesirable in biopharmaceutical
production.
A cooperation project of the Bio Pilot Plant
with the WACKER Biotech GmbH Jena aimed
at the development of new fermentation strategies to prevent the misincorporation of noncanonical amino acids.
As analytical basis for the project, a novel
ultra-high performance liquid chromatography technique was developed allowing for simultaneous analysis of norvaline, norleucine
and 15 standard amino acids (Biermann et al.
2013a).
The main focus of the project was the impact
of the overflow metabolism related trace elements molybdenum, nickel and selenium.
We analysed the trace element associated
response of recombinant antibody producing
E. coli to oxygen limitation at high glucose
concentration with special focus on non-canonical amino acids. Current physiological
studies revealed strong evidence for the connection of glucose overflow metabolism and
pyruvate accumulation to the occurrence of
norvaline in wildtype E. coli fermentations.
The trace elements molybdenum, nickel and
selenium form essential parts of the formate
hydrogen lyase (FHL) metalloprotein complex, which is a key enzyme of anaerobic
pyruvate metabolism in E. coli and could
therefore represent a crucial connection to
the pyruvate accumulation associated biosynthesis of rare amino acids.
During fed-batch cultivation with provoked
oxygen limitation and glucose excess, norleucine and norvaline were accumulated only in
the absence of molybdenum, nickel and selenium. In contrast, trace element supplemented stress fermentation showed significantly reduced concentrations of these rare
amino acids and the major signature fermentation product formate, supporting the correlation between a functional FHL metalloprotein complex and low unspecific amino acid
synthesis under oxygen limitation at high
glucose concentration.
In conclusion, the formation of norleucine
and norvaline by recombinant E. coli at cultivation with provoked oxygen limitation and
glucose excess can be reduced to levels at
the detection limit by adding the trace elements molybdenum, selenium and nickel to
the fermentation medium. Even under the
metabolic burden during induction phase
the physiologically available concentrations
of non-canonical amino acids remained low
(Biermann et al. 2013b).
3
Life science meets physics - Microbial adhesion and biofilm formation
on biomaterials
Group Leader: Claudia Lüdecke, Martin Roth
(in cooperation with Klaus D. Jandt,
Otto Schott Institute of Materials Research,
Friedrich Schiller University Jena)
Modern medicine increasingly uses biomaterials for implant purposes, e. g., to restore
human body functions. Infections related to
these biomaterials are most frequently associated with microbial colonisation of the
indwelling medical devices. Understanding
mechanisms of bacterial adhesion and biofilm formation is thus a crucial prerequisite
to develop strategies for the prevention of
biomaterial-associated infections.
149
Figure 6
In vitro test system for investigation of microbial adhesion and
biofilm formation at constant
and reproducible conditions.
The experimental setup includes a
continuous culture (chemostat), a
biofilm reactor (non-constant-depth
film fermenter; nCDFF), microscopy
and image analysis. (from Lüdecke
et al. 2014)
A new standard testing system for bacterial adhesion and biofilm formation
Various investigations were made to influence bacterial adhesion and biofilm formation by materials’ surface properties, such
as surface topography and morphology or
chemistry and wettability of the biomaterials. However, these studies are hardly comparable and reproducible since there are no
standard tests available for adequately studying microbial adhesion and biofilm formation
and all studies differ in experimental conditions and parameters. The first aim of this
highly interdisciplinary project was, therefore, to develop an in vitro testing system for
the investigation of microbial adhesion and
biofilm formation on biomaterials surfaces at
constant and reproducible conditions.
To realise the new testing system, a biofilm
reactor was coupled to a continuous culture
(chemostat) of Escherichia coli. For evaluation of the system, biofilms were cultivated
in several repeated experimental runs on titanium surfaces for up to 9 days (Fig. 6).
Using a continuous culture for inoculation
of the biofilm reactor provided intact and viable microbial cells for adhesion and biofilm
cultivation compared to the most often used
Figure 7
Adhesion and biofilm formation of
Escherichia coli on titanium surfaces
after 24 hours (A), 2 days (B), 3 days
(C) and 6 days (D), respectively, cultivated with the new in vitro testing
system. (from Lüdecke et al. 2014)
150
Figure 8
PhD student Claudia Lüdecke investigates the adhesion of Escherichia coli
on nanorough titanium surfaces using a scanning electron microscope
at the Otto Schott Institute of Materials Research (OSIM), Friedrich Schiller
University, Jena. (Photo by Jan-Peter
Kasper, FSU Jena)
microtiter well plate based approaches where
cells are weakened by centrifugation and resuspension procedures are used.
The cultivated biofilms showed equal and reproducible growth with a typical four-stagesdevelopment including initial bacterial adhesion, micro colony formation and early and
late maturation (Fig. 7).
Inoculation of the biofilm reactor with a
continuous culture minimised variations
between different experimental runs. The
new in vitro testing system thus allows the
investigation of microbial adhesion and reproducible cultivation of biofilms at constant
experimental conditions. The system will
support developing and adequate testing of
biomaterial’s surface modifications for prevention of biomaterial-associated infections
(Lüdecke et al. 2014).
Microbial adhesion on nanorough
titanium surfaces
It is well known that surface topographical features of biomaterials in the micrometer range strongly affect the adhesion
of microorganisms and subsequent biofilm
formation. To date, the impact of materials
surface topography on the nanometer range
was much less investigated and fundamental
mechanisms have not been identified. The
second aim of this project was to identify fundamental mechanisms involved in bacterial
adhesion on nanorough titanium surfaces.
The adhesion of the pathogenic microorganisms Staphylococcus aureus and Escherichia
coli most often involved in biomaterial-associated infections was investigated on titanium surfaces with different nanoroughness
using the new in vitro testing system (Fig. 8).
Titanium surface coverage by E. coli and
S. aureus was decreased by approximately
151
50% with increasing surface nanoroughness
from 2 to 6 nm. Two main mechanisms were
identified to be most probably responsible for
the reduced number of adhered bacteria with
increasing titanium surface nanoroughness:
With increasing roughness, the size of titanium grains at the surface increased and,
accordingly, the relative number of surface
grains decreased. For first contact of bacterial cells with the titanium surface (early
adhesion), fewer contact points due to the
lower number of surface grains were available with increasing roughness resulting in
a reduced number of adhered cells.
A scanning electron microscope equipped
with a focused ion beam (FIB-SEM) provided
direct insight into the interface between bacterial cell and nanorough titanium surface.
After 3 h of adhesion (early adhesion), bacterial cells were attached to the nanorough surfaces only at few points. After 9 h of adhesion
(late adhesion), bacterial cells produced extracellular polymeric substances (e.g. carbohydrates) and were in complete contact with
the titanium surface. With increasing titanium surface roughness, the specific surface
area available for contact between the bacterial cell and the titanium decreased resulting also in a reduced number of adhered
cells (Fig. 9).
In this project it was shown that surface
roughness even on the very low nanometer
scale influences bacterial adhesion behavior.
These findings can give new momentum to
biomaterials research and will support the
development of biomaterials surfaces with
anti-infectious surface properties (Lüdecke
et al. 2013).
This interdisciplinary project, a cooperation
between the HKI Bio Pilot Plant and the Otto
Schott Institute of Materials Research (OSIM)
at the Friedrich Schiller University, Jena, is
supported with a PhD fellowship awarded by
the Graduate School “Jena School for Microbial Communication (JSMC)”. The successful
collaboration will be continued in a second
JSMC PhD project.
152
4
Production of microbial biomass
and natural products for research
projects of HKI groups and external
academic partners
Group Leaders: Uwe Horn, Martin Roth
A large number of microbial products from
different microorganisms were produced for
projects of research groups at HKI. 620 fermentations at the 1- to 7-litre-laboratory-scale
and 155 fermentations at the 10 to 500-litrepilot-scale were performed within 2012-2013
including preparation of inoculum cultures,
fermentation and monitoring of the fermentations by data acquisition and analysing samples. The following downstream processing
procedures have been carried out: separation
of supernatant and biomass, fractionation of
supernatant by preparative HPLC, capture of
natural products by adsorption on synthetic
resins or by solvent extraction, and concentration and freeze drying of the extracts. In
2012-2013, products from about 5,500 litres
fermentation broth were prepared.
Selected projects
Isolation and structural elucidation of
bioactive natural products and genetics
of biosynthetic pathways
(Department of Biomolecular Chemistry)
41 pilot scale fermentations (30 – 300 L) with
different Streptomyces and Burkholderia
strains and mutants, and with Aspergillus oryzae were performed. Downstream
processing, especially solvent extraction and
preparative HPLC, was done to isolate the
natural products.
Discovery of new drugs and metabolites
(Junior Research Group Secondary Metabolism of Predatory Bacteria)
Predatory and phytopathogenic bacteria
were cultivated in pilot scale fermentations
(30 – 75 L) in order to discover and to isolate
new natural products.
Figure 9
Staphylococcus aureus cells adhered
to nanorough titanium (after 3 h
of adhesion (A, C) and after 9 h of
adhesion (B, D)) cross-sectioned with
a focused ion beam. Arrows indicate the adhesion points between
microbial cell and titanium surface.
With increasing time, the cells are in
complete contact to the surface.
Genome mining in clostridia for natural
product discovery (Department of Biomolecular Chemistry)
More than 400 fermentations with strictly
anaerobic Clostridium species were performed in lab-scale bioreactors (1 – 7 L) using
a variety of media for the isolation of novel
natural products (Fig. 10).
Molecular biology of natural products
(Department of Molecular and Applied
Microbiology)
40 pilot scale fermentations (30 L) of Aspergillus fumigatus mutants were performed to
enable the elucidation of the biosynthesis of
the toxin gliotoxin.
Analysis of the Aspergillus fumigatus
response to low atmospheric oxygen
levels (Department of Molecular and
Applied Microbiology)
31 laboratory fermentations in the 7-L-scale
were performed to investigate the dynamic
changes of the A. fumigatus transcriptome
and proteome in hypoxia utilising an oxygen-controlled bioreactor system.
Identification of protein antigens in the
secretome of Candida albicans for the
diagnosis of fungal sepsis (Department of
Molecular and Applied Microbiology)
This projcet aims at characterising the secretome of the two morphotypes of C. albicans, yeast cell and hypha. The cultivation of
both cell types in 10-litre-fermenters and the
sample preparation were established and improved in order to obtain sufficient amounts
of the proteins for the secretome analyses,
because C. albicans secretes only low protein
concentrations in contrast to saprophytic
fungi. By using this approach it was possible
to identify 43 proteins excreted by the yeast
cell type, and 114 proteins secreted by the
hypha type.
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Figure 10
Anaerobic cultivation of clostridia in
1 liter bioreactors (BIOSTAT Qplus,
Sartorius Stedim).
Use of heavy metal resistant streptomycetes in bioremediation (Friedrich Schiller
University Jena, Institute of Microbiology,
Microbial Communication)
Pilot scale fermentation (30 – 300 L) of heavy
metal resistant Streptomyces strains and
preparation of biomass for bioremediation
studies at the former uranium mining site in
Eastern Thuringia.
5
Development of high performance
bioprocesses in cooperation with
academic and industrial partners
Group Leaders: Bettina Bardl, Klaus-Dieter
Menzel, Gundela Peschel, Martin Roth
The development of integrated fermentation
and downstream processes for the production of antibiotics, enzymes, toxins and biopolymers in one of the main tasks of the Bio
Pilot Plant. Our research is aimed at GMP-
154
compliant processes in order to transfer
them to GMP production facilities. Integrated
bioprocesses for the following products were
developed or improved:
Corallopyronin A
(in cooperation with the University Hospital
Bonn (A. Hörauf), the Institute of Pharmaceutical Biology, University Bonn (G. König),
funded by the German Center for Infection
Research (DZIF))
Corallopyronin A (CorA) is an antibiotic from
the myxobacterium Corallococcus coralloides
that inhibits the bacterial DNA dependent
RNA polymerase. CorA is effective against
Gram-positive bacteria, e.g. Staphylococcus
aureus, but has recently been shown to also
be effective against the intracellular Gramnegative bacteria Wolbachia spp., endosymbionts in filarial worms. However, it is ineffective against Mycobacterium spp. The mode
of action (MoA) of CorA is different to that
of rifampicin. Thus CorA is effective against
multi-resistant S. aureus. The MoA also allows the use of CorA against global cases of
filaria infections in which rifampicin would
be indicated, but is not used due to tuberculosis co-endemicity in order to avoid selecting rifampicin resistant tuberculosis.
For preclinical trials CorA is needed in gram
amounts. We improved the culture media
and developed a GMP-compliant fermentation and purification process. The process
was scaled-up to the 500-litre-pilot-scale.
Gram quantities of pure CorA can now be
produced and provided for preclincal trials.
Alpha-amanitin
(in collaboration with Heidelberg Pharma
GmbH, Ladenburg)
A general objective in oncology drug discovery and development is to achieve tumour cell eradication, while maintaining at
the same time a high level of tolerability to
healthy cells and tissues. Antibody-drug conjugates (ADCs) combine the well-established
principle of targeted antibodies (safety and
tolerability) with the effectiveness of potent
toxins (anti-tumour efficacy). They consist of
a monoclonal antibody conjugated to toxins
by chemical linkers. The function of the antibody part of the ADC molecule is to ‘guide’
the toxin specifically to the target tumour
cells, and to allow healthy cells to be left
untouched. Current opinion however is, that
tumour relapse and tumour metastases may
be related to ‘dormant or quiescent tumour
cells’ that are insensitive to most – if not all
- currently approved drugs for the treatment
of cancer. This explains the tremendous demand for toxins and coupling techniques that
result in new ADCs with entirely new properties, in particularly to meet the challenges in
treating tumour metastases and preventing
tumour relapses.
In this environment, the Heidelberg Pharma
GmbH (HDP) concentrates on combining the
targeting properties of antibodies with the
effectiveness of the ultra-potent basidiomycete toxin alpha-amanitin, a specific inhibi-
tor of RNA polymerase II. Amanitin differs
from other toxins used in today’s ADC therapeutics by its unique intracellular target and
its systemic and proteolytic stability. ADCs
with amanitin (AMA-ACs) have the potential
to treat multiresistant tumours and ‘dormant
or quiescent’ tumour cells, which are regarded as a main reason for metastasis and
resistance.
The availability of sufficient amounts of alpha-amanitin is a prerequisite for the development of AMA-ACs and clinical trials to test
their efficiency. Therefore it was the aim of a
project funded by the BMBF in the programme
‘KMU-innovativ’ to develop a biotechnological
process for the production of alpha-amanitin.
A saprophytic basidiomycete was selected as
production strain. The developed fermentation and downstream process was scaledup to the 500-litre-pilot-scale. The complete
production process is conforming to GMP
standards. The transfer of the production
process to a GMP certified industrial partner
of HDP is in preparation in order to provide
the toxin for the production of AMA-ACs for
clinical trials.
Purified alpha-amanitin is produced in gram
quantities at HKI and delivered to HDP for
the development of antibody-amanitin conjugates.
Polyhydroxyalkanoates
In a continued collaboration with Tepha Medical Devices Inc., Lexington, USA we develop
and improve fermentation processes for the
production of polyhydroxyalkanoates with
different specific properties using recombinant E. coli strains.
These biocompatible, absorbable polyesters
are used at Tepha for developmental work
addressing a variety of products for medical
applications, e. g. surgical monofilament sutures and meshes, surgical films, composite
meshes, absorbable stents and stent coatings,
absorbable microspheres and versatile drug
release matrices.
155
Group members
External funding
Collaborations
Head
Dr. Uwe Horn
Phone
+49 3641 532-1500
Fax
+49 3641 532-2500
[email protected]
DiNaMid – Genom-basierte Findung neuer
antimikrobieller Naturstoffe in mikrofluidischen
Chips; Identifizierung neuer Stoffwechselwege
und Naturstoff-Isolierung
Markus Nett, Martin Roth
B.R.A.I.N. AG, Zwingenberg, Germany
Dr. Klaus Liebeton
Scientists
Dr. Bettina Bardl
Christine Hoffmeier
Uwe Knüpfer
Karin Martin
Klaus-Dieter Menzel
Dr. Jörg-Hermann Ozegowski
Dr. Gundela Peschel
Dr. Martin Roth
Deutsches Zentrum für Infektionsforschung
New natural compounds – Neue Naturstoffe
Martin Roth, Uwe Horn
Project: Exploiting the metagenome of microbial communities on a microfluidic platform
Uwe Horn
Ph.D. Students
Heike Arnold
Magdalena Bereza
Michael Biermann (until 09/2013)
Alexander Funk
Jana Funk
Christian Haupt
Liesa Heinrich
Claudia Lüdecke
Lisa Mahler
Miguel Tovar
Thomas Weber
Emerson Zang
Biermann M, Linnemann J, Knüpfer U, Vollstädt S, Bardl B, Seidel G, Horn U (2013) Trace
element associated reduction of norleucine
and norvaline accumulation during oxygen
limitation in a recombinant Escherichia coli fermentation. Microbial Cell Factories 12, 116-124.
Thomas Krauße
Annelie Pohle
Marco Säuberlich (08/2012–03/2013)
Astrid Pflieger (04/2012)
Sebastian Tennstedt (04/2012)
Miguel Tovar (03/2012–09/2012)
Research Assistents
Patrick Berthel (until 10/2013)
Michael Cyrulies
Dirk Femerling
Christian Heiden (until 10/2012)
Gudrun Krauter
Karin Perlet
Renate Presselt (until 05/2012)
Jan Schönemann
Armin Siering
Matthias Steinacker
Thomas Weber (since10/2012)
Karsten Willing
Trainees
Laura Geilman (06/2012)
Verena Hammer (09/2012)
Kwon Jisoo (03/2013–04/2013)
Michael Meyer (11/2012)
Astrid Pflieger (03/2012–04/2012)
Stephan Riesenberg (03/2012)
Pimolpong Sriyapai (07/2013–09/2013)
Sebastian Tennstedt (03/2012–04/2012)
Tobias Voigt (02/2012-03/2012)
156
Zang E, Brandes S, Tovar M, Martin K, Mech
F, Horbert P, Henkel T, Figge MT, Roth M (2013)
Real-time image processing for label-free
enrichment of Actinobacteria cultivated in
picolitre droplets. Lab Chip 13, 3707–3713.
Prof. Dr. Erika Kothe
Prof. Dr. Klaus D. Jandt
PD Dr. Jörg Bossert
Heidelberg Pharma GmbH,
Ladenburg, Germany
Dr. Jan Anderl
Dr. Marcel Linssen
Institut für Bioprozess- und Analysenmesstechnik, Heilbad Heiligenstadt, Germany
Dr. Gunter Gastrock
Leibniz Institute for Photonic Technologies,
Jena, Germany
Dr. Thomas Henkel
Merck KgaA, Darmstadt, Germany
Dr. Björn Hock
Dr. Lars Toleikis
Dr. Andreas Stein
Andrè, Kiesewetter
Miltenyi Biotec GmbH Teterow, Germany
Dr. Bernd Schröder
OFS Online Fluid Sensoric GmbH,
Ronneburg, Germany
Wilried Schellbach
Lüdecke C, Bossert J, Roth M, Jandt K D (2013)
Physical vapor deposited titanium thin films
for biomedical applications: Reproducibility
of nanoscale surface roughness and microbial
adhesion properties. Applied Surface Science
280, 578-589.
Sanofi-Aventis Deutschland GmbH,
Frankfurt/M., Germany
Dr. Luigi Toti
Biermann M, Bardl B, Vollstaedt S,
Linnemann J, Knuepfer U, Seidel G, Horn U
(2013) Simultaneous analysis of the non-canonical amino acids norleucine and norvaline
in biopharmaceutical-related fermentation
processes by a new ultra-high performance
liquid chromatography approach. Amino Acids
44, 1225–1231.
Tepha Medical Devices, Inc., Lexington, USA
Dr. David Martin
Andrew Joiner
Scharf DH, Chankhamjon P, Scherlach K,
Heinekamp T, Willing K, Brakhage A A,
Hertweck C (2013) Epidithiodiketopiperazine
biosynthesis: A four-enzyme cascade converts
glutathione conjugates into transannular
disulfide bridges. Angew Chem Int Ed 52, 1109211095.
Technical University Ilmenau, Germany
Prof. Dr. J. Michael Köhler
University Hospital Bonn, Germany
Prof. Dr. Achim Hörauf
Prof. Dr. Gabriele König
Wacker Biotech GmbH, Jena, Germany
Dr. Guido Seidel
International Leibniz Research School
for Microbial and Biomolecular Interactions
157
International Leibniz Research School
for Microbial and Biomolecular Interactions
158
Graduate training at HKI is organised within
the “International Leibniz Research School
for Microbial and Biomolecular Interactions” (ILRS Jena). ILRS Jena was established
in 2005 and initially financed by the competitively granted means of the Joint Initiative
for Research and Innovation. Since 2008, the
graduate school has been included in the HKI
budget. It is headed by the HKI in close collaboration with the Friedrich Schiller University Jena (FSU) and the Max Planck Institute
for Chemical Ecology (MPI CE).
ate school and umbrella organisation for
microbiological research in Jena funded by
the Excellence Initiative of the German Research Foundation. The close network of research activities with a focus on microbial
interactions and communication creates an
environment with excellent research condidtion. The doctoral researchers benefit from
the scientific exchange and extended choice
of courses offered by the close network with
other graduate schools in Jena and the Jena
Graduate Academy.
Since 2006, ILRS is a member of the Jena
School for Microbial Communication, a gradu-
In 2012 and 2013, 10 ILRS doctoral researchers successfully graduated and moved on to
Die strukturierte Doktorandenausbildung
am HKI findet im Rahmen der „International Leibniz Research School for Microbial
and Biomolecular Interactions“ (ILRS Jena)
statt. Die ILRS Jena wurde nach Bewilligung
des Antrages im Pakt für Forschung und
Innovation der Leibniz-Gemeinschaft im
Herbst 2005 ins Leben gerufen und ist seit
2008 im Haushalt des HKI verstetigt. Neben
dem HKI sind die Friedrich-Schiller-Universität Jena (FSU) und das Max Planck Institut
für chemische Ökologie (MPI CE) beteiligt.
Communication (JSMC). Die enge Vernetzung der Forschungsaktivitäten auf dem
Gebiet der Mikrobiologie mit Schwerpunkt
auf dem Aspekt Wechselwirkung und Kommunikation von Mikroorganismen schafft
die Voraussetzungen für herausragende
wissenschaftliche Leistungen. Die Doktoranden profitieren vom wissenschaftlichen Austausch und erweiterten Kursangebot durch
die enge Zusammenarbeit der ILRS mit anderen Graduiertenschulen und der Graduiertenakademie Jena.
Seit 2006 ist die ILRS Mitglied der mit Mitteln der Exzellenz-Initiative geförderten
Dachorganisation Jena School for Microbial
Im Zeitraum von 2012 bis 2013 schlossen
10 Doktoranden ihre Promotion erfolgreich
ab und nahmen verantwortungsvolle Tätig-
HKI Research Report 2012 / 2013 International Leibniz Research School
responsible jobs in industry or academia.
The first joint recruitment of ILRS and JSMC
was carried out in 2012 and attracted close
to 1500 applicants from 82 countries. Currently, 38 doctoral researchers of eleven
different nationalities are working towards
their PhD degree within ILRS. Prof. Dr. Marc
Thilo Figge (systems biology) and Prof. Dr.
Matthias Gunzer (infection biology of human
pathogenic fungi) joined the faculty of ILRS
in 2012.
Prof. Gabriele Diekert and Prof. Bernhard
Hube. The doctoral researchers are represented by Juliane Fischer and Felix Mingo
who succeeded Daniel Schwenk and Sarbani
Sarkar as representatives.
Prof. Peter F. Zipfel has been the Speaker of
ILRS since 2008; the other members of the
Steering Committee are Prof. Ian T. Baldwin,
keiten in der akademischen oder industriellen Forschung auf. 2012 wurde erstmals
ein Auswahlverfahren gemeinsam mit der
JSMC durchgeführt. Auf die ausgeschriebenen Stellen bewarben sich knapp 1500
Studenten aus 82 Ländern. Derzeit arbeiten
in der ILRS 38 Promovierende aus elf verschiedenen Nationalitäten an ihrer Promotion. Die Fakultät der ILRS wurde 2012 durch
die Aufnahme von Prof. Dr. Marc Thilo Figge
im Bereich Systembiologie und durch Prof.
Matthias Gunzer im Bereich Infektionsbiologie humanpathogener Pilze verstärkt.
Prof. Ian T. Baldwin, Prof. Gabriele Diekert
und Prof. Bernhard Hube. Die Doktoranden
werden durch Juliane Fischer und Felix Mingo vertreten, die Ende 2013 die bisherigen
Sprecher (Daniel Schwenk und Sarbani Sarkar) ablösten.
Sprecher der ILRS ist seit 2008 Prof. Peter
F. Zipfel, die stellvertretenden Sprecher sind
International Leibniz Research School HKI Research Report 2012 / 2013
159
160
Scientific Topics
In their natural habitat, microorganisms
do not occur in isolation but live in close
association with other organisms of different species and developmental stages.
Their interactions can take many forms,
ranging from mutual symbiosis to parasitic
interactions. How these complex, multiorganismic networks are regulated by microbial communication is still poorly understood. The ILRS PhD projects therefore aim
to gain insight into the microbial and biomolecular interactions underlying the communication processes among microorganisms
in diverse habitats with interdisciplinary
approaches, combining methods of microbiology, natural product chemistry, chemical
ecology, bioinformatics and systems biology.
Structured PhD Training
The implementation of a curriculum during the course of the PhD programme is an
integral part of the structured PhD training. This curriculum consists of advanced
methods courses offered by the ILRS Faculty
Members. Special soft skill courses such as
“Scientific Writing”, “Poster and Oral Presentations”, “Research Funding”, as well as
Public Relations Activities and German and
English Language courses complement the
training on a non-scientific level.
In addition, there are regular ILRS group
seminars and an annual international symposium.
All means of education follow a program of
study specifically developed for ILRS. Thus,
an efficient qualification of the jun-ior researchers within a tight time frame is ensured.
Wissenschaftliche Themenschwerpunkte
Mikroorganismen kommen in der Natur in
der Regel nicht als Einzelzellen vor, sondern
leben in den verschiedensten Habitaten im
Verbund mit anderen Organismen, z. B. in
mutualistischen Symbiosen oder aber in parasitären Interaktionen. In der Umwelt stehen Mikroorganismen in Wechselwirkung
mit vielen anderen Mikroorganismen (Bakterien / Bakterien, Bakterien/Pilze, Pilze/
Pilze) und auch mit höheren Organismen
wie Pflanzen und Tieren. Die an dieser Interaktion beteiligten Kommunikationsmechanismen sind weitgehend unverstanden.
Die Forschungsprojekte der ILRS versuchen
daher, die zugrunde liegenden mikrobiellen
und biomolekularen Interaktionen mit Methoden der Mikrobiologie, Naturstoffchemie,
chemischen Ökologie, Bioinformatik und
Systembiologie zu entschlüsseln.
Strukturierte Doktorandenausbildung
und Lehre
Wesentlicher Bestandteil der strukturierten
Doktorandenausbildung ist die Implementierung von Lehrveranstaltungen in die
Dissertationszeit. Dazu wird ein vielfältiges
Angebot an Methodenpraktika durch die Mitglieder der ILRS Fakulät angeboten. Spezielle
soft skill-Kurse, wie „Scientific Writing“, „Poster and Oral Presentations“, „Research Funding“, sowie “Public Relations Activities“ und
Sprachkurse in Deutsch und Englisch bieten
Qualifizierungsmöglichkeiten über die rein
fachliche Weiterbildung hinaus.
Außerdem finden regelmäßig Gruppenseminare und jährlich ein internationales Symposium statt. Alle ausbildungsrelevanten
Maßnahmen werden von den Doktoranden
entsprechend einem für die ILRS entwickelten Program of Study absolviert. Dies
sichert eine effiziente Qualifikation des wissenschaftlichen Nachwuchses in einem stringenten Zeitschema.
PhD Student
Title of the PhD Project
Supervisor
Van Thi Luu
Mutualistic microbial associates of Nicotiana attenuata
Ian T. Baldwin
Arne Weinhold
Microbial Interactions relevant for the fitness of Nicotiana
attenuata in the native environment
Ian T. Baldwin
Pol Alonso
Melolontha hippocastani gut specialized bacterial community:
function and structure elucidation
Wilhelm Boland
Huijuan Guo
Isolation, purification, and structural elucidation of active
compounds from the tissue of insect
Wilhelm Boland
Juliane Fischer
Post-translational modifications and secondary metabolism
of Aspergillus nidulans
Axel A. Brakhage
Christoph Heddergott
Secretome analysis / pathogenicity mechanisms of Arthroderma
benhamiae
Axel A. Brakhage
Andreas Thywißen
Molecular mechanisms of the interaction between Aspergillus
fumigatus and alveolar macrophages
Axel A. Brakhage
Shruthi Ramachandra
Host pathogen interactions of human-pathogenic yeast
Matthias Brock /
Bernhard Hube
Nayla Jbeily
Ceramide formation in host response to bacterial and fungal
infection and development of organ failure
Ralf A. Claus /
Konrad Reinhart
Felix Mingo
The role of Desulfitobacterium spp. in the global network of
O-demethylation in soil
Gabriele Diekert
Anita Mac Nelly
Expression control and biosynthesis of dehalogenating enzymes
from anaerobic soil bacteria in response to the interaction with
aerobic halogenating fungi
Gabriele Diekert
Continuation Ú
161
PhD Student
Supervisor
Susanne Brandes
Automated analysis of dynamic properties in biological systems
from image data
Marc Thilo Figge
Pegah Seddigh
Identifying the pulmonary phagocytic network of mice infected
with Aspergillus fumigatus
Matthias Gunzer
Fabian Horn
Prediction of gene regulatory networks involved in the differentiation, secondary metabolism and cross talk of Aspergillus nidulans
Reinhard Guthke
Himanshu Manchanda
Optimization of the influenza neuraminidase inhibitor therapy
by use of secondary bacterial infections
Reinhard Guthke
Sebastian Müller
Integrated genome-wide data analysis by ensemble learning
methods to understand infection processes
Reinhard Guthke
Thomas Wolf
Bioinformatic investigation and prediction of gene regulatory
elements in pathogenic fungi
Reinhard Guthke
Swantje Behnken
Genome mining of Gram-positive bacteria for secondary metabolites
Christian Hertweck
Katharina Graupner
Molecular basis of bacterial-mushroom interactions
Christian Hertweck
Srividhya Sundaram
Elucidation of complex biosynthetic pathways in microorganisms
Christian Hertweck
Alexander Funk
Regulation of polyketide synthase gene clusters in Aspergillus
nidulans
Uwe Horn
Daniel Braga de Lima
Investigation of polyketide biosynthesis in agaricomycetes
Uwe Horn /
Dirk Hoffmeister
Daniel Schwenk
Natural products from an unidentified homobasidiomycete which
control wood-deteriorating microorganisms
Uwe Horn /
Dirk Hoffmeister
Franziska Gerwien
Intracellular survival of Candida glabrata in phagocytes
Bernhard Hube
Sarah Höfs
Candida albicans – The pathway to epithelial damage
Bernhard Hube
Francois Mayer
Identification and characterization of infection-associated genes
in Candida albicans
Bernhard Hube
Volha Skrahina
The role of micronutrients during fungal infections
Bernhard Hube
Kristin Kroll
The hypoxic (low-oxygen) response of the pathogenic mould
Aspergillus fumigatus and its relevance to pathogenicity
Olaf Kniemeyer
Elena Shekhova
Investigation of the stress response system of A. fumigatus by
redox proteomics
Olaf Kniemeyer
162
PhD Student
Supervisor
Reyna Murry
Phosphatidylinositol (PI) signaling in basidiomycete S. commune
Erika Kothe
Dominik Senftleben
A surface hydrophobin in ectomycorrhiza interaction
Erika Kothe
Wei Li
The entrainment of the circadian clock by light-dark and temperature cycles: Functional analysis of components of involved signaling
pathways
Maria Mittag
Karthik Mohan
An insertional mutagenesis approach to understand temperature
entrainment of the circadian clock in Chlamydomonas reinhardtii
Maria Mittag
Christine Lembke
Induced resistance of phytoplankton against algicidal bacteria
Georg Pohnert
Michaela Mauß
Unraveling the (bio)chemical processes involved in phytoplankton-virus interactions
Georg Pohnert
Shayista Amin
Host Pathogen interactions, Aspergillus fumigatus and PET/CT
Technology
Hans Peter Saluz
Sara Mohebbi
Measuring interacellular pH during the apoptosis induction and
inhibition regarding the Aspergillus fumigatus infection
Hans Peter Saluz
Denise Buhlmann
Functional characterization of the soluble complement receptor
type II (CR2/CD21) in infection
Christine Skerka
Tina Enghardt
Complement evasion of human pathogenic microorganisms
Christine Skerka
Sandra Gusewski
Assessing DNA-binding specificity of MADS-domain transcription
factors
Günter Theißen
Khushboo Jetha
Interactions and regulatory networking of MADS-domain transcription factors in model plants (Arabidopsis thaliana)
Günter Theißen
Isabell Kopka
Complement regulation in kidney cells
Gunter Wolf
Qian Chen
The Role of complement in autoimmune disease
Peter F. Zipfel
Hannes Eberhardt
The role of CFHR proteins in human autoimmune diseases
Peter F. Zipfel
Selina Icke
Functional characterizations of variant forms of the human
complement regulator CFHR1
Peter F. Zipfel
Justus Linden
Immunevasion of the human pathogenic yeast Candida albicans
Peter F. Zipfel
Christiane Pilz
Complement evasion strategies of Candida albicans
Peter F. Zipfel
Sarbani Sarkar
Complement escape mechanisms of Streptococcus pneumoniae
Peter F. Zipfel
163
164
165
166
The Jena School of Microbial Communication (JSMC) was founded in September
2006 as an umbrella organization for three
pre-existing Research Training Groups, the
“International Leibniz Research School for
Microbial and Biomolecular Interactions”
(ILRS) of the HKI, the “International Max
Planck Research School“ (IMPRS) of the Max
Planck Institute for Chemical Ecology (MPI
CE) and the DFG Research Training Group
GRK 1257 “Alteration and Element Mobility
at Microbe-Mineral Interfaces” of the Friedrich Schiller University. Axel Brakhage, director of the HKI, founded the JSMC as its
main coordinator together with Wilhelm
Boland, Director of the Department of Bioorganic Chemistry at the MPI-CE, and Erika
Kothe, coordinator of the Research Training
Group GRK 1257. They act as the Executive
Board together with the JSMC management
team and two doctoral representatives. In
November 2007, the JSMC has been awarded
funding by the DFG excellence initiative as
the only project in Thuringia, and has been
able to secure continued funding for another
five years in November 2012 after a highly
competitive selection process. Within its
structured and interdisciplinary PhD programme, JSMC efficiently educates doctoral
researchers to become internationally competitive and versatile independent scientists,
aiming at a more detailed and holistic view
on microbial communication in all its facets.
This only becomes feasible through the inter-
Die “Jena School for Microbial Communication” (JSMC) wurde im September 2006
als Dachorganisation der bestehenden strukturierten Doktorandenausbildungen „International Leibniz Research School for Microbial and Biomolecular Interactions“ (ILRS)
des HKI, der „International Max Planck Research School“ (IMPRS) des Max-Planck-Instituts für chemische Ökologie (MPI CE)
sowie des DFG-Graduiertenkollegs 1257
“Alteration and Element Mobility at Microbe-Mineral Interfaces” (GRK 1257) der Friedrich-Schiller-Universität Jena gegründet.
HKI-Direktor Axel Brakhage zeichnete sich
damals gemeinsam mit Wilhelm Boland,
Direktor der Abteilung „Bioorganische Chemie“ des MPI-CE, und Erika Kothe, Sprecherin des GRK 1257, für die Gründung der
JSMC verantwortlich und fungiert seitdem
als deren Sprecher und Koordinator, mit W.
Boland und E. Kothe als Stellvertretern. Im
November 2007 wurde die JSMC als einziges Thüringer Projekt im Rahmen der DFG
Exzellenzinitiative bewilligt und finanziert,
im November 2012 folgte dann nach einem
hochkompetitiven Auswahlverfahren die
erfolgreiche Bewilligung zu einer Fortsetzungsfinanzierung für weitere 5 Jahre. Im
Zentrum der JSMC steht die strukturierte
und fächerübergreifende Ausbildung von
Doktoranden zu interdisziplinären und
international konkurrenzfähigen Wissenschaftlern, deren fachliche Ausrichtung zu
einem detaillierten und holistisch Verständnis der mikrobiellen Kommunikation führen
soll. Dies kann erst durch die fachübergreifende Integration der Disziplinen Biologie,
Chemie, Medizin, Geologie, Physik und Systembiologie erreicht werden und führt auf
diese Weise alle in Jena lokalisierten mikro-
HKI Research Report 2012 / 2013 Jena School for Microbial Communication
disciplinary integration of biology, chemistry,
medicine, geology, physics and systems biology and as such connects all research groups
in Jena working on microbiological topics.
JSMC doctoral researchers complete an ambitious training programme, which familiarises them with state-of-the-art methods
and research concepts while simultaneously
providing them with vital transferable skills,
an indispensable feature for their later career
paths. The most important elements of doctoral training at JSMC are:
•Cross-disciplinary PhD research projects
situated in the field of tension between basic
research and industrial application.
biologisch arbeitenden Gruppen zusammen.
Die JSMC-Doktoranden absolvieren ein ambitioniertes Lehrprogramm, in welchem sie
transdisziplinär mit den neuesten Methoden
und Forschungskonzepten vertraut gemacht
werden. Wichtigste Elemente dieser Doktoranden-Ausbildung sind:
•ein fächerübergreifender Ansatz der Forschungsprojekte, angesiedelt im Spannungsfeld zwischen Grundlagenforschung
bis hin zur industriellen Anwendung
•frühe Internationalisierung und Netzwerkbildung der Doktoranden durch Konferenzteilnahmen, Auslandsaufenthalte und Kooperationsprojekte
•umfangreiche Ausbildung in sogenannten „Transferable Skills“, wie z. B. wissen-
•Promotion of research stays abroad, as well
as of participation in international conferences and in collaborative projects, to give
doctoral researchers early international experience and to enhance their networking
skills.
•Comprehensive training in ‘transferable
skills’, e.g. scientific publication and presentation, acquiring research funds, etc.
•Close supervision by a team of mentors to
ensure that the students achieve the goals
of their PhD projects within three years.
schaftliches Publizieren und Präsentieren,
Einwerben von Fördergeldern durch gezielte Anträge, etc.
•intensive Betreuung durch Mentoren-Teams
für eine effiziente Umsetzung der Ziele der
Promotionsprojekte innerhalb von 3 Jahren.
Die erbrachten Leistungen werden in einem
Credit Point System erfasst und zertifiziert.
Damit werden hochqualifizierte Nachwuchswissenschaftler herangebildet, die in Jena
ein wissenschaftlich äußerst kreatives Netzwerk bilden welches international wettbewerbsfähige und richtungsweisende Forschungsleistungen hervorbringt.
2012 – 2013 verteidigten 31 der insgesamt
über 130 JSMC Doktoranden Ihre Doktorarbeit erfolgreich. Auf die offenen Promo-
Jena School for Microbial Communication HKI Research Report 2012 / 2013
167
Credit Points are awarded for each achievement and component of the JSMC PhD training, ensuring the conversion to highly qualified young academics, who build an extremely
creative scientific network in Jena conducting
internationally competitive and seminal research on microbial communication.
In 2012 – 2013, 31 of currently over 130 JSMC
doctoral students successfully defended their
theses. Over 7000 applicants from around 90
different nations worldwide competed in total
for available PhD positions, 24 of which were
ultimately selected after a rigorous selection
procedure and multiple recruiting events as
new JSMC PhD students. Dr. Ákos T. Kovács
tionsstellen der JSMC bewarben sich insgesamt über 7000 Kandidaten aus rund 90
verschiedenen Nationen, von denen nach rigorosen Selektionsverfahren und mehrtägigen Rekrutierungstreffen schließlich die 24
am besten geeigneten in die JSMC als neue
Doktoranden aufgenommen wurden. Dr.
Ákos T. Kovács wurde als neuer JSMC Junior
Gruppenleiter mit Schwerpunkt „terrestrische Biofilme“ ausgewählt, und ist mit seiner
Forschungsgruppe im neu renovierten JSMC
Gebäude in der Neugasse 23 tätig welches
168
has been selected as new JSMC junior research group leader, working on terrestrial
biofilms at the renovated building in Neugasse 23, which now also houses the JSMC
management. Katja Präfcke succeeded Ulrike
Schleier as JSMC management assistant in
November 2012, and Dr. Jan Büllesbach succeeded Dr. Carsten Thoms as scientific manager of the JSMC in November 2013. Silvio
Waschina, Zerrin Üzüm, Alessio Garrone and
Juliane Fischer succeeded the previous JSMC
doctoral student speakers Markus Bohnert,
Martin Föge and Daniel Schwenk in June
2013.
nun auch die JSMC Geschäftsführung beherbergt. Katja Präfcke löste Ulrike Schleier als
Assistentin der JSMC Geschäftsführung im
November 2012 ab, und Dr. Jan Büllesbach
trat im November 2013 die Nachfolge von Dr.
Carsten Thoms als wissenschaftlicher Geschäftsführer der JSMC an. Silvio Waschina,
Zerrin Üzüm, Alessio Garrone und Juliane
Fischer übernahmen im Juni 2013 die Funktion als JSMC Doktorandensprecher von Ihren Vorgängern Markus Bohnert, Martin
Föge and Daniel Schwenk.
HKI Research Report 2012 / 2013 Jena School for Microbial Communication
Cross-sectional Unit
Jena Microbial Resource Collection
169
Cross-sectional Unit Jena Microbial Resource Collection
The Jena Microbial Resource Collection
(JMRC) was established in October 2010 as
a joint institution of the HKI and the Friedrich-Schiller-University Jena and represents
a cross-sectional unit within the HKI. The
JMRC manages a collection of living microorganisms comprising about 15,000 fungal
and 30,000 bacterial strains. This collection
functions as a microbial diversity bank in
which the strains serve as microbial resources for academic and industrial research into
natural products and infection biology. As regards research, the staff of the JMRC headed
by Kerstin Voigt put their focus on pathogenesis, host-pathogen interactions and biodiversity of zygomycetes. Hereby, the main
focus is on the evolution of pathogenicity
in human pathogenic zygomycetes, namely
species of the genera Lichtheimia, Rhizopus
and Conidiobolus. Our aim is to decode the
mechanisms of infections using evolutionary, cell and infection biology and physiology
in combination with Omics-approaches and
complex infection models.
Head:
Die Jena Microbial Resource Collection
(JMRC) wurde im Oktober 2010 als gemeinsame Einrichtung des HKI und der Friedrich-Schiller-Universität Jena gegründet
und repräsentiert eine Querschnittseinrichtung innerhalb des HKI. Die JMRC unterhält
eine Sammlung von lebenden Mikroorganismen, die aus 15.000 Pilzstämmen sowie
30.000 Bakterienstämmen besteht. Diese
Sammlung fungiert als mikrobielle Diversitätsbank, bei der die Stämme als mikrobielle Ressourcen für die akademische und
industrielle Forschung an Naturstoffen und
Infektionsbiologie dienen. In der Forschung
170
widmen sich die Mitarbeiter der JMRC unter der Leitung von PD Dr. Kerstin Voigt der
Pathogenese, der Wirt-Pathogen Interaktion
und der Biodiversität von Zygomyceten. Der
Fokus liegt hierbei auf der Evolution der Pathogenität human-pathogener Zygomyceten,
namentlich Arten der Gattungen Lichtheimia, Rhizopus und Conidiobolus. Unser Ziel
ist es, die Mechanismen der Infektion mit
Hilfe von evolutions-, zell- und infektionsbiologischen sowie physiologischen Ansätzen in
Kombination mit Omics-Verfahren und komplexen Infektionsmodellen zu entschlüsseln.
HKI Research Report 2012 / 2013 Cross-sectional Unit Jena Microbial Resource Collection
We are especially interested in the interaction of Lichtheimia with the host’s immune
system in disseminated infections in order
to unravel the pathogenicity mechanisms
and to define targets for prevention or protection against the disease. In this context,
projects address the microscopic observation
of the interaction of Lichtheimia with murine
and human phagocytes (macrophages), the
determination of the genome, transcriptome
and proteome of Lichtheimia corymbifera, the
establishment of a mouse model and the species recognition of Lichtheimia and Rhizopus,
which are the main causative agents of zygo-
mycoses, based on molecular systematics approaches to aid rapid diagnosis in comparative conjunction to the diversity of zygomycetes.
Wir sind im Besonderen an der Interaktion
von Lichtheimia mit dem Immunsystem des
Wirts in disseminierenden Infektionen interessiert, um die Pathogenitätsmechanismen
im Hinblick auf die Definition von Targets
für die Prävention von und Schutz gegen Infektionen mit Zygomyceten zu untersuchen.
In diesem Kontext befassen sich unsere Projekte mit der mikroskopischen Beobachtung
des Zusammenspiels von Lichtheimia mit
murinen und humanen Phagozyten (Makrophagen), der Entschlüsselung des Genoms,
Transkriptoms und Proteoms von Lichtheimia corymbifera, der Etablierung eines
Maus-Modells sowie der Artbildung inner-
halb der beiden Haupterreger von Mucormykosen Lichtheimia und Rhizopus mit Hilfe
von molekular-systematischen Ansätzen mit
dem Ziel der schnellen Diagnose in vergleichender Konjunktion mit der Diversität von
Zygomyceten.
In 2012 to 2013, our research was supported by
the Jena School for Microbial Communication
(JSMC), the Friedrich Schiller University Jena
and the Deutsche Forschungsgemeinschaft
(DFG). Projects are integrated into the DFG
Collaborative Research Center / Transregio
CRC124 “Pathogenic fungi and their human
host: Networks of interaction – FungiNet”.
Die Forschungsarbeit der JMRC wurde in den
Jahren 2012 und 2013 durch die Jena School
for Microbial Communication (JSMC), die
Friedrich-Schiller-Universität Jena und die
Deutsche Forschungsgemeinschaft (DFG) unterstützt. Die JMRC ist am SFB /TR 124 “Pathogenic fungi and their human host: Networks of interaction – FungiNet” beteiligt.
Cross-sectional Unit Jena Microbial Resource Collection HKI Research Report 2012 / 2013
171
Scientific Projects
Figure 1
Schematic diagram of the phagocytosis assay.
1
The interaction of the human pathogenic zygomycete Lichtheimia with
macrophages
Group Leader: Kerstin Voigt
The number of cases of zygomycosis has
increased over the past few years. Lichtheimia species are the 2nd and 3rd most common causative agents for zygomycosis in
Europe and USA respectively. The main route
of infection is via the respiratory tract where
alveolar macrophages (AM) act as the immune system’s first line of defense. A literature
review was carried out focusing on the interaction of pathogenic zygomycetes with
innate immune cells in order to determine
the current knowledge of the interplay at the
cellular level between both counterparts. In
order to assess the phagocytic efficiency of
murine alveolar macrophages (MH-S) (provided by Hans-Martin Dahse, Department
Infection Biology), co-incubation of MH-S
172
and two strains of L. corymbifera; FSU 9682
(fully virulent) and 10164 (attenuated) was
carried out for an hour with MOI of 5 and
fluorescence images were taken for the calculation Phagocytic index (PI). Three conditions; resting, pre-swollen, and opsonised
spores were taken for the analysis (Fig. 1).
The major findings were: In contrast to the
other conditions, FSU 9682 and 10161 showed
significant difference in PI in opsonisation.
Quantification of C3b (a by-product of C3
which plays a central role in complement
system) was followed on two strains via
Fluoresence-Activiated Cell Sorting (FACS).
Interestingly, complement component C3b
was more abundant on FSU 9682 than that of
FSU 10164 despite of the similarity in their
sizes. These data show for the first time the
PI of L. corymbifera by MH-S cells which was
never measured previously. We also demonstrated that L. corymbifera binds to main components of the complement system.
In addition to the manual analysis, automated image analysis was also carried out
in collaboration with Kaswara Kraibooj
and Thilo Figge (Research Group Applied
Systems Biology).
As a perspective we aim to study the interaction of AM and L. corymbifera in more detail
at the transcriptomic level using RNA-seq
analyses. Various methods such as investigation of binding to complement regulators
like factor H, killing assay and transcriptomic analysis of L. corymbifera will be used
to extend the current knowledge of pathogensis of L. corymbifera. For transcriptomic analysis, we will be using primary macrophages
provided by Kathleen Wagner and Marie von
Lilienfeld-Toal from Associated Group Infections in Hematology/Oncology (IHO).
This project is funded by the JSMC and performed in close collaboration with Axel A.
Brakhage (MAM) and Peter F. Zipfel (IB).
2
De novo sequenced genome from
Lichtheimia corymbifera, an ancient
human pathogenic basal lineage
fungus causing mucormycoses and
its functional genomics in the context of evolution of pathogenicity of
zygomycetes
Group Leaders: Kerstin Voigt, Axel Brakhage
Mucoralean fungi are able to cause severe
infections in animals and humans. Although
these mucormycoses are regarded as uncommon infections the number of cases increased during the last decades. The majority of mucormycotic infections is caused by
Rhizopus, Mucor or Lichtheimia species, comprising 70 to 80 % of all clinical cases of zygomycoses. While the genera Rhizopus and
Mucor are closely related mucoralean fungi,
Lichtheimia represents a more basal group of
mucoralean fungi which also includes other
pathogenic species such as Rhizomucor pusillus. In addition, the three genera show differences in growth kinetics and morphology
(e.g. dimorphism), thermotolerance and resistance to antifungals.
The genomes of Rhizopus oryzae and Mucor
circinelloides were already sequenced, whereas no genomic data of more basal mucoralean
pathogens was available. Here we present
first insights into the genome L. corymbifera.
Based on 454 and Illumina data contigs with
a total length of 33.6 MB were assembled. To
aid prediction of protein-coding genes, RNAseq analyses were performed for three different growth conditions (standard growth,
hypoxia, iron depletion). The use of different
conditions should ensure a higher number of
expressed genes, thereby allowing evidencebased gene predictions for many gene models. Prediction of protein-coding genes was
performed using AUGUSTUS, resulting in
12,379 predicted genes. The L. corymbifera
genome provides first insights into the basal
mucoralean pathogens and will contribute to
the understanding of these pathogens at the
level of genome architecture and its evolution.
L. corymbifera represents the most basal
mucoralean pathogen sequenced so far. Comparison of the L. corymbifera genome to other
mucoralean genomes revealed that less than
40 % of the gene families are shared with
all other mucoralean genomes representing
53.7 % of L. corymbifera genes. Conserved
regions, in terms of gene order, between mucoralean genomes were examined and a total
of 230 regions with a minimum of 3 conserved genes of L. corymbifera were found
that were present in at least one of the other
genomes. These regions covered only 7.6 %
of the L. corymbifera genome reflecting the
high dissimilarity between the mucoralean
genomes.
Comparable to the situation in R. oryzae a
high amount of duplicated genes was found
in the L. corymbifera genome. In addition,
several Lichtheimia-specific gene expansions
were found including transcription factors
and transporters. Interestingly, gene expansions often overlapped with tandem duplica-
173
tions of the genes. Thus retention of tandem
duplicated genes could be an explanation
for the high incidence of mostly speciesspecific gene duplications in mucoralean
genomes. Expression analyses of duplicated
genes revealed that the different copies
of the genes were differently regulated in
many cases. Thus, gene duplication seems
to result in functionally diverse genes. Functional genomics will be performed using the
genomes of R. oryzae and M. circinelloides,
both causing mucormycoses in immunocompromised patients in conjunction with
the genome of Condiobolus coronatus, which
causes entomophthoromycosis in immuncompetent patients. The genome of C. coronatus was determined in collaboration with
the Leibniz Institute for Age Research – Fritz
Lipmann Institute (FLI, Matthias Platzer).
This project is in part funded by FungiNet and
performed in close collaboration with Olaf
Kniemeyer and Axel A. Brakhage (MAM).
3
Development of a pulmonary
mouse infection model for
Lichtheimia spp.
Group Leaders: Kerstin Voigt,
Ilse D. Jacobsen
Although the number of mucormycosis cases
has increased during the last decades, little
is known about the pathogenesis of mucoralean fungi. While Rhizopus oryzae represents
the most-common cause of mucormycosis
worldwide, the frequency of Lichtheimia infections differs between geographic regions.
Lichtheimia species represent the secondmost common cause of mucormycosis in
Europe but play only a minor role in the USA.
The primary route of infection is believed to
be the inhalation of spores, and pulmonary
infection is one of the most-common presentations. Patients with diabetes mellitus and
immunocompromised patients following
transplantation represent major risk groups.
Although different infection models have
been used to study Lichtheimia infections, a
174
standardised model has not been established
yet. Mostly spores of Lichtheimia were applied via intravenous infection which does
not represent the natural route of infection.
Based on intranasal infection models for
R. oryzae, a model for L. corymbifera was developed and used to test the effect of different risk factors.
No clinical disease was observed after infection with L. corymbifera spores in healthy
mice. However, immunosuppression with
cortisone acetate resulted in development of
disease and mortality. The infection was associated with massive tissue destruction in
lungs and the kidneys. Additionally, also dissemination into liver, spleen and the brain
was observed. Viable spores were found to be
present in lungs of immunocompetent and
immunocompromised surviving animals 28
days post infection.
Although described as a major risk factor for
mucormycoses, diabetic mice were not susceptible for L. corymbifera infection. Thus,
Lichtheimia species seem to differ from other
mucoralean pathogens, e.g. R. oryzae in risk
factors.
4
Species recognition and diagnostics
of human pathogenic zygomycetes
Group Leaders: Grit Walther, Kerstin Voigt
Taxonomy and diagnostics of the Rhizopus
arrhizus (syn. R. oryzae) species complex
Rhizopus arrhizus (syn. R. oryzae) causes by
far the most mucormycoses worldwide. On
the other hand the species has been used
for centuries for the production of fermented
food in Asia. Recently, molecular phylogenetic analyses based on four markers revealed
the existence of two phylogenetic species,
R. arrhizus s. s. and R. delemar differing by
the production of lactic acid or fumaric-malic
acid, respectively. The phylogenetic species
R. arrhizus s. s. contains two copies of Lactate dehydrogenase, LDHA and LDHB, while
R. delemar has only a single copy of this
gene (LDHB). However, the genetic distance
between R. arrhizus s. s. and R. delemar is
extremely low compared to distances among
other species in the genus Rhizopus. In addition, the formation of zygospores between
strains of R. arrhizus s. s. and R. delemar as
an indicatior for sexual reproduction have
been reported. In cooperation with Somayeh
Dolatabadi and Sybren de Hoog from the CBSKNAW Fungal Biodiverstiy Centre (Utrecht,
The Netherlands) we currently try to answer
the questions if R. arrhizus s. s. and R. delemar should be treated as single or as separate
species and if they differ in their pathogenic
potential. Therefore we study a set of about 80
strains belonging two both putative species
and originating from different sources (clinical, environmental, food). We performed a
comprehensive molecular phylogenetic study
based on five different markers, AFLPs and
MALDI-TOF MS analyses. In order to assess
biological species boundaries we performed
mating tests.
Preliminary results revealed that two out
of five phylogenetic gene trees show polyphylies of R. arrhizus and R. delemar within
the R. oryzae complex suggesting a single
species. Currently the sequencing is repeated in order to exclude that this deviating position of the strains in the trees is only
caused by the confusion of strain numbers.
Mating tests were successful only in two
pairings, one of which represents a pairing between R. arrhizus sensu stricto and
R. delemar, a fact that is furthermore supporting the hypothesis of a single species.
other genera in the fungal kingdom. Recent
molecular phylogenetic studies based only
on a small number of strains revealed that
the morphologically defined species Syncephalastrum racemosum consists of at least
two distinct groups that might represent
independent species. In cooperation with
numerous partners we are currently studying the Syncephalastrum racemosum species
complex molecular phylogenetically, morphologically, physiologically and by mating
tests for biological species recognition. Central questions are: how many species to this
complex? Do they differ in their pathogenic
potential and in their susceptibility for antifungals? And how can they be diagnosed?
Preliminary results of the molecular phylogenetic study revealed a surprisingly high biodiversity. To date we recognized five phylogenetic species that differ neither in their
morphology nor in their susceptibility profiles. Mating tests to recognise the biological
species boundaries were still not successful.
The project is performed in close collaboration with the National Reference Center for
Invasive Mycoses (NRZMyk) and is funded
by grant of the DFG to Dr. Grit Walther.
References
Grützmann K1, Szafranski K, Pohl M,
Voigt K, Petzold A, Schuster S (2014)
Fungal alternative splicing is associated with multicellular complexity
and virulence: a genome-wide multispecies study. DNA Res 21, 27-39.
Schwartze VU, Hoffmann K, Nyilasi I,
Papp T, Vágvölgyi C, de Hoog S, Voigt
K, Jacobsen ID (2012) Lichtheimia species exhibit differences in virulence
potential. PLoS One 7, e40908.
Schrödl W, Heydel T, Schwartze VU,
Hoffmann K, Walther G, AlastrueyIzquierdo A, Rodriguez-Tudela JL,
Olias P, Jacobsen ID, de Hoog GS,
Voigt K (2012) Direct analysis and
identification of opportunistic Lichtheimia species by Matrix Assisted
Laser Desorption Ionization (MALDI)
- Time-Of-Flight (TOF) analyzermediated mass spectrometry. J Clin
Schoch CL, Seifert KA, Huhndorf S,
Robert V, Spouge JL, Levesque CA,
Chen W; Bolchacova E, Voigt K and
the Fungal Barcoding Consortium
(2012) Nuclear ribosomal internal
transcribed spacer (ITS) region as a
universal DNA barcode marker for
Fungi. Proc Natl Acad Sci U S A 109,
6241-6246.
Hoffmann K, Pawłowska J, Walther
G, Wrzosek M, Hoog GS de, Benny
GL, Kirk PM, Voigt K (2013) The family
structure of the Mucorales: a synoptic
revision based on comprehensive
multigene-genealogies. Persoonia
30, 57–76.
Taxonomy and diagnostics of the Syncephalastrum racemosum species complex
Syncephalastrum racemosum is a thermotolerant saprotropic fungus belonging to the
order Mucorales with a main distribution
in the tropics and subtropics. It is able to
cause infections in humans with impaired
immunity but these mucormycoses are rare
and restricted to its main distribution area.
The species is also of interest because it possesses two distinctly deviating types of ITS
sequences which are known only from two
175
Group members
Collaborations
Head
Phone
+49 3641 532-1395
Fax
+49 3641 532-2395
[email protected]
Prof. Dr. Erika Kothe
Ph.D. Students
Volker Schwartze
Hea Reung Park
Fritz Lipmann Institute, Jena, Germany
Max Planck Institute for Chemical Ecology, Jena
Freie Universität Berlin, Germany
Research Assistents
Christiane Weigel
Caroline Semm
Leibniz Institute of Freshwater Ecology and
Inland Fisheries (IGB), Berlin, Germany
External funding
Centre for Genomic Regulation (CRG),
Barcelona, Spain
SFB/TR FungiNet
Project Z01: Die Rolle der Regulation putativer
Pathogenitäts-assoziierter Gene in der Virulenz
des humanpathogenen Mucormykose-Erregers Lichtheimia corymbifera: Vergleichende
RNAseq-Expressionsanalyse von attenuiertem
versus virulentem Stamm
Kerstin Voigt
Universitat Pompeu Fabra (UPF),
Barcelona, Spain
Centre Nacional d'Anàlisi Genòmica (CNAG),
Barcelona, Spain
Laboratório Nacional de Computação Científica
(LNCC), Rio de Janeiro, Brazil
Ruhr University Bochum, Germany
Schwartze VU, Winter S, Shelest E, MarcetHouben M, Horn F, Wehner S, Linde J, Valiante
V, Sammeth M, Riege K, Nowrousian M,
Kaerger K, Jacobsen ID, Marz M, Brakhage AA,
Gabaldón T, Böcker S, Voigt K (2014) Gene
expansion shapes genome architecture in the
human pathogen Lichtheimia corymbifera: an
evolutionary genomics analysis in the ancient
terrestrial Mucorales (Mucoromycotina). PLoS
Genetics, in press.
Institut Pasteur, Paris, France
Schoch CL, Robbertse B, Robert V, …, Voigt
K, …, Federhen S (2014) Finding needles in
haystacks: linking scientific names, reference
specimens. Database (Oxford), 1-21.
Leibniz Institute DSMZ-German Collection
of Microorganisms and Cell Cultures Braunschweig, Germany
University of Szeged, Hungary
Institute of Animal Physiology and Genetics,
Czech Academy of Sciences,
Prague, Czech Republic
Botanische Staatssammlungen München,
Germany
CBS Utrecht, The Netherlands
Hoffmann K, Pawlowska J, Walther G, Wrzosek
M, de Hoog GS, Benny GL, Kirk PM, Voigt K
(2013) The family structure of the Mucorales:
a synoptic revision based on comprehensive
multigene-genealogies. Persoonia 30: 57–76.
Federal Rural University of Pernambuco –
Academic Unit of Serra Talhada, Pernambuco,
Brazil
NIH/NLM/NCBI Bethesda, Maryland, USA
Schrödl W, Heydel T, Schwartze VU, Hoffmann
K, Walther G, Alastruey-Izquierdo A, RodriguezTudela JL, Olias P, Jacobsen ID, de Hoog GS,
Voigt K (2012) Direct analysis and identification
of opportunistic Lichtheimia species by Matrix
Assisted Laser Desorption Ionization (MALDI) Time-Of-Flight (TOF) analyzer-mediated mass
spectrometry. J Clin Microbiol 50, 419-427.
Royal Botanic Gardens Kew, Mycology section,
UK
University of Gainsville Florida, USA
Schoch CL, Seifert KA, Huhndorf S, Robert V,
Spouge JL, Levesque CA, Chen W; Bolchacova E,
Voigt K and the Fungal Barcoding Consortium
(2012) Nuclear ribosomal internal transcribed
spacer (ITS) region as a universal DNA barcode
marker for Fungi. Proc Natl Acad Sci USA 109,
6241-6246.
176
Collaborative Research Centre / Transregio 124
FungiNet
177
Collaborative Research Centre / Transregio 124 – FungiNet
178
Pathogenic fungi and their human host:
Networks of interaction
The Collaborative Research Centre (CRC) /
Transregio 124 “FungiNet” started its work,
funded by the German Research Foundation
(DFG) in October 2013. It brings together
natural scientists, clinicians and bioinformaticians from Jena and Würzburg, who will
work in 18 different research projects. They
join forces to study the two major pathogenic
fungi Candida albicans and Aspergillus fumigatus and their interaction with the human
host. The aim of FungiNet is to apply a sytems
biological approach to decipher the complex
pathobiology of the two human-pathogenic
fungi and the response of the host’s immune
system. This approach will uncover novel
regulatory and metabolic pathways that are
important in host-pathogen interactions and
may help to redefine the basic concepts of
virulence and pathogenicity of human-pathogenic fungi. The aims of FungiNet are:
•to identify pathogenic determinants specific for A. fumigatus or C. albicans
•to investigate the roles of epithelial barriers, the innate and adaptive immune system for the pathogenesis of fungal infections
Pathogene Pilze und ihr menschlicher
Wirt: Netzwerke der Interaktion
Der SFB / Transregio 124 „FungiNet“, der
von der Deutschen Forschungsgemeinschaft
(DFG) gefördert wird, nahm im Oktober 2013
seine Arbeit auf. In 18 Teilprojekten arbeiten Naturwissenschaftler, Mediziner und
Bioinformatiker aus Jena und Würzburg zusammen daran, die beiden bedeutenden human-pathogenen Pilze Aspergillus fumigatus
und Candida albicans zu untersuchen und
die Interaktionen mit dem menschlichen
Wirt zu entschlüsseln. Gemeinsames Ziel
ist es, die komplexen Mechanismen der Pil-
zinfektionen zu verstehen und mittels eines
systembiologischen Ansatzes ein „virtuelles
Infektionsmodell” zu generieren, das als Ausgangspunkt zur Entwicklung effektiver Präventions- und Therapiestrategien dient.
FungiNet will
•Spezifische Pathogenitätsfaktoren für A.
fumigatus und C. albicans identifizieren
•Die Rolle von Gewebeschranken sowie
des angeborenen und adaptiven Immunsystems bei der Pathogenese pilzlicher Infektionen untersuchen
•Die komplexen Interaktionsmechanismen während des Infektionsprozesses
HKI Research Report 2012 / 2013 Collaborative Research Centre / Transregio 124 – FungiNet
•to elucidate the complex mechanisms of
interactions during the infection process
and to develop virtual models and tools,
which can describe these interplays
•to use the gained knowledge for the development of therapeutic approaches
•Candida albicans: from commensal to
pathogen.
Additionally, the CRC comprises central projects which provide services to the involved
scientists. Axel Brakhage (HKI) is the speaker of FungiNet.
FungiNet is divided in three interlinked research areas, namely
•Aspergillus fumigatus: from environmental
microorganisms to pathogen,
•Bioinformatics / Computational systems
biology of infection; creation of a „virtual
infection model“
entschlüsseln und virtuelle Modelle und
Werkzeuge entwickeln, die dieses Zusammenspiel beschreiben
•Das erworbene Wissen dafür einsetzen,
neue Therapiestrategien für Pilzinfektionen zu entwickeln
•Candida albicans: vom Kommensalen zum
Pathogen
Außerdem beinhaltet der SFB / Transregio
noch Zentralprojekte, die Services für die beteiligten Wissenschaftler liefern. Axel Brakhage ist der Sprecher des SFB / Transregio.
Dazu kommen in FungiNet drei miteinander
verknüpfte Ansätze zum Einsatz:
•Aspergillus fumigatus: vom allgegenwärtigen Mikroorgansimus zum Pathogen
•Bioinformatik / Systembiologie der Infektion: Erstellung eines „virtuellen Infektionsmodells”
Collaborative Research Centre / Transregio 124 – FungiNet HKI Research Report 2012 / 2013
179
180
Associated Group
Infections in Hematology and Oncology
181
Associated Group Infections in Hematology and Oncology
The Associated Group Infections in Hematology and Oncology (IHO) was founded in
summer 2013 with the appointment of Marie
von Lilienfeld-Toal and consists of clinical
scientists based at the University Hospital
Jena and of natural scientists in the laboratory based within the HKI. Our research
interest comprises all aspects of infectious
complications in patients with malignancies
with a special focus on atypical infections
like invasive fungal infections. We form a
link between the clinical departments and
institutes of the medical faculty on one side
and the HKI on the other side which enables
interdisciplinary research of clinically relevant questions (Fig. 1).
Infections are the leading cause of treatment-related mortality in patients with
malignant diseases. This is commonly believed to be due to a profound immunosuppression, which makes patients susceptible
to opportunistic infections. Good examples
for such opportunistic infections are invasive fungal infections, which almost never
occur in healthy individuals and which are
Head:
Prof. Dr. Marie von Lilienfeld-Toal
182
Die assoziierte Gruppe Infektionen in der
Hämatologie und Onkologie (IHO) entstand
im Sommer 2013 mit der Berufung von
Marie von Lilienfeld-Toal und besteht aus
wissenschaftlich tätigen Ärzten im Universitätsklinikum Jena-Lobeda sowie Naturwissenschaftlern in den Laborräumen im HKI.
Wir beschäftigen uns mit allen Gesichtspunkten von Infektionen bei Patienten mit
malignen Systemerkrankungen mit einem
speziellen Schwerpunkt im Bereich atypischer Infektionen wie zum Beispiel invasiver Pilzerkrankungen. Mit dem Standort
Universitätsklinikum Jena-Lobeda auf der
einen Seite und dem Standort Hans-Knöll-Institut auf der anderen Seite bilden wir eine
Verbindung zwischen klinischen und theoretischen Instituten und ermöglichen so die
interdisziplinäre Bearbeitung klinisch relevanter Fragestellungen (Fig. 1).
Infektionen sind die wichtigste Ursache für
therapieassoziierte Mortalität bei Patienten
mit Krebserkrankungen. Man geht davon
aus, dass dies vor allem der ausgeprägten
Immunsuppression, die die Patienten für
opportunistische Infektionen prädisponiert,
geschuldet ist. Invasive Pilzinfektionen sind
HKI Research Report 2012 / 2013 Associated Group Infections in Hematology and Oncology
exceptionally difficult to diagnose. In addition, treatment of invasive fungal infections
is often toxic and not very successful. For
these reasons, invasive fungal infections in
patients with hematological malignancies
have a very high mortality rate. Besides opportunistic infections, cancer patients are
at risk of common infections, which often
take a more severe clinical course than in
healthy individuals. This is believed to be
the case for infections with respiratory viruses like influenza or adenovirus. We aim
at understanding the pathogenesis and clini-
ein gutes Beispiel für solche opportunistischen Infektionen, da sie bei gesunden Menschen fast nie vorkommen. Sie sind schwierig
zu diagnostizieren und erfordern eine relativ
toxische Therapie, die oft nicht ausreichend
wirkt. Aus diesen Gründen sind invasive Pilzinfektionen mit einer hohen Mortalitätsrate
assoziiert. Neben dem Risiko für opportunistische Infektionen besteht außerdem das
Risiko für „normale“ Infektionen, die bei
Krebspatienten oft schwerer verlaufen als
bei Gesunden. Infektionen mit respiratorischen Viren wie Influenza oder Adenovirus
sind Beispiele, für die schwerere Verläufe bei
cal course of some of these infections with
the goal to improve diagnostics and clinical
treatment of these patients.
In 2013 our research was supported by the
Federal Ministry of Education and Research
(BMBF) within the Center for Sepsis Control
and Care (CSCC) and by the Interdisciplinary Centre for Clinical Research (IZKF) of
the Medical Faculty of Friedrich Schiller
University Jena.
Krebspatienten beschrieben werden. Unser
Ziel ist es, das Verständnis für die Entstehung
dieser Infektionen und ihren klinischen Verlauf zu vertiefen, um so die Diagnostik und
Therapie verbessern zu können.
2013 erhielt unsere Gruppe finanzielle Unterstützung vom Bundesministerium für Bildung und Forschung (BMBF) innerhalb des
Verbundes Center for Sepsis Care and Control
(CSCC)und vom Interdisziplinären Zentrum
für Klinische Forschung (IZKF) der medizinischen Fakultät der Friedrich-Schiller-Universität Jena.
Associated Group Infections in Hematology and Oncology HKI Research Report 2012 / 2013
183
Scientific Projects
IHO takes part in a number of ongoing multicentre clinical trials which will not be
discussed in detail. The following projects
have been developed over the last six months
within our group in Jena.
1
Establishment of registry of infections in patients with hematological
malignancies
Group Leader: Tobias Rachow
Prior research has been partly based on
a large registry of clinical cases from the
university hospital at Bonn (Schwab et al.
2014, Hahn-Ast et al. 2010). We are now in
the process of establishing a registry of infections in patients with hematological malignancies treated in the University Hospital
Jena. A retrospective documentation of the
years 2012 and 2013 is the foundation for the
prospective documentation of the following
years. We document the clinical courses of
patients receiving myelosuppressive chemotherapy for acute leukaemia with a special
focus on all aspects of infectious complications including the respective diagnostic and
therapeutic interventions. We will enlarge
the registry to include additional types of
malignant diseases apart from leukaemia in
the future. In particular, we focus on the following aspects:
a) Efficacy of antimicrobial prophylaxis
and therapy
Guidelines on the management of infectious complications in cancer patients are
mostly based on the results of prospective
clinical trials. However, for better comparability these trials include only very selected
patient populations. Thus, the results often
do not reflect clinical reality as they cannot
184
be transferred from the homogenous trial
population (often only one disease included, no co-morbidities, median age low, very
specified co-medication allowed) to the more
heterogeneous patient population (different diseases, many co-morbidities, median
age higher, large variety of co-medication)
inhabiting the wards. It is therefore necessary to acquire knowledge about the clinical
course and the treatment efficacy of infections in clinical reality by descriptive analysis of patient cohorts (Schwab et al. 2014).
In cooperation with the University Hospital
Bonn (Karin Mayer), we conducted a first
analysis of 286 patients with acute myeloid
leukaemia with regard to the efficacy of two
different regimens of antibiotic prophylaxis
and found, in contrast to published trials, an
equivalent efficacy of the newer drugs (fluorochinolones) and the older drug (cotrimoxazole). Current efforts will lead to enlargement
of this patient cohort and additional aspects
like empirical antimicrobial treatment in the
first or second line will be analysed.
b) Incidence, risk factors and clinical
course of invasive fungal infections
As part of the registry, patients with suspected or diagnosed invasive fungal infections
are also documented. Diagnosis of Aspergillus infection in these patients is based on the
detection of the galactomannan-antigen specific for Aspergillus spp. in the serum or in
bronchial-lavage fluid. Importantly, for these
patients we collect left-over serum samples
from test tubes collected for detection of
the Aspergillus antigen. Over the past four
months, more than 400 samples have been
collected and stored. This allows the testing of novel diagnostic tools on serum with
known results in the conventional diagnostic
methods. Here, we collaborate in particular
with the Department Molecular and Applied
IHO Infec?ons in Haematology and Oncology Respiratory Viruses in Healthy Donors Sample Storage Hans Knöll Ins*tute Associated Research Group IHO University Hospital Jena Research Group IHO Sample and Data Collec*on PCR Registry Infec?ons in Haematology and Oncology Sample Storage Clinical Trials C. diff, CMV, ESBL Immune response against Aspergillus Cell Culture, Flow Cytometry, Cytokine Response Infec*on Biology Microbiology. In addition, we are able to provide fresh samples of patients with proven
fungal disease or with specified immune defects to other research groups, in particular
to Fungal Septomics and the Jena Microbial
Resource Collection (JMRC).
c) Epidemiology, diagnosis and treatment of atypical mycobacteria
Infections with atypical mycobacteria such
as Mycobacterium avium intracellulare are
rare events in patients with hematological
malignancies. Nonetheless, they do occur
and little is known about their relevance in
terms of morbidity and mortality. Most treatment recommendations have been deduced
from patients with HIV as these patients are
believed to be similarly immunosuppressed.
However, it has been recognised that patients
with hematological malignancies might differ in their susceptibility to infection and in
their clinical course, but there are still not
enough epidemiological data to draw any
conclusions. We set out to document patient
records with proven or suspected infection
with atypical mycobacteria and describe
the severity of the infection as well as the
response to treatment (if administered). This
project is being conducted in collaboration
with B. Hermann from the Institute of Medical Microbiology, University Hospital Jena.
Figure 1
Depiction of the Research Group IHO
and its collaborations within the HKI.
185
References
Schwab KS, Hahn-Ast C, Heinz WJ,
Germing U, Egerer G, Glasmacher A,
Leyendecker C, Marklein G, Nellessen
CM, Brossart P, von Lilienfeld-Toal M
(2014) Tigecycline in febrile neutropenic patients with haematological
malignancies: a retrospective case
documentation in four university
hospitals. Infection 42, 97-104.
Hahn-Ast C, Glasmacher A, Mückter
S, Schmitz A, Kraemer A, Marklein
G, Brossart P, von Lilienfeld-Toal M
(2010) Overall survival and fungal
infection-related mortality in patients with invasive fungal infection
and neutropenia after myelosuppressive chemotherapy in a tertiary care
centre from 1995 to 2006. J Antimicrob Chemother 65, 761-768.
von Lilienfeld-Toal M, Frank S,
Leyendecker C, Feyler S, Jarmin S,
Morgan R, Glasmacher A, Märten A,
Schmidt-Wolf IG, Brossart P, Cook G
(2010) Reduced immune effector cell
NKG2D expression and increased
levels of soluble NKG2D ligands in
multiple myeloma may not be causally linked. Cancer Immunol Immunothe. 59, 829-839
2
Immune response against Aspergillus infections in patients with hematological malignancies (IMPACT),
CSCC
Group Leader: Marie von Lilienfeld-Toal
Group Leader: Kathleen Wagner
Patients with hematological malignancies
are believed to be susceptible to invasive fungal infections mainly because of their therapy-induced neutropenia (i.e. low number of
immune cells). However, it is likely that they
also exhibit a functional defect of the immune response, as other patient populations
with low cell numbers (for example patients
receiving treatment with interferon) do not
show the same risk and because of observed
defects in the tumour immune response as
described by us and others (von LilienfeldToal et al. 2010). In this project, we analyse
the innate immune response to Aspergillus
fumigatus in healthy subjects compared to
patients with interferon-therapy and patients
with acute myeloid leukaemia prior to, during and after regeneration of chemotherapyinduced neutropenia. Immune cells from the
peripheral blood are characterised by flowcytometry and cytokine release to determine
the activation profile (pro-inflammatory versus anti-inflammatory). In addition, freshly
sampled immune cells and germinating conidia of Aspergillus spp. are co-incubated to
investigate the interaction between immune
cells and conidia with regard to phagocytosis, viability and activation of immune cells.
This project is funded by the BMBF/Center
for Sepsis Control and Care (CSCC) and the
experiments are performed in close collaboration with the Departments Molecular and
Applied Microbiology, Infection Biology and
ZIK Fungal Septomics.
186
3
Respiratory viruses in healthy individuals – Prospective surveillance
with a multiplex PCR
Recently, infections with respiratory viruses
such as respiratory syncytial virus or influenza A have received much attention. This
is partly because of the 2009 pandemic, but
may also be due to the fact that refined diagnostic techniques like multiplex PCR are increasingly used to examine nasopharyngeal
aspirates or swabs. With these nucleic-acid
tests, viruses are now detected which have
no clearly defined role such as metapneumovirus, bocavirus or parainfluenzavirus.
However, case reports describe potentially
fatal courses of cancer patients with pneumonia and evidence of metapneumovirus or
parainfluenzavirus, which are then assumed
to be causally linked. As there are no data
whatsoever on the prevalence of these newly
described viruses in healthy individuals, it
is impossible to draw any firm conclusions
on these findings. We have started a prospective diagnostic study to determine the
prevalence and relevance of 21 respiratory
pathogens in healthy individuals. The study
started in November 2013 and has included
85 individuals so far. PCR measurements
take place in our laboratory with a commercial multiplex PCR kit. We plan to recruit altogether 225 individuals over the course of
two winters to be able to detect a prevalence
of 10 % in the whole population in a statistically valid fashion.
The study is conducted under the patronage of the Infectious Disease Working Party
of the German Society for Haematology and
Oncology (AGIHO) and is funded in part by
the Interdisciplinary Centre for Clinical Research (IZKF) of the Medical Faculty, FSU
Jena.
Group members
Head
Prof. Dr. Marie von Lilienfeld-Toal
Phone +49 3641 532-1720
Fax
+49 3641 532-0815
[email protected]
Scientists
Dr. Kathleen Wagner
Medical Doctors
Dr. Beate Hermann
Dr. Tobias Rachow
Neumann S, Krause SW, Maschmeyer G, Schiel
X, von Lilienfeld-Toal M, Infectious Diseases
Working Party (AGIHO), German Society of
Hematology and Oncology (DGHO) (2013)
Primary prophylaxis of bacterial infections and
Pneumocystis jirovecii pneumonia in patients
with hematological malignancies and solid
tumors: guidelines of the Infectious Diseases
Working Party (AGIHO) of the German Society
of Hematology and Oncology (DGHO). Ann
Hematol 92, 433-442.
Fleischhacker M, Schulz S, Jöhrens K, von
Lilienfeld-Toal M, Held T, Fietze E, Schewe
C, Petersen I, Ruhnke M (2012) Diagnosis of
chronic disseminated candidosis from liver
biopsies by a novel PCR in patients with haematological malignancies. Clin Microbiol Infect 18,
1010-1016.
MD. Students
Jana Kalkreuth
Julia Kurrek
Stephanie Kurze
Katharina Meckel
Verena Schlüter
Maria Schuwirth
Franziska Wittig
Mayer K, Kapelle M, Kaeferstein A, Weßling M,
Bekeredjian-Ding I, Leutner C, von LilienfeldToal M, Brossart P, Wolf D (2013) Successful
management of Candida krusei monoarthritis
after allo-SCT. Bone Marrow Transplant 48,
1585-1586.
Research Assistants
Pia Konowski
Collaborations
External funding
Project: IMPACT: Immune response against
mould species in patients with acute myeloid
leukaemia
Marie von Lilienfeld-Toal
Universitätsklinikm Bonn, Germany
Prof. Dr. Georg Baumgarten,
Dr. Karin Mayer
Universitätsklinikum Jena, Germany
Prof. Dr. Frank Brunkhorst
PD Dr. Michael Kiehntopf
Prof. Dr. Wolfgang Pfister
Prof. Dr. Matthias Pletz
Schwab KS, Hahn-Ast C, Heinz WJ, Germing
U, Egerer G, Glasmacher A, Leyendecker C,
Marklein G, Nellessen CM, Brossart P, von
Lilienfeld-Toal M (2014) Tigecycline in febrile
neutropenic patients with haematological malignancies: a retrospective case documentation
in four university hospitals. Infection 42, 97-104.
Epub 2013 Aug 25.
Lo-Coco F, Avvisati G, Vignetti M, Thiede C, Orlando SM, Iacobelli S, Ferrara F, Fazi P, Cicconi L,
Di Bona E, Specchia G, Sica S, Divona M, Levis A,
Fiedler W, Cerqui E, Breccia M, Fioritoni G, Salih
HR, Cazzola M, Melillo L, Carella AM, Brandts
CH, Morra E, von Lilienfeld-Toal M, Hertenstein B, Wattad M, Lübbert M, Hänel M, Schmitz
N, Link H, Kropp MG, Rambaldi A, La Nasa G,
Luppi M, Ciceri F, Finizio O, Venditti A, Fabbiano
F, Döhner K, Sauer M, Ganser A, Amadori S,
Mandelli F, Döhner H, Ehninger G, Schlenk RF,
Platzbecker U; Gruppo Italiano Malattie Ematologiche dell'Adulto; German-Austrian Acute
Myeloid Leukemia Study Group; Study Alliance
Leukemia (2013) Retinoic acid and arsenic trioxide for acute promyelocytic leukemia. N Engl J
Med 11, 111-121.
187
188
Associated Group
Microbial Biochemistry and Physiology
189
Associated Group Microbial Biochemistry and Physiology
The Associated Group Microbial Biochemistry and Physiology (MBP) investigates fungal nutrition during pathogenesis, the effect
of environmental conditions on secondary
metabolite production and the real-time visualisation of fungal infections by bioluminescence imaging. Currently, we focus our
research on the filamentous ascomycetes
Aspergillus fumigatus and Aspergillus terreus
and the dimorphic yeast Candida albicans.
way in A. fumigatus, Aspergillus nidulans
and Saccharomyces cerevisiae. Here, a special
interest was given to the isomerisation reaction between homocitrate and homoisocitrate
that is required for α-aminoadipate synthesis, which is, in some fungi, the precursor
for penicillins and cephalosporins. Furthermore, α-oxidation of fatty acids was studied
in C. albicans to investigate its importance
during infection.
In terms of nutrient acquisition during infection, we elucidated the synthesis of the amino acid lysine via the α-aminoadipate path-
A. terreus is a potent source of secondary
metabolites, since its genome contains a
great variety of secondary metabolite gene
Die Asoziierte Gruppe Mikrobielle Biochemie und Physiologie beschäftigt sich
maßgeblich mit der Nährstoff-Verwertung
pathogener Pilze im Infektionsprozess, der
Bedeutung der Sekundärstoff-Produktion in
Abhängigkeit von Umweltbedingungen und
der Echtzeit-Visualisierung von Pilzinfektionen am lebenden Versuchstier. Hierbei stehen insbesondere die filamentösen Pilze Aspergillus fumigatus und Aspergillus terreus,
sowie die dimorphe Hefe Candida albicans
im Zentrum der Studien.
Head:
Im Rahmen der Nährstoff-Verwertung wurde die Synthese der Aminosäure Lysin anhand von A. fumigatus, Aspergillus nidulans
und Saccharomyces cerevisiae über den
190
α-Aminoadipatweg näher untersucht. Hierbei stand die Isomerisierung von Homocitrat
zu Homoisocitrat im Vordergrund, die für
den Vorläufer α-Aminoadipat essentiell ist.
Dieser Vorläufer wird von einigen Pilzen
gleichzeitig für die Synthese von Penicillinen
und Cephalosporinen benötigt. Des Weiteren
wurden Untersuchungen zur Fettsäure-Verwertung von Candida albicans angestellt,
um den Einfluss dieser Nährstoffquelle auf
den Infektionsprozess zu beleuchten.
A. terreus ist eine bedeutende Quelle für
Sekundärstoffe, da das Genom zahlreiche
Gencluster für solche Metabolite besitzt.
Hierbei ist A. terreus besonders für die Produktion des HMG-CoA Reduktase-Inhibi-
HKI Research Report 2012 / 2013 Associated Group Microbial Biochemistry and Physiology
clusters. Due to its ability to produce the
HMG-CoA reductase inhibitor lovastation,
this fungus is currently used in white biotechnology. However, A. terreus is also found
as a pathogen of humans and is, similar to
A. fumigatus, able to provoke life-threatening invasive aspergillosis. However, in contrast to A. fumigatus, invasive aspergillosis
caused by A. terreus is frequently associated
with dissemination. Additionally, due to an
intrinsic Amphotericin B resistance, one of
the most important first-line drugs to combat
fungal infections is not available. Therefore,
we established infection models to compare
invasive aspergillosis caused by both fungi
and are currently investigating the requirement of secondary metabolite production for
environmental competition of A. terreus.
tors Lovastatin bekannt und wird zur biotechnologischen Produktion dieses Statins
eingesetzt. Daneben ist A. terreus aber in
der Lage, lebensbedrohliche invasive Aspergillosen hervorzurufen, die insbesondere
durch den verwandten Vertreter A. fumigatus bekannt sind. Im Gegensatz zu A. fumigatus sind Infektionen mit A. terreus häufig von einer Disseminierung der Infektion
begleitet. Zusätzlich zeigt A. terreus eine
intrinsische Resistenz gegenüber dem bedeutenden Antimykotikum Amphotericin B,
was die Behandlung zusätzlich erschwert.
Daher wurden Infektionsmodelle etabliert,
die es erlauben, den Infektionsverlauf über
A. fumigatus und A. terreus zu vergleichen.
Weiterhin wird zurzeit der Einfluss der Se-
kundärstoff-Produktion auf die Kompetition
von A. terreus mit der Umwelt studiert.
Last but not least, bioluminescence imaging
has been optimised to monitor onset and progression of invasive fungal infections under
in vivo conditions. This technique has now
been applied to study the effect of immunosuppression on disease development and to
elucidate the in vivo efficacy of antifungal
drugs. This technique allows the visualisation of temporal and spatial progression of
Außerdem wurde die Methode des Biolumineszenz-Imaging optimiert, um Beginn und
Verlauf invasiver Pilzinfektionen unter in
vivo-Bedingungen zu untersuchen. Diese
Technik wurde angewendet, um den Effekt
von Immunsuppressionen auf den Krankheitsverlauf zu studieren und die Wirksamkeit antifungaler Therapien im lebenden Tier zu dokumentieren. Da sowohl der
zeitliche als auch der räumliche Verlauf von
Pilzinfektionen sichtbar gemacht werden
kann, ist es mit dieser Technik möglich, die
Strategien zur Therapie von Pilzinfektionen
zu verbessern.
Associated Group Microbial Biochemistry and Physiology HKI Research Report 2012 / 2013
191
fungal infections and can, thus, help to identify new therapeutic strategies against fungal infections.
In 2012 to 2013, our research was supported
by the International Leibniz Research School
(ILRS) in a joint project with the department
of Microbial Pathogenicity Mechanisms, the
Deutsche Forschungsgemeinschaft (DFG)
and the DFG Collaborative Research Center / Transregio CRC124 “Pathogenic fungi
and their human host: Networks of interaction – FungiNet”.
In 2012 und 2013 wurden die Forschungsarbeiten durch ein gemeinsames Projekt mit
der Abteilung Mikrobielle Pathogenitätsmechanismen im Rahmen der “International Leibniz Research School” (ILRS), durch
Einzelförderung der Deutschen Forschungsgemeinschaft (DFG), dem DFG SFB/TR 124
“Pathogenic fungi and their human host: Networks of interaction – FungiNet” gefördert.
192
Scientific Projects
1
Nutrients and fungal pathogenesis
Nutrient acquisition is important for fungal pathogens, as for all microorganisms,
to proliferate within the host. In recent studies we focused on the synthesis of the amino acid lysine, which is an essential amino
acid for humans, but is synthesised via the
α-aminoadipate pathway in fungi. Since this
pathway is also important for production of
the antibiotics Penicillin and Cephalosporin,
a main focus was given to reactions leading
to the common precursor α-aminoadipate.
In addition, fatty acids are very abundant in
the host environment, since they are major
components of membranes and used as energy storage compounds in the form of lipids.
Literature on the impact of lipid metabolism
on pathogenesis of C. albicans and enzymes
involved in this metabolic path has remained
confusing and was, therefore, revisited in
our investigations.
1) Synthesis of the amino acid lysine
Fungi synthesise lysine via the α-aminoadipate pathway and the precursor α-aminoadipate is additionally used by some fungi
to produce beta-lactam antibiotics. Despite
the great importance of α-aminoadipate
production, not all reactions leading to this
intermediate have been studied in detail.
Thus, we concentrated on the isomerisation
between homocitrate and homoisocitrate, a
reaction that is similar to the aconitase reaction in the citrate cycle. However, while
the citrate cycle only requires a single enzyme for this isomerisation, two enzymes are
essentially required in the α-aminoadipate
pathway. Unexpectedly, we discovered that
the citrate cycle aconitase contributes to the
α-aminoadipate pathway by performing the
dehydration of homocitrate to homoaconitate.
Subsequently, the rehydration of homoaconitate to homoisocitrate is performed by a pa-
thway specific homoaconitase. The requirement for two enzymes in this isomerisation
is due to the extended aliphatic chain-length
in homocitrate compared citrate (Fig. 1). Isomerisation requires a 180° rotation of the
substrate, which is characterised by a citrate
and isocitrate binding mode in aconitases.
The additional methylene-group in homocitrate prevents correct orientation of the substrate in the isocitrate mode. Thus, aconitase
dehydrates homocitrate in the citrate mode,
releases homoaconitate that is subsequently
hydrated by homoaconitase in the isocitrate
mode. Thus, the catabolic enzyme aconitase
is directly linked with the anabolism of lysine. However, a second unexpected observation resulted from a difference between
filamentous fungi and yeasts. Although both
species contain two aconitase isoenzymes,
the aconitase B is inactive in filamentous
fungi, but has specifically adapted to serve
for the α-aminoadipate pathway in yeasts by
displaying high affinity for homocitrate but
not citrate. This allows fermenting yeasts to
separate anabolic lysine synthesis from the
catabolic citrate cycle, which is dispensable
for yeasts on fermentable nutrient sources.
In contrast, filamentous fungi rely on an active citrate cycle since they are not able to
ferment. Thus, differences in the life-style
of fungi have manifested in the adaptation
of isoenzymes to specific metabolic needs.
While these discoveries shed new light on
the fungal synthesis of lysine, they are also
important for optimisation of β-lactam antibiotics production in biotechnological processes due to the tight link of cata- and anabolism in α-aminoadipate synthesis.
2) Fatty acid oxidation in Candida albicans: The role of thiolases
Genes involved in β-oxidation are highly
upregulated during the interaction of C. albicans with immune cells. The final reaction of
α-oxidation is the release of acetyl-CoA units
193
Figure 1
Scheme of substrate conversion
by aconitase and homoaconitase.
In the citrate cycle aconitase binds
citrate in the “citrate mode” and
performs a dehydration to aconitate.
After a substrate flip and binding
of aconitate in the isocitrate mode
water is added by aconitase to yield
isocitrate. Homocitrate can also
bind to the citrate cycle aconitase,
but binding is limited to the citrate
mode. Homoaconitate resulting from
the dehydration cannot be stabilised
in the isocitrate mode due to the
extended aliphatic chain. Therefore,
rehydration in the isocitrate mode
requires homoaconitase, which, in
turn, is not able to bind the substrate
in the citrate mode.
from the oxidised fatty acid via a β-ketothiolase. While filamentous fungi degrade fatty acids in mitochondria and peroxisomes,
yeasts only posses a peroxisomal path. Nevertheless, the dimorphic yeast C. albicans contains three isoenzymes of β-ketothiolases,
whereas the yeast S. cerevisiae only contains
a single enzyme. Therefore, we analysed the
contribution of each isoenzyme to fatty acid
degradation and virulence. Two of the three
isoenzymes in C. albicans (Pot1 and Fox3)
showed close phylogenetic relation to the
single thiolase from S. cerevisiae, whereas
the third enzyme (Pot13) was only distantly related and clustered with mitochondrial
thiolases from filamentous fungi. Analyses
of single, double and triple deletion mutants
revealed that Pot1 is the major thiolase in
C. albicans and generally required for utilisation of fatty acids. Interestingly, while
Pot13 did not contribute to fatty acid utilisation – which is in agreement with a lacking
mitochondrial β-oxidation cycle ‑ Fox3 was
194
required for wild-type growth at elevated
temperatures. Thus, Fox3 seems to have adapted to support growth of C. albicans when
growing as a commensal or pathogen inside
the mammalian host. However, analyses of
the virulence revealed that none of the β-ketothiolases was essential for virulence in a
hen’s egg infection model. Although this
does not exclude an importance of β-oxidation in the commensal state of the fungus, it
supports the assumption that other nutrients
such as proteins and/or glucose may serve as
the primary nutrients during infection.
2
Pathogenesis of A. terreus
infections
A. terreus is an important source of natural
products, but also an emerging cause of life-threatening invasive aspergillosis. Thus,
in collaboration with Ilse Jacobsen, Research
Group Microbial Immunology and Chri-
A Lung Histology
B Tissue homogenate
Figure 2
Aspergillus terreus persistence in
infected lung tissues.
(A) Lung histology (PAS staining) of
mice infected with A. terreus conidia
reveals persisting conidia (indicated
by arrows). (B) Plating of tissue homogenates yields a dense lawn of
A. terreus colonies, indicating that
conidia remained in a viable state.
stian Hertweck, Department Biomolecular
Chemistry, we are investigating virulence
of A. terreus in comparison to A. fumigatus
and analyse natural products specifically
produced by A. terreus. Although leukopenic
mice showed a similar outcome of infection
in comparison to A. fumigatus, a 102 to 103
fold higher infectious dose was required for
100% lethality. Interestingly, in corticosteroid treated animals, all mice revealed an initial acute infection, whereby approximately
50% of infected mice survived and showed no
signs of infection at the end of the observation period. However, the surviving animals
revealed a persistence of ungerminated, but
viable conidia in the lung tissue (Fig. 2).
Furthermore, all mice that succumbed to infection and regardless the immunosuppression regimen showed a fatty liver degeneration, suggesting production of toxic secondary
metabolites. Persistence of A. terreus in mice
implied a fundamental difference between
A. terreus and A. fumigatus infections that
was assumed to derive from differences in
the interaction with primary immune cells.
A comparative study between both fungi revealed a substantially higher exposure of the
surface antigens beta-1,3-glucan and galactomannan on A. terreus conidia, which was
responsible for fast phagocytosis by macrophages. Additionally, despite acidification of
phagolysosomes, A. terreus conidia were not
inactivated, which contrasts A. fumigatus conidia that prevent phagocytosis and rapidly
escape by germination. A major difference
between both species derived from the lack
of a DHN-melanin derivative in A. terreus
conidia, which inhibits phagolysosome acidification in other Aspergillus species. Accordingly, recombinant production of the
respective polyketide in A. terreus inhibited
acidification, enhanced escape from macrophages and increased virulence. Further
studies now focus on the identification of the
pigment produced by A. terreus conidia and
secondary metabolites provoking liver da-
195
Figure 3
Bioluminescence of Aspergillus
fumigatus reporter strains.
(A) Dark-field photograph of an
A. fumigatus colony expressing a
codon-optimised luciferase. (B) In
vivo imaging of a mouse infected
with the bioluminescent A. fumigatus
reporter strain. Photons emitted by
the reporter strain from the infected
lung are detected by a highly sensitive CCD camera and visualised in the
overlay image.
mage in infected mice to elucidate different
pathogenicity mechanisms among Aspergillus species.
3
Monitoring of therapy efficacy by
bioluminescence imaging
Bioluminescence imaging (BLI) allows the visualisation of infections in living animals by
collecting photons from the site of infection
that are emitted by a luminescent pathogen.
In our studies, we optimised A. fumigatus
reporter strains (Fig. 3) with the aim to monitor drug efficacy under in vivo conditions.
Intranasal infection of leukopenic mice with
the reporter strain resulted in rapid increase in the pulmonary luminescence signal
accompanied with 100 % mortality. Amphotericin B (AmB) treatment was effective to
reduce mortality, but imaging implied a low
efficacy of AmB in the sinus region, which
led to lethal infections in some animals. In
196
contrast, Anidulafungin (Anid) was not sufficient to prevent pulmonary aspergillosis, but
no signal increase was observed from the sinus. Thus, a combination of AmB and Anid
was applied, which resulted in 100 % survival of mice with a control of infection in both,
sinus and pulmonary region. Thus, bioavailabilty of AmB and Anid may differ in sinus
and lung, which results in a synergistic therapeutic effect when given in parallel. Thus,
BLI enables to visualise disease progression
in temporal and spatial resolution and allows
to follow the efficacy of antifungal drugs at
diverse body sites. This results in an increased resolution of the outcome of therapy
approaches by simultaneously reducing the
number of animals in such investigations.
Group members
Collaborations
Head
Phone +49 3641 532-1710
Fax
+49 3641 532-0809
[email protected]
Goethe University Frankfurt,
Frankfurt/Main, Germany
Prof. Dr. Thomas Lehrnbecher
Scientists
Dr. Felicitas Schöbel (until 10/2012)
Ph.D. Students
Markus Gressler
Christian Otzen
Shruthi Ramachandra (until 09/2013)
Amélie Savers (since 10/2013)
Institute Pasteur, Paris, France
Dr. Oumaima Ibrahim-Granet
Dr. Grégory Jouvion
Max Planck Institute for Chemical Ecology,
Jena, Germany
Dr. Almuth Hammerbacher
Research Institute of Vall d´Hebron Hospital,
Barcelona, Spain
Dr. Joan Gavaldà
Katrin Emmelmann (11/2012–01/2013)
St George's University of London, UK
Dr. Kai Hilpert
Research Assistants
Daniela Hildebrandt
Universidade Federal de Goiás, Goiás, Brazil
Prof. Dr. Maristela Pereira
Prof. Dr. Célia Maria de Almeida Soares
External funding
University of Leipzig, Germany
Prof. Dr. Gottfried Alber
Aspergillus terreus as a cause of invasive aspergillosis: Impact of secondary metabolites on
pathogenesis
Matthias Brock
SFB/TR FungiNet
Project A03: In vivo Bildgebung der Ausbreitung und immunologischen Kontrolle von
Aspergillus fumigatus und Candida albicans
Infektionen
Matthias Brock
Otzen C, Müller S, Jacobsen ID, Brock M (2013)
Phylogenetic and phenotypic characterisation
of the 3-ketoacyl-CoA thiolase gene family from
the opportunistic human pathogenic fungus
Candida albicans. FEMS Yeast Res 13, 553-564.
Galiger C*, Brock M*, Jouvion G, Savers A,
Parlato M, Ibrahim-Granet O (2013) Assessment
of efficacy of antifungals against Aspergillus
fumigatus: the value of real-time bioluminescence imaging. Antimicrob Agents Chemother
57, 3046-3059. (*equal contribution)
Fazius F, Zaehle C, Brock M (2013) Lysine biosynthesis in microbes: relevance as drug target
and prospects for β-lactam antibiotic production. Appl Microbiol Biotechnol 97: 3763-3772
197
198
Associated Group Pharmaceutical Microbiology
199
Associated Group Pharmaceutical Microbiology
The Associated Group Pharmaceutical Microbiology is affiliated with the Pharmaceutical
Institute of the Friedrich Schiller University
and, at the same time, associated with the
HKI. The department thus represents another excellent example for the HKI’s close
interaction with the university. As a group
focusing on pharmaceutical research our
objective is to isolate and analyse microbial
small molecule natural products as potential
new pharmaceuticals or as chemical signals in microbial communication processes.
Along with this endeavour, we elucidate the
biochemical and genetic basis of natural
product biosyntheses. A particular emphasis
is placed on basidiomycete secondary metabolism, however, our projects also include bacterial small molecules. As a university-affiliated group, the Department Pharmaceutical
Microbiology actively engages in teaching
within the curriculum of the Pharmaceutical Sciences. Students can elect to fulfil the
requirements for diploma theses or laboratory internships in the department and gain
hands-on experience in natural product research early on.
Die assoziierte Gruppe Pharmazeutische
Mikrobiologie ist Teil des Institutes für
Pharmazie der Friedrich-Schiller-Universität Jena, jedoch gleichzeitig dem HKI assoziiert. Sie stellt damit ein weiteres Beispiel
für die enge Verzahnung des HKI mit der
universitären Forschung dar. Als eine pharmazeutisch ausgerichtete Gruppe betreibt
sie Forschung über Naturstoffe mit dem
Ziel der Isolation und Analyse von kleinen
mikrobiellen Sekundärmetaboliten als potentielle neue Wirkstoffe oder Informationsträger für mikrobielle Kommunikation.
Ein Schwerpunkt ist die Erforschung der
genetischen und biochemischen Grundlagen, auf denen die jeweiligen zellulären Bio-
Head:
Prof. Dr. Dirk Hoffmeister
200
synthesewege dieser Sekundärmetabolite
aufbauen. Hauptsächlich werden Ständerpilze (Basidiomyceten) untersucht, jedoch ist
auch der bakterielle Stoffwechsel durch ein
Projekt in der Gruppe repräsentiert. Als universitäre Gruppe ist die assoziierte Gruppe
Pharmazeutische Mikrobiologie auch aktiv
in Lehraufgaben zur Ausbildung von Apothekern eingebunden. Durch die Vergabe
von Praktikums- und Diplomarbeiten sowie
Plätzen für Wahlpflichtpraktika gemäß der
Approbationsordnung für Apotheker kann
der pharmazeutische Nachwuchs auf diese
Weise frühzeitig an Naturstoff-Forschung
herangeführt werden.
HKI Research Report 2012 / 2013 Associated Group Pharmaceutical Microbiology
Scientific Projects
SidE
A T C GreA
A T TE LnaA/
LnbA
1
A T C Figure 1
Domain layout and products of the
peptide synthetase SidE and peptide
synthetase-like enzymes GreA and
LnaA/LnbA. Enzyme domain abbreviations are A: adenylation; T: thiolation; C: condensation; TE: thioesterase; R: reductase.
A T R Multi-domain enzymes in fungal
natural product biosyntheses
Fungal peptide synthetases and related
enzymes
A major focus of our scholarly work lies
on the non-ribosomal peptide synthetases
(NRPSs), i.e., multi-domain enzymes that catalyse the assembly of complex natural products from a large variety of monomeric building blocks. Through their inherent logic of
self-activating their substrates via specific
adenylation, NRPSs are not limited to the
usual proteinogenic L-amino acids. The cumulative genomic sequence data shows that
fungi usually encode numerous NPRS biosynthetic pathways. However, the majority of
their products is still unknown. If it was possible to accurately predict substrate specificities of NRPSs from their primary amino acid
sequence, we could anticipate product structures and, ultimately, fungal secondary metabolomes from genomic data in silico. This
would help accelerate discovery of drugs and
other potentially useful compounds. Currently, such predictions are, to a degree, possible but by far not as accurate as for their
bacterial counterparts. Still, progress toward
deciphering and refining such specificities
has been made, and the results by the Department Pharmaceutical Microbiology contributed to this objective.
The Aspergillus fumigatus bimodular NRPS
SidE (Fig. 1) is encoded by a gene that was
previously shown to be strongly induced during initiation of murine lung infection. Using
heterologously produced full-length SidE,
fumarylalanine formation was observed in
vitro, thereby disproving the hypothesis that
SidE was related to siderophore formation.
Therefore, SidE allows, for the first time, insight how NRPSs select fumaric acid, i.e., a
dicarboxylic non-amino acid substrate. Furthermore, fumarylalanine was also identified in A. fumigatus culture supernatants
Associated Group Pharmaceutical Microbiology HKI Research Report 2012 / 2013
201
Figure 2
Structure of dehydroarmillylorsellinate (left) and ArmB-catalysed
cross-coupling during melleolide
biosynthesis (right).
under inducing conditions, unless sidE was
genetically inactivated. This project was accomplished in close collaboration with Prof.
Hubertus Haas (Innsbruck Medical University). Our current hypothesis includes that
fumarylalanine production could result in
local immunosuppression, so the results may
help better understand the processes behind
infection by A. fumigatus.
The basidiomycete Suillus grevillei (“larch
bolete”) encodes the gene greA for a monomodular NRPS-like biosynthesis enzyme
that lacks a condensation (C) domain but is
composed of three domains for adenylation,
thiolation, and thioesterase (Fig. 1). GreA
was biochemically characterized in vitro and
shown to possess atromentin synthetase
activity. Specificity-conferring amino acid
residues were identified which showed an
unprecedented combination specific for aromatic α-keto acid substrates.
202
In cooperation with Prof. Nancy Keller’s group
at the University of Wisconsin - Madison, a
second class of C-domainless NRPS-like enzyme has recently been found relevant for
the biosynthesis of small natural products.
These enzymes, LnaA and LnbA of Aspergillus flavus, feature an adenylation, thiolation,
and reductase domain arrangement (Fig.
1). Therefore, they resemble α-aminoadipic
acid reductases, a unique class of fungal
enzymes that serve fungal L-lysine biosynthesis, i.e., primary metabolism. LnaA and
LnbA are functionally redundant and govern
the production of piperazines and morpholines via a reductive mechanism whose details are not yet understood. Consistent with
the chemical structure of the final product,
in vitro work demonstrated that L-tyrosine
is the preferred substrate of either enzyme.
A double mutant (deletion and silencing, respectively, of both lnaA and lnbA) showed an
altered phenotype as the strain was defec-
tive in the formation of sclerotia. This result
therefore suggests that natural products also
play a role for the producer itself as its own
small molecules help it complete its life cycle
properly.
Fungal polyketide synthases – Large-scale
genome sequencing efforts (see also section
3) have helped anticipate the capacity of basidiomycetes to synthesize polyketides. A
phylogenetic analysis of 111 type I polyketide
synthase genes from 35 sequenced basidiomycete genomes was carried out. The resulting phylogeny suggests that all main types
of fungal iterative PKS had already evolved
before the Ascomycota and Basidiomycota
diverged during evolution. We expect this
study supports genomic mining projects
aimed at discovering structurally diverse
mushroom-derived polyketides.
In cooperation with Prof. Barbara Howlett’s
group at the University of Melbourne, Australia, the fungus Leptosphaeria maculans, a
serious pathogen on canola, was investigated
regarding its capacity to produce the phytotoxin phomenoic acid. It was shown that the
polyketide synthase PKS2 catalyses the biosynthesis of this long-chain aliphatic carboxylic acid.
2
Melleolide natural products from
Armillaria species
Many species within the basidiomycete
genus Armillaria (“honey mushroom”) are
known as notorious butt and root rot agents
and globally present as pathogens both in
managed and unmanaged hardwood and
conifer forests. Armillaria species produce
melleolides (e.g., dehydroarmillylorsellinate,
Fig. 2) which are composed of a sesquiterpenoid protoilludene whose alcohol functionality at position 5 engages in ester bond formation with orsellinic acid. This tetraketide
is a common microbial metabolite, and protoilludenes are frequently found in basidiomycetes. However, the esterification of these
two building blocks into a more complex metabolite is a unique biosynthetic capacity of
Armillaria. Further, a permutation-like biosynthetic combination of methylation, chlorination, and oxidation/reduction generates in
excess of 50 melleolide analogs which make
them one of the most diverse groups of fungal natural products and an attractive model
to study structure-activity-relationships.
An Armillaria mellea non-reducing polyketide synthase, termed ArmB, was identified as
orsellinic acid synthase. Further, cross-coupling activity of the ArmB thioesterase domain, i.e., esterification of orsellinic acid
with various alcohols, among them a tricyclic Armillaria sesquiterpene alcohol, was
shown (Fig. 2). Therefore, the biosynthesis of
the melleolide melledonol was reconstituted
in vitro.
The pharmaceutical aspect of research on
melleolides stems from their antifungal activity and their inhibitory activities against
human cell lines. Five new melleolides were
isolated and their structure elucidated. Using
these new and a series of known melleolides,
the ∆2,4 -double bond in the protoilludene moiety was identified as a key structural feature
for antifungal activity, but turned out irrelevant for cytotoxicity against human cells.
Further, treatment with an antifungally active melleolide impacts upon transcription of
Aspergillus nidulans natural product genes.
Transcriptomic studies were accomplished
in close cooperation with the Department
of Molecular and Applied Microbiology (A.
Brakhage). After melleolide treatment, the
most intriguing transcriptional up-regulation pertained to genes in a silent cluster
which represents a portion of an extended
genetic locus that had previously been recognized by HKI members as responsive to the
presence and physical contact of streptomycetes. Therefore, our findings add to the notion of this Aspergillus locus as critical for intermicrobial communication. This study was
integrated into the Jena School of Microbial
Communication (JSMC).
203
3
Basidiomycete genome projects
The associated group Pharmaceutical Microbiology has been involved in various multi-PI, international consortia to annotate the
genomic sequences of basidiomycetes whose
DNA was sequenced at the Joint Genome Institute (JGI) in Walnut Creek, CA, USA. The
group provided expertise to annotate genes
that encode biosynthetic capacity for natural products in the respective species and
to modify gene models, if necessary. In the
2012/2013 biennium, the group primarily
focused on
i) the white-rot fungus Ceriporiopsis subvermispora, which is a model basidiomycete for
selective ligninolysis, as this fungus readily
depolymerizes lignin with relatively little
cellulose degradation;
ii) the tree pathogen Heterobasidion annosum, the causative agent of root rot and a
cosmopolitan pathogen in coniferous forests;
iii) and Phlebiopsis gigantea, which degrades
all components of plant cell walls and is
uniquely able to colonize freshly exposed
conifer sapwood. Spore suspensions of this
fungus are marketed as a biological pesticide
to control root rot.
The genomic sequences of the former two species, including the contributions regarding
the natural products, have been published.
A two-fold benefit results from this work. Regarding the goals of the HKI and discovery of
new compounds, such projects help identify
species which encode a particularly diverse
secondary metabolism or which harbour unusual biosynthesis genes. These species then
can be given priority to search for new biochemistry and unprecedented chemical compounds and bioactivities. Seen from a more
ecological angle, this work contributes to the
focus area “microbial communication”, as
204
interorganismal interactions are frequently mediated by small molecule natural products. The careful genetic analysis provides
us with a map to the secondary metabolome
of a species.
4
Secondary metabolism of Ralstonia
solanacearum
Ralstonia solanacearum is a Gram-negative
bacterium and notorious plant pathogen as
it causes a lethal wilt disease. The host range of this organism is remarkably broad and
includes plants used for staple food production, such as banana and potato, and species
of significant commercial value, including
tomato, eggplant, and tobacco. Previous studies identified the global regulatory proteins
VsrAD and PhcA as central for both virulence and for biosynthesis of ralfuranones,
a small family of phenylpyruvate-derived
aryl-substituted furanones.
The ralfuranone synthetase RalA was characterised biochemically and several sulfur-containing derivatives were isolated,
among them the methyl thioether-containing
ralfuranone D. The project included stable
isotope feeding, headspace analyses of volatile compounds, gene inactivation, as well
as bioinformatic analyses of the ralfuranone biosynthesis. A non-enzymatic one-step
mechanism for the transfer of an intact methylthio group from L-methionine or α-keto-γ-methylthiobutyric acid was established.
The methylthio acceptor molecule ralfuranone I, a previously postulated biosynthetic
intermediate, was isolated and characterized by NMR (Fig. 3). The highly reactive
Michael acceptor system of this intermediate
readily reacts with various thiols, including
glutathione. These results may point to the
actual biological role of ralfuranones in interference with glutathione-mediated signalling in host plant cells. These results, again,
may contribute to the focus area “microbial
communication”.
γ
α
α
Figure 3
Mechanism of methylthioether
formation during ralfuranone D
biosynthesis.
This work was accomplished in cooperation
with i) a partner group at the University of
Wisconsin-Madison (transcriptome analysis), ii) the Max Planck Institute for Chemical Ecology in Jena (headspace analysis) and
iii) HKI’s Junior Research Group Secondary
Metabolism of Predatory Bacteria (NMR). In
return, the latter group was supported with
biochemical experiments and in vitro enzyme characterisations during their research
on micacocidin, i.e., a thiazoline-containing
antibiotic with significant activity against
Mycoplasma pneumoniae.
205
Group members
Kalb D, Lackner G, Hoffmeister D (2013) Fungal peptide synthetases – an update on functions and specificity signatures. Fungal Biol Rev
27, 43-50.
Head
Prof. Dr. Dirk Hoffmeister
Phone +49 3641 949-850
Fax
+49 3641 949-852
[email protected]
Steinchen W, Lackner G, Yasmin S, Schrettl M,
Dahse HM, Haas H, Hoffmeister D (2013)
Bimodular peptide synthetase SidE produces
fumarylalanine in the human pathogen Aspergillus fumigatus. Appl Environ Microbiol 79,
6670-6676.
Scientists
Dr. Gerald Lackner
Ph.D. Students
Markus Bohnert
Jana Braesel
Daniel Braga de Lima (since 05/2013)
Daniel Kalb
Julia Pauly
Daniel Schwenk
Collaborations
Max-Planck-Institute for Chemical Ecology,
Jena, Germany
Dr. Jeanine Linz
Philip Brandt (since 10/2013)
Matthias Böhm (since 10/2013)
Wieland Steinchen (until 05/2013)
Jonas Wick (until 11/2013)
Research Assistants
Julia Greßler
Elke Hänert (until 01/13)
External funding
Project: Chemical ecology of the honey mushroom Armillaria mellea: Investigations on the
mode of action of melleolide antibiotics
Dirk Hoffmeister
The University of Melbourne, Australia
Prof. Dr. Barbara Howlett
Dr. Candace Elliott
University of Wisconsin, Madison, WI, USA
Prof. Dr. Caitilyn Allen
Prof. Dr. Nancy Keller
United States Department of Agriculture –
Forest Products Laboratory, Madison, WI, USA
Dr. Daniel Cullen
Forseth RR, Amaike SA, Schwenk D, Affeldt KJ,
Hoffmeister D, Schroeder FC, Keller NP (2013)
Homologous non-canonical NRPS gene clusters mediate redundant small-molecule biosynthesis and sclerotia formation in Aspergillus
flavus. Angew Chem Intl Ed 52, 1590-1594.
Lackner G, Bohnert M, Wick J, Hoffmeister D
(2013) Assembly of melleolide antibiotics
involves a polyketide synthase with cross-coupling activity. Chem Biol 20, 1101-1106.
Pauly J, Spiteller D, Linz J, Jacobs JM, Allen C,
Nett M, Hoffmeister D (2013) Ralfuranone
thioether production by the plant pathogen
Ralstonia solanacearum. ChemBioChem 14,
2169-2178.
206
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Tokarski C, Hummert S, Mech F,
Figge MT, Germerodt S, Schroeter
A, Schuster S (2012) Agent-based
modeling approach of immune
defense against spores of opportunistic human pathogenic fungi.
Research Group Fungal
Septomics
Bauer R, Mezger M, Blockhaus C,
Schmitt AL, Kurzai O, Einsele H,
Löffler J (2012) 40-O -[2-Hydroxyethyl]rapamycin modulates human
dendritic cell function during
exposure to Aspergillus fumigatus.
J Basic Microbiol 52, 269-276.
Cottier F, Raymond M, Kurzai
O, Bolstad M, Leewattanapasuk
W, Jimenez-Lopez C, Lorenz MC,
Sanglard D, Vachova L, Pavelka N,
Palkova Z, Mühlschlegel FA (2012)
The bZIP transcription factor
Rca1p is a central regulator of a
novel CO(2) sensing pathway in
yeast. PLoS Pathog 8, e1002485.
Escalante-Perez M, Jaborsky M,
Reinders J, Kurzai O, Hedrich R,
Ache P (2012) Poplar extrafloral
nectar is protected against plant
and human pathogenic fungus. Mol
Plant 5, 1157-1159.
Heddergott C, Bruns S, Nietzsche
S, Leonhardt I, Kurzai O, Kniemeyer O, Brakhage AA (2012) The
Arthroderma benhamiae hydrophobin HypA mediates hydrophobicity and influences recognition
by human immune effector cells.
Eukaryot Cell 11, 673-682.
Miramón P, Dunker C, Windecker H, Bohovych IM, Brown AJ,
Kurzai O, Hube B (2012) Cellular
responses of Candida albicans to
phagocytosis and the extracellular
activities of neutrophils are critical
to counteract carbohydrate starvation, oxidative and nitrosative
stress. PLoS One 7, e52850.
Research Group Systems
Biology and Bioinformatics
Altwasser R, Linde J, Buyko E,
Hahn U, Guthke R (2012) Genomewide scale-free network inference
for Candida albicans. Front Microbiol 3, 51.
Baumgart M, Groth M, Priebe S,
Appelt J, Guthke R, Platzer M,
Cellerino A (2012) Age-dependent
regulation of tumor-related microRNAs in the brain of the annual
fish Nothobranchius furzeri. Mech
Ageing Dev 133, 226-233.
Kremling A, Goehler A, Jahreis
K, Nees M, Auerbach B, SchmidtHeck W, Kökpinar Ö, Geffers R,
Rinas U, Bettenbrock K (2012)
Analysis and design of stimulus
response curves of E. coli. Metabolites 2, 844-871.
Boettger D, Bergmann H, Kuehn
B, Shelest E, Hertweck C (2012)
Evolutionary imprint of catalytic domains in fungal PKS-NRPS
hybrids. ChemBioChem 13, 23632373.
Linde J, Hortschansky P, Fazius
E, Brakhage AA, Guthke R, Haas
H (2012) Regulatory interactions
for iron homeostasis in Aspergillus
fumigatus inferred by a Systems
Biology approach. BMC Syst Biol
6, 6.
Druzhinina IS, Shelest E, Kubicek
CP (2012) Novel traits of Trichoderma predicted through the analysis
of its secretome. FEMS Microbiol
Lett 337, 1-9.
Erdmann S, Freihorst D, Raudaskoski M, Schmidt-Heck W, Jung
EM, Senftleben D, Kothe E (2012)
Transcriptome and functional
analysis of mating in the basidiomycete Schizophyllum commune.
Eukaryot Cell 11, 571-589.
Fazius F, Shelest E, Gebhardt P,
Brock M (2012) The fungal alphaaminoadipate pathway for lysine
1508-1530.
Fuellen G, Dengjel J, Hoeflich A,
Hoeijemakers J, Kestler HA, Kowald A, Priebe S, Rebholz-Schuhmann D, Schmeck B, Schmitz U,
Stolzing A, Suhnel J, Wuttke D,
Vera J (2012) Systems biology and
bioinformatics in aging research: a
workshop report. Rejuvenation Res
15, 631-641.
Gerke J, Bayram O, Feussner K,
Landesfeind M, Shelest E, Feussner I, Braus GH (2012) Breaking
the silence: protein stabilization
uncovers silenced biosynthetic
gene clusters in the fungus Aspergillus nidulans. Appl Environ Microbiol 78, 8234-8244.
Hecker M, Goertsches RH, Fatum
C, Koczan D, Thiesen HJ, Guthke
R, Zettl UK (2012) Network analysis of transcriptional regulation
in response to intramuscular
interferon-beta-1a multiple sclerosis treatment. Pharmacogenomics J
12, 134-146.
Kastner S, Müller S, Natesan
L, König GM, Guthke R, Nett M
(2012) 4-Hydroxyphenylglycine
biosynthesis in Herpetosiphon
aurantiacus: a case of gene duplication and catalytic divergence. Arch
Microbiol 194, 557-566.
Martinez DA, Oliver BG, Gräser Y,
Goldberg JM, Li W, Martinez-Rossi
NM, Monod M, Shelest E, Barton
RC, Birch E, Brakhage AA, Chen
Z, Gurr SJ, Heiman D, Heitman J,
Kosti I, Rossi A, Saif S, Samalova
M, Saunders CW, Shea T, Summerbell RC, Xu J, Young S, Zeng
Q, Birren BW, Cuomo CA, White
TC (2012) Comparative genome
analysis of Trichophyton rubrum
and related dermatophytes reveals
candidate genes involved in infection. MBio 3, e00259-00212.
Müller S, Baldin C, Groth M,
Guthke R, Kniemeyer O, Brakhage
AA, Valiante V (2012) Comparison
of transcriptome technologies in
the pathogenic fungus Aspergillus
fumigatus reveals novel insights
into the genome and MpkA dependent gene expression. BMC Genomics 13, 519.
Rauchfuss F, Lambeck S, Claus
RA, Ullmann J, Schulz T, Weber M,
Katenkamp K, Guthke R, Bauer
M, Settmacher U (2012) Sustained
liver regeneration after portal vein
embolization - a human molecular
pilot study. Dig Liver Dis 44, 681688.
160, 64-71.
Simon S, Guthke R, Kamradt T,
Frey O (2012) Multivariate analysis
of flow cytometric data using decision trees. Front Microbiol 3, 114.
Tierney L, Linde J, Müller S,
Brunke S, Molina JC, Hube B,
Schock U, Guthke R, Kuchler K
(2012) An interspecies regulatory
network inferred from simultaneous RNA-seq of Candida albicans
invading innate immune cells.
Vlaic S, Schmidt-Heck W, MatzSoja M, Marbach E, Linde J, MeyerBaese A, Zellmer S, Guthke R,
Gebhardt R (2012) The extended
TILAR approach: a novel tool for
dynamic modeling of the transcription factor network regulating the
adaption to in vitro cultivation of
murine hepatocytes. BMC Syst Biol
6, 147.
Wartenberg D, Vödisch M, Kniemeyer O, Albrecht-Eckardt D,
Scherlach K, Winkler R, Weide
Werner S, Schroeter A, Schimek C,
Vlaic S, Wöstemeyer J, Schuster S
(2012) Model of the synthesis of trisporic acid in Mucorales showing
bistability. IET Syst Biol 6, 207-214.
Zarse K, Schmeisser S, Groth M,
Priebe S, Beuster G, Kuhlow D,
Guthke R, Platzer M, Kahn CR,
Ristow M (2012) Impaired insulin/
IGF1 signaling extends life span by
promoting mitochondrial L-proline
catabolism to induce a transient
ROS signal. Cell Metab 15, 451-465.
Secondary Metabolism of
Predatory Bacteria
Kastner S, Müller S, Natesan
L, König GM, Guthke R, Nett M
(2012) 4-Hydroxyphenylglycine
biosynthesis in Herpetosiphon
aurantiacus: a case of gene duplication and catalytic divergence. Arch
Microbiol 194, 557-566.
Kreutzer MF, Kage H, Nett M
(2012) Structure and biosynthetic
assembly of cupriachelin, a photoreactive siderophore from the
bioplastic producer Cupriavidus
necator H16. J Am Chem Soc 134,
5415-5422.
Kreutzer MF, Nett M (2012)
Genomics-driven discovery of taiwachelin, a lipopeptide siderophore
from Cupriavidus taiwanensis. Org
Biomol Chem 10, 9338-9343.
Cellular Immunobiology
This group ceased in 2012.
Head: Dr. Mihály Józsi
Castiblanco-Valencìa MM, Fraga
TR, Silva LB, Monaris D, Abreu PA,
Strobel S, Józsi M, Isaac L, Barbosa
AS (2012) Leptospiral immunoglobulin-like proteins interact with human complement regulators factor
H, FHL-1, FHR-1, and C4BP. J Infect
Dis 205, 995-1004.
Hebecker M, Józsi M (2012) Factor H-related protein 4 activates
complement by serving as a platform for the assembly of alternative pathway C3 convertase via its
interaction with C3b protein. J Biol
Chem 287, 19528-19536.
Kopp A, Hebecker M, Svobodová
E, Józsi M (2012) Factor H: A complement regulator in health and
disease, and a mediator of cellular
interactions. Biomolecules 2, 46-75.
Kopp A, Strobel S, Tortajada A,
Rodríguez de Córdoba S, SánchezCorral P, Prohászka Z, LópezTrascasa, Józsi M (2012) Atypical
hemolytic uremic syndrome-associated variants and autoantibodies
impair binding of factor H and factor H-related protein 1 to pentraxin
3. J Immunol 189, 1858-1867.
Krmar RT, Skattum L, Barany P,
Józsi M (2012) Screening for antifactor B autoantibody in a patient
with acute renal injury due to
dense deposit disease. Clin Nephrol
77, 85-86.
Nozal P, Strobel S, Ibernon M,
López D, Sánchez-Corral P, Rodríguez de Córdoba S, Józsi M, LópezTrascasa M (2012) Anti-factor H
antibody affecting factor H cofactor
activity in a patient with dense
deposit disease. Clin Kidney J 5,
133-136.
Svobodová E, Staib P, Losse J,
Hennicke F, Barz D, Józsi M (2012)
Differential interaction of the two
related fungal species Candida
albicans and Candida dubliniensis
with human neutrophils. J Immunol
189, 2502-2511.
Fundamental Molecular Biology
of Pathogenic Fungi
Head: Dr. habil. Peter Staib
Hof H, Eigner U, Maier T, Staib P
(2012) Differentiation of Candida
dubliniensis from Candida albicans
by means of MALDI-TOF mass
spectrometry. Clin Lab 58, 927-931.
Svobodová E, Staib P, Losse J,
Hennicke F, Barz D, Józsi M (2012)
Differential interaction of the two
related fungal species Candida
albicans and Candida dubliniensis
with human neutrophils. J Immunol
189, 2502-2511.
Bio Pilot Plant
Ding L, Maier A, Fiebig HH, Lin
WH, Peschel G, Hertweck C (2012)
Kandenols A-E, eudesmenes from
an endophytic Streptomyces sp. of
211
the mangrove tree Kandelia candel.
J Nat Prod 75, 2223-2227.
Ding L, Peschel G, Hertweck C
(2012) Biosynthesis of archetypal
plant self-defensive oxylipins by
an endophytic fungus residing in
mangrove embryos. ChemBioChem
13, 2661-2664.
Gläser SP, Galatis H, Martin K,
Kämpfer P (2012) Luteolibacter
cuticulihirudinis sp. nov., isolated
from Hirudo medicinalis. Antonie
Van Leeuwenhoek 102, 319-324.
Graupner K, Scherlach K,
Bretschneider T, Lackner G, Roth
M, Gross H, Hertweck C (2012)
Imaging mass spectrometry and
genome mining reveal highly
antifungal virulence factor of
mushroom soft rot pathogen. Angew
Chem Int Ed 51, 13173-13177.
Kämpfer P, Arun AB, Young CC,
Rekha PD, Martin K, Busse HJ,
Chen WM (2012) Microbulbifer
taiwanensis sp. nov., isolated from
coastal soil. Int J Syst Evol Microbiol
62, 2485-2489.
Kämpfer P, Falsen E, Lodders N,
Martin K, Kassmannhuber J, Busse
HJ (2012) Paenibacillus chartarius
sp. nov., isolated from a paper mill.
Int J Syst Evol Microbiol 62, 13421347.
Kämpfer P, Lai WA, Arun AB,
Young CC, Rekha PD, Martin
K, Busse HJ, Chen WM (2012)
Paracoccus rhizosphaerae sp. nov.,
isolated from the rhizosphere of the
plant Crossostephium chinense (L.)
Makino (Seremban). Int J Syst Evol
Microbiol 62, 2750-2756.
Kämpfer P, Lodders N, Grün-Wollny
I, Martin K, Busse HJ (2012) Nocardia grenadensis sp. nov., isolated
from sand of the Caribbean Sea. Int
J Syst Evol Microbiol 62, 693-697.
Kämpfer P, Lodders N, Martin K,
Avendano-Herrera R (2012) Flavobacterium chilense sp. nov. and
Flavobacterium araucananum sp.
nov., isolated from farmed salmonid
fish. Int J Syst Evol Microbiol 62,
1402-1408.
Kämpfer P, Lodders N, Martin K,
Falsen E (2012) Massilia oculi sp.
nov., isolated from a human clinical
specimen. Int J Syst Evol Microbiol
62, 364-369.
Kämpfer P, Schäfer J, Lodders N,
Martin K (2012) Microbacterium
murale sp. nov., isolated from an
indoor wall. Int J Syst Evol Microbiol
62, 2669-2673.
212
Kämpfer P, Wellner S, Lohse K,
Martin K, Lodders N (2012) Duganella phyllosphaerae sp. nov., isolated
from the leaf surface of Trifolium
repens and proposal to reclassify
Duganella violaceinigra into a novel
genus as Pseudoduganella violceinigra gen. nov., comb. nov. Syst
Appl Microbiol 35, 19-23.
Martinez-Ramirez JA, Voigt K,
Peters FT (2012) Studies on the
metabolism of five model drugs
by fungi colonizing cadavers using LC-ESI-MS/MS and GC-MS
analysis. Anal Bioanal Chem 404,
1339-1359.
Möbius N, Ross C, Scherlach K,
Rohm B, Roth M, Hertweck C
(2012) Biosynthesis of the respiratory toxin bongkrekic acid in the
pathogenic bacterium Burkholderia
gladioli. Chem Biol 19, 1164-1174.
Chen W, Fungal Barcoding Consortium (2012) Nuclear ribosomal
internal transcribed spacer (ITS)
region as a universal DNA barcode
marker for Fungi. Proc Natl Acad
Sci U S A 109, 6241-6246.
Morgado I, Wieligmann K, Bereza
M, Ronicke R, Meinhardt K, Annamalai K, Baumann M, Wacker
J, Hortschansky P, Malesevic M,
Parthier C, Mawrin C, SchieneFischer C, Reymann KG, Stubbs
MT, Balbach J, Gorlach M, Horn U,
Fändrich M (2012) Molecular basis
of beta-amyloid oligomer recognition with a conformational antibody
fragment. Proc Natl Acad Sci USA
109, 12503-12508.
VU, Hoffmann K, Große-Herrenthey A, Walther G, AlastrueyIzquierdo A, Rodriguez-Tudela JL,
419-427.
160, 64-71.
Nyilasi I, Papp T, Vagvolgyi C, de
Hoog S, Voigt K, Jacobsen ID (2012)
Lichtheimia species exhibit differences in virulence potential. PLoS
One 7, e40908.
Scharf DH, Chankhamjon P, Scherlach K, Heinekamp T, Roth M,
Epidithiol formation by an unprecedented twin carbon-sulfur lyase in
the gliotoxin pathway. Angew Chem
Int Ed 51, 10064-10068.
Jena Microbial Resource
Collection
Choi JS, Gräser Y, Walther G,
Peano A, Symoens F, de Hoog S
(2012) Microsporum mirabile and
its teleomorph Arthroderma mirabile, a new dermatophyte species
in the M. cookei clade. Med Mycol
50, 161-169.
Ebersberger I, de Matos Simoes R,
Kupczok A, Gube M, Kothe E, Voigt
K, von Haeseler A (2012) A consistent phylogenetic backbone for the
fungi. Mol Biol Evol 29, 1319-1334.
ME, Miadlikowska J, Wu S, Voigt
K, Walther G, Anishchenko IM,
Vilgalys R (2012) Molecular phylogeny of the Entomophthoromycota.
Mol Phylogenet Evol 65, 682-694.
Vitale RG, de Hoog GS, Schwarz P,
Dannaoui E, Deng S, Machouart
M, Voigt K, van de Sande WW,
Dolatabadi S, Meis JF, Walther G
(2012) Antifungal susceptibility
and phylogeny of opportunistic
members of the order mucorales. J
Clin Microbiol 50, 66-75.
Associated Group Microbial
Biochemistry and Physiology
Brock M (2012) Application of bioluminescence imaging for in vivo
monitoring of fungal infections. Int
J Microbiol 2012, 956794.
Citiulo F, Jacobsen ID, Miramón P,
Schild L, Brunke S, Zipfel P, Brock
M, Hube B, Wilson D (2012) Candida albicans scavenges host zinc via
Pra1 during endothelial invasion.
PLoS Pathog 8, e1002777.
Fazius F, Shelest E, Gebhardt
P, Brock M (2012) The fungal
α-aminoadipate pathway for lysine
1508-1530.
Fekkar A, Pionneau C, Brossas
JY, Marinach-Patrice C, Snounou
G, Brock M, Ibrahim-Granet O,
Mazier D (2012) DIGE enables the
detection of a putative serum biomarker of fungal origin in a mouse
model of invasive aspergillosis. J
Proteomics 75, 2536-2549.
Jouvion G, Brock M, Droin-Bergere
S, Ibrahim-Granet O (2012) Duality
of liver and kidney lesions after
systemic infection of immunosuppressed and immunocompetent
mice with Aspergillus fumigatus.
Virulence 3, 43-50.
Slesiona S, Greßler M, Mihlan M,
Zaehle C, Schaller M, Barz D, Hube
PLoS One 7, e31223.
Slesiona S, Ibrahim-Granet O,
Olias P, Brock M, Jacobsen ID
(2012) Murine infection models for
Aspergillus terreus pulmonary aspergillosis reveal long-term persistence of conidia and liver degeneration. J Infect Dis 205, 1268-1277.
Research Group Pharmaceutical
Microbiology
Fernandez-Fueyo E, Ruiz-Duenas
FJ, Ferreira P, Floudas D, Hibbett DS, Canessa P, Larrondo LF,
James TY, Seelenfreund D, Lobos
S, Polanco R, Tello M, Honda Y,
Watanabe T, Watanabe T, Ryu JS,
Kubicek CP, Schmoll M, Gaskell
J, Hammel KE, St John FJ, Vanden
Wymelenberg A, Sabat G, Splinter
BonDurant S, Syed K, Yadav JS,
Doddapaneni H, Subramanian V,
Lavin JL, Oguiza JA, Perez G, Pisabarro AG, Ramirez L, Santoyo F,
Master E, Coutinho PM, Henrissat
B, Lombard V, Magnuson JK, Kues
U, Hori C, Igarashi K, Samejima M,
Held BW, Barry KW, LaButti KM,
Lapidus A, Lindquist EA, Lucas
SM, Riley R, Salamov AA, Hoffmeister D, Schwenk D, Hadar Y,
Yarden O, de Vries RP, Wiebenga A,
Stenlid J, Eastwood D, Grigoriev IV,
Berka RM, Blanchette RA, Kersten
P, Martinez AT, Vicuna R, Cullen
D (2012) Comparative genomics
of Ceriporiopsis subvermispora
and Phanerochaete chrysosporium
provide insight into selective ligninolysis. Proc Natl Acad Sci U S A
109, 5458-5463.
Lackner G, Misiek M, Braesel J,
Hoffmeister D (2012) Genome mining reveals the evolutionary origin
and biosynthetic potential of basidiomycete polyketide synthases.
Fungal Genet Biol 49, 996-1003.
Misiek M, Hoffmeister D (2012)
Sesquiterpene aryl ester natural
products in North American Armillaria species. Mycol Prog 11, 7-15.
Olson A, Aerts A, Asiegbu F,
Belbahri L, Bouzid O, Broberg A,
Canbäck B, Coutinho PM, Cullen D, Dalman K, Deflorio G, van
Diepen LT, Dunand C, Duplessis S,
Durling M, Gonthier P, Grimwood
J, Fossdal CG, Hansson D, Henrissat B, Hietala A, Himmelstrand K,
Hoffmeister D, Högberg N, James
TY, Karlsson M, Kohler A, Kües U,
Lee YH, Lin YC, Lind M, Lindquist
E, Lombard V, Lucas S, Lundén K,
Morin E, Murat C, Park J, Raffaello
T, Rouzé P, Salamov A, Schmutz J,
Solheim H, Ståhlberg J, Vélëz H, de
Vries RP, Wiebenga A, Woodward
S, Yakovlev I, Garbelotto M, Martin
F, Grigoriev IV, Stenlid J (2012) Insight into trade-off between wood
decay and parasitism from the genome of a fungal forest pathogen.
New Phytol 194, 1001-1013.
Wackler B, Lackner G, Chooi
YH, Hoffmeister D (2012) Characterization of the Suillus grevillei
quinone synthetase GreA supports
a nonribosomal code for aromatic
alpha-keto acids. ChemBioChem 13,
1798-1804.
Peer Reviewed Articles 2013
Originalarbeiten 2013
Chemistry
Baunach M, Ding L, Bruhn T,
Bringmann G, Hertweck C (2013)
Regiodivergent N-C and N-N aryl
coupling reactions of indoloterpenes and cycloether formation
mediated by a single bacterial
flavoenzyme. Angew Chem Int Ed
52, 9040-9043.
Bretschneider T, Heim JB, Heine
D, Winkler R, Busch B, Kusebauch B, Stehle T, Zocher G, Hertweck C (2013) Vinylogous chain
branching catalysed by a dedicated
polyketide synthase module. Nature 502, 124-128.
Busch B, Ueberschaar N, Behnken S, Sugimoto Y, Werneburg
M, Traitcheva N, He J, Hertweck
C (2013) Multifactorial control
of iteration events in a modular
polyketide assembly line. Angew
Chem Int Ed 52, 5285-5289.
Coyne S, Chizzali C, Khalil MN,
Litomska A, Richter K, Beerhues L,
Hertweck C (2013) Biosynthesis of
the antimetabolite 6-thioguanine
in Erwinia amylovora plays a key
role in fire blight pathogenesis. Angew Chem Int Ed 52, 10564-10568.
pathway in the pathogenic fungus
Aspergillus fumigatus. ChemBioChem 14, 938-942.
Fazius F, Zähle C, Brock M (2013)
Lysine biosynthesis in microbes:
relevance as drug target and prospects for beta-lactam antibiotics
production. Appl Microbiol Biotechnol 97, 3763-3772.
Letzel AC, Pidot SJ, Hertweck C
(2013) A genomic approach to the
cryptic secondary metabolome of
the anaerobic world. Nat Prod Rep
30, 392-428.
Franke J, Ishida K, Ishida-Ito M,
Hertweck C (2013) Nitro versus
hydroxamate in siderophores of
pathogenic bacteria: effect of missing hydroxylamine protection in
malleobactin biosynthesis. Angew
Chem Int Ed 52, 8271-8275.
Gatte-Picchi D, Weiz A, Ishida K,
Hertweck C, Dittmann E (2014)
Functional analysis of environmental DNA-derived microviridins
provides new insights into the
diversity of the tricyclic peptide
family. Appl Environ Microbiol 80,
1380-1387. Epub 2013 Dec 13.
Grumbt M, Monod M, Yamada
T, Hertweck C, Kunert J, Staib
P (2013) Keratin degradation by
dermatophytes relies on cysteine
dioxygenase and a sulfite efflux
pump. J Invest Dermatol 133, 15501555.
Hennicke F, Grumbt M, Lermann
U, Ueberschaar N, Palige K,
Böttcher B, Jacobsen ID, Staib C,
Morschhäuser J, Monod M, Hube B,
Hertweck C, Staib P (2013) Factors
supporting cysteine tolerance and
sulfite production in Candida albicans. Eukaryot Cell 12, 604-613.
Hölscher D, Dhakshinamoorthy S, Alexandrov T, Becker M,
Bretschneider T, Buerkert A,
Crecelius AC, De Waele D, Elsen A,
Heckel DG, Heklau H, Hertweck C,
Kai M, Knop K, Krafft C, Maddula
RK, Matthäus C, Popp J, Schneider
B, Schubert US, Sikora RA, Svatoš
A, Swennen RL (2014) Phenalenone-type phytoalexins mediate
resistance of banana plants (Musa
spp.) to the burrowing nematode
Radopholus similis. Proc Natl Acad
Sci USA 111, 105-110. Epub 2013
Dec 9.
Kloss F, Pidot S, Goerls H, Friedrich T, Hertweck C (2013) Formation of a dinuclear copper(I) complex from the clostridium-derived
antibiotic closthioamide. Angew
Chem Int Ed 52, 10745-10748.
König CC, Scherlach K, Schroeckh
V, Horn F, Nietzsche S, Brakhage
AA, Hertweck C (2013) Bacterium
induces cryptic meroterpenoid
Mayeku PW, Hassanali A, Kiremire
BT, Odalo JO, Hertweck C (2013)
Anti-bacterial activities and phytochemical screening of extracts of
different parts of Thallictrum rhynchocarpum. Afri J Tradit Complement Altern Med, 10, 341-344.
Meyer F, Ueberschaar N, Dahse
HM, Hertweck C (2013) Synthesis
and biological evaluation of hydrazidomycin analogues. Bioorg Med
Chem Lett 23, 6043-6045.
Meyer F, Ueberschaar N,
Hertweck C (2013) Concise total
synthesis of hydrazidomycin A, a
rare hydrazide metabolite of Eur J
Org Chem 2013, 4242-4244.
Nützmann HW, Fischer J, Scherlach K, Hertweck C, Brakhage AA
(2013) Distinct amino acids of histone H3 control secondary metabolism in Aspergillus nidulans. Appl
Environ Microbiol 79, 6102-6109.
Scherlach K, Heinekamp T, Willing K, Brakhage AA, Hertweck C
(2013) Epidithiodiketopiperazine
biosynthesis: a four-enzyme cascade converts glutathione conjugates into transannular disulfide
bridges. Angew Chem Int Ed 52,
11092-11095.
Scherlach K, Lackner G,
Graupner K, Pidot S, Bretschneider T, Hertweck C (2013) Biosynthesis and mass spectrometric
imaging of tolaasin, the virulence
factor of brown blotch mushroom
disease. ChemBioChem 14, 24392443.
Ueberschaar N, Dahse HM,
Bretschneider T, Hertweck C
(2013) Rational design of an apoptosis-inducing photoreactive DNA
intercalator. Angew Chem Int Ed 52,
6185-6189.
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gene clusters. 2nd International
Workshop on Pattern Recognition
in Proteomics, Structural Biology
and Bioinformatics. In: ICIAP 2013
Workshops, Lecture Notes in Computer Science (LNCS) 8158, Petrosino A, Maddalena L, Pala P (eds),
pp. 389–398, Springer-Verlag,
Berlin, Heidelberg.
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(2012) 1. Gut Fungi: Classification,
Evolution, Life Style and Application. In: Fungi: Types, Environmental Impact and Role in Disease.
Series: Environmental Health
- Physical, Chemical and Biological
Factors. Silva AP, Sol M (eds), Nova
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(2013) Ecological and economical
importance of parasitic and zoosporic true Fungi In: The Mycota
Vol. XI: Agricultural Applications.
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243-270, Springer Verlag, Berlin,
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monitoring of fungal infections. Int
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relevance as drug target and prospects for beta-lactam antibiotics
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Józsi M (2012) Anti-Factor H autoantibodies in kidney disease:
Detection, pathogenic role, and relevance for treatment. In: Autoantibodies: Detection, Pathogenicity
and Health Implications. Jenkins
GE, Hall JI (eds), Hauppauge, Nova
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Memberships in Editorial Boards
2012/2013
Mitgliedschaften in Editorial
Boards 2012/2013
Axel A Brakhage
Applied Environmental
Microbiology
Applied Microbiology and
Biotechnology
Current Genetics
eLife
Eukaryotic Cell
Frontiers in Fungi and Their
Interactions
Frontiers in Microbiology
Matthias Brock
FEMS Microbiology Letters
Pathogens
Oliver Kurzai
Medical Mycology Case Reports
Journal of Basic Microbiology
Christine Skerka
Journal of Genetic Syndromes and
Gene Therapy
Kerstin Voigt
Frontiers in Evolutionary and
Population Genetics
Journal of Basic Microbiology
MycoKeys
Persoonia (Guest editor)
Peter F Zipfel
Molecular Immunology
Frontiers in Molecular Innate
Immunity
Marc Thilo Figge
Cytometry Part A, Special Issue
Image-based Systems Biology
FIAS Interdisciplinary Science
Series, Springer Int. Publishing
Frontiers in Computational Physics
Frontiers Special Topic Systems
Biology of Microbial Infection
ISRN Biomathematics
Journal of Computational Medicine
Lectures at the HKI 2012
Kolloquien am HKI 2012
Reinhard Guthke
BMC Systems Biology
Frontiers Special Topic Systems
Biology of Microbial Infection
Eduard Reithmeier
Leibniz-Universität Hannover
3D-Object-/Surface-Measurement
using a SEM with 4 EverhartThornley Detectors
07.03.2012
Host: Brakhage AA
Christian Hertweck
Bioorganic Chemistry
ChemBioChem
Chemistry & Biology
Organic and Biomolecular
Chemistry
The Journal of Antibiotics
Kerstin Hoffmann
Frontiers in Evolutionary and
Population Genetics
Uwe Horn
Microbiological Research
Bernhard Hube
BMC Microbiology
BMC Research Notes
Cellular Microbiology
FEMS Yeast Research
Frontiers in Fungi and Their
Interactions
Frontiers in Molecular Innate
Immunity
mBio
Pathogens
Ilse Jacobsen
Medical Mycology Case Reports
PLoS One
Olaf Kniemeyer
Archives of Microbiology
Christoph M. Tang
Sir William Dunn School of
Pathology, Oxford
Complement and infection of Neisseria meningitidis
24.01.2012
Host: Zipfel PF
Matthias Mörgelin
Lund University, Schweden
Antimicrobial properties of the
extracellular matrix
20.03.2012
Host: Zipfel PF
Candace Elliott
Faculty of Science School of Botany,
Melbourne
Unravelling the roles of peptide
synthetases and polyketide synthases in the canola pathogen
Leptosphaeria maculans
27.03.2012
Host: Hoffmeister D
Frank Martin Brunkhorst
Universitätsklinikum Jena
Die Gefährlichkeit von Krankenhausinfektionen
03.04.2012
Host: Brakhage AA, Stelzner A,
Zipfel PF
Kai Naumann
Leibniz-Institut für Pflanzenbiochemie, Halle
Label-free shotgun proteomics as
versatile tool in biological research
08.05.2012
Host: Kniemeyer O
Hortense Slevogt
Danger signals in der Pathogenese
der Sepsis
15.05.2012
Zipfel PF
Chambers C. Hughes
University of California, San Diego
The discovery and development of
marine microbial natural products
as therapeutic leads and cellular
probes
29.05.2012
Anja Forche
Bowdoin College, Brunswick, ME,
USA
Increase of genetic and phenotypic
diversity as an adaptation mechanism to changing host environments
05.06.2012
Host: Hube B, Brakhage AA
Mathias Pletz
Antibiotikaresistenzen
17.07.2012
Zipfel PF
Pierre Stallforth
Harvard Medical School
Metabolomic analysis of the social
amoeba’s bacterial symbionts reveals a highly adapted mutualistic
association
25.09.2012
Host: Habel A
Oliver Werz
Friedrich-Schiller-Universität Jena
Molekulare Mechanismen geschlechts-spezifischer Unterschiede bei entzündlichen Reaktionen
09.10.2012
Zipfel PF
Christine Beemelmanns
Harvard Medical School
Symbiotic and associated bacteria:
big players in evolution and rich
sources of new natural compounds
16.10.2012
Host: Habel A
Lectures at the HKI 2013
Kolloquien am HKI 2013
Thomas Lehrnbecher
Universitätsklinikum Frankfurt
Immunotherapeutic approaches to
invasive fungal infections 05.02.2013
Host: Brock M
Gregers Rom Andersen
University of Aarhus, Denmark
Structural basis for proteolytic
cleavage of complement C4 and C5
19.02.2013
Host: Zipfel PF
Brandon Jutras
University of Kentucky, College of
Medicine
20.03.2013
Host: Zipfel PF
Richard Pott
Leibniz-Universität Hannover
Biodiversitätskrise – Das sechste
Massensterben auf der Erde?
16.04.2013
Host: Brakhage AA
Walter Doerfler
Friedrich-Alexander Universität
Erlangen-Nürnberg
Epigenetics – a different way
of looking at genetics
23.04.2013
Zipfel PF
Thorsten Heinzel
Lysine acetylation as a regulatory
protein modification
14.05.2013
Zipfel PF
Frank Ebel
Max von Pettenkofer-Institut, LMU
München
Woronin bodies - their anchoring
at the septal pore and contribution
to the stress resistance of Aspergillus fumigatus
23.07.13
Host: Köster-Eiserfunke N
Mike Bromley
University of Manchester
Antifungal resistance and drug
discovery
22.08.13
Utz Reichard
Universitätsmedizin Göttingen
Aspergillus fumigatus and vaccines
- is there hope?
05.12.13
Andreas Holzenburg
Microscopy and Imaging Center,
Texas A&M University
Microscopy and Imaging in the
Life Sciences
19.12.13
Host: Brakhage AA
Scientific Awards 2012
Preise und Auszeichnungen 2012
Böhm, Sascha
Posterpreis
XXIV International Complement
Workshop, Kreta, Griechenland
2012
Bretschneider, Tom
Posterpreis
Irseer Naturstofftagung
2012
Chankhamjon, Pranatchareeya
medac-Forschungspreis
medac GmbH, Wedel
2012
Chen, Qian
Else Körner-Fresenius Posterpreis
43. Jahrestagung der Gesellschaft
für Pädiatrische Nephrologie
2012
Chen, Qian
Vortragspreis
2012
Eberhardt, Hannes
Posterpreis
2012
Greßler, Markus
Posterpreis
European Fungal Genetics Conference, Marburg
2012
Hallström, Teresia
Vortragspreis
2012
Heinekamp, Thorsten
medac GmbH, Wedel
2012
Horn, Fabian
Vortragspreis, 5. ILRS Symposium
ILRS
2012
Kasper, Lydia
Vortragspreis
DFG Schwerpunkt-Meeting 1580,
Bonn
2012
Kreutzer, Martin
Vortragspreis MiCom
JSMC
2012
Kreutzer, Martin
Vortragspreis VAAM International
Workshop Biology and Chemistry
of Antibiotic-Producing Bacteria
and Fungi
VAAM
2012
Letzel, Anne-Catrin
Posterpreis, VAAM-Naturstofftagung
Liebig-Vereinigung der Gesellschaft deutscher Chemiker
2012
Roth, Martin
medac GmbH, Wedel
2012
Scharf, Daniel
medac GmbH, Wedel
2012
Scherlach, Kirstin
medac GmbH, Wedel
2012
Seccareccia, Ivana
Posterpreis MiCom
JSMC
2012
Seider, Katja
Forschungspreis der DGHM Fachgruppe Eukaryontische Krankeitserreger
DGHM
2012
Thywißen, Andreas
Posterpreis
Keystone Symposium, Santa Fe,
USA
2012
Wolf, Thomas
Vortragspreis MiCom
JSMC
2012
Scientific Awards 2013
Preise und Auszeichnungen 2013
Behnken, Swantje
DECHEMA Doktorandenpreis
DECHEMA
2013
Brunke, Sascha
Vortragspreis FEBS Young Investigator Award
5th FEBS Advanced Lecture Course
2013
Buhlmann, Denise
Posterpreis
14th European Meeting on Complement in Human Disease, Jena
2013
Chankhamjon P, Scherlach K,
Hertweck C
Publikationspreis der DMykG
Stiftung der Deutschsprachigen
Mykologischen Gesellschaft
2013
221
Chen, Qian
Vortragspreis
14th European Meeting on Complement in Human Disease, Jena
2013
Citiulo, Francesco
2013
Kästner, Julia
Young Scientist Presentation
Award
2nd European Meeting on Animal
Chlamydioses and Zoonotic Implications (EMAC-2), Jena
2013
Kloss F, Pidot S, Goerls H, Friedrich T, Hertweck C
Posterpreis des VAAM Workshop
Biology of Bacteria Producing
Natural Products
VAAM
2013
Mayer, Francois
Young Investigator Award der
DGHM Fachgruppe Eukaryontische
Krankeitserreger
DGHM
2013
Figge MT
Jena, Germany
2012
3rd International Student Conference on Microbial Communication
(MiCom 2012)
Pollmächer J, Sarkar S
Jena, Germany
2012
5th ILRS Symposium
Vogler C, Zipfel PF
Jena, Germany
2012
11th European Congress on Fungal
Genetics (ECFG 11)
Brakhage AA
Marburg, Germany
2012
11th European Conference on
Computational Biology
Figge MT
Basel, Switzerland
2012
11th International Conference on
Artifical Immune Systems
Figge MT
Taormina, Italy
2012
18th Congress of the International
Society for Human and Animal
Mycology (ISHAM) 2012
Hube B, Brock M
Berlin, Germany
2012
Scharf, Daniel
medac Forschungspreis
medac GmbH, Wedel
2013
Scharf, Daniel, Chankhamjon P,
Scherlach K, Hertweck C
2013
Scharf, Daniel
Promotionspreis
Beutenberg Campus e.V.
2013
BMT 2012 – 46 Jahrestagung der
Deutschen Gesellschaft für Biomedizinische Technik
(section on imaging and pattern
recognition)
Saluz HP
Jena, Germany
2012
German Conference on Bioinformatics 2012
Figge MT, Guthke R
Jena, Germany
2012
Scharf, Daniel
Promotionspreis
VAAM
2013
Ueberschaar N, Dahse H-M,
Bretschneider T, Hertweck C
Posterpreis, VAAM Workshop Biology of Bacteria Producing Natural
Products
VAAM
2013
Meetings, Workshops,
Symposia 2012/2013
Wissenschaftliche
Veranstaltungen 2012/2013
1 International Workshop
“Image-based Systems Biology”
st
222
Irseer Naturstofftage
Hertweck C
Irsee, Germany
2012
Marie-Curie ITN ARIADNE
Summer School
Kniemeyer O et al.
Jena, Germany
2012
1st International Conference
on Natural Products
Brakhage AA
Frankfurt, Germany
2013
2nd International Workshop
Systems Biology of Microbial
Infection, S2B0M1I3
Figge MT, Guthke R
Jena, Germany
2013
6th ILRS Symposium
Vogler C, Zipfel PF
Jena, Germany
2013
14th European Meeting on
Complement in Human Disease
Zipfel PF, Skerka C,
Hallström T et al.
Jena, Germany
2013
CBS Workshop Emerging
Zygomycetes, a new problem
in the clinical lab
A workshop of the ECMM/ISHAM
Working Group on Zygomycetes
Voigt K et al.
Utrecht, The Netherlands
2013
FEBS Advanced practical course
“State-of-the-art infection models
for human pathogenic fungi”
Hube B, Jacobsen ID
Jena, Germany
2013
Irseer Naturstofftage
Hertweck C
Irsee, Germany
2013
Jahrestagung der Deutschsprachigen Mykologischen
Gesellschaft DMykG
Kurzai O
Tübingen, Germany
2013
Saluz HP
10/2008 – 01/2012
Teilprojekt 39: Analysis of the interaction of the human pathogenic
fungus Aspergilllus fumigatus with
immune effector cells by functional
genomics
Brakhage AA
09/2009 – 12/2012
Teilprojekt 52: The interaction
of Candida glabrata with human
Neutrophils
Kurzai O
11/2010 – 07/2014
Teilprojekt 55: Development of a
multi-scale simulator to model
host-pathogen interactions
Figge MT
10/2011 – 09/2015
Teilprojekt 56: Transcriptome
data analysis and modeling of
Aspergillus fumigatus - phagocytes
interaction
Guthke R
08/2011 – 07/2014
Teilprojekt 58: Molecular basis for
predatory interactions of Cupriavidus necator wich ist prey bacteria
Nett M
10/2011 – 09/2014
Teilprojekt 59: Functional analysis
of major transcriptional regulators
in pathogenic dermatophytes
Brakhage AA
01/2013 – 09/2014
Teilprojekt 67: Bacterial endosymbionts in plant-pathogenic fungi
Hertweck C
11/2012 – 10/2015
Participation in Research
Networks 2012/2013
Beteiligung an Netzwerken und
Verbundprojekten 2012/2013
Teilprojekt 72: Interactions of the
fungal pathogen Aspergillus fumigatus with human macrophages
Brakhage AA
11/2012 – 10/2015
Exzellenzgraduiertenschule
Jena School for Microbial
Communication
Teilprojekt 77: Exploiting the metagenome of microbial communities
on a microfluidic platform
Horn U
03/2013 – 02/2016
Teilprojekt 6: Molecular Basis of
Bacterial-Fungal Symbioses
Hertweck C
11/2007 – 10/2012
Teilprojekt 21: MicroCom – Host
pathogen interactions of human
pathogenic yeast
Hube B
10/2008 – 01/2012
Teilprojekt 31: Comparative
genomics of host/chlamydiales
interactions
Teilprojekt 78: Infection-associated
genes of Candida glabrata
Hube B
02/2013 – 02/2016
Teilprojekt 79: Immunevasion
of the Gram positive bacterium
Streptococcus pneumoniae
Zipfel PF
10/2013 – 09/2016
Teilprojekt 83: Pathogen/host communication: dual RNA expression
dynamics to investigate differential patterns of virulence
Saluz HP
10/2013 – 10/2016
Teilprojekt 87
Shelest E
10/2013 – 09/2016
Teilprojekt 89: Soil amoeba interactions with filamentous fungi as
driving forces for pathogenicity
Hillmann F
10/2013 – 01/2017
Teilprojekt 92: Mechanisms of
Candida albicans colonization and
translocation
Jacobsen ID
02/2013 – 01/2017
Sonderforschungsbereich/
Transregio 124 FungiNet
Teilprojekt B03: Prädiktive Modellierung von Wirth-Pathogenen-Interaktionen durch Rekonstruktion
genregulatorischer Netzwerke
Guthke R, Shelest E
10/2013 – 06/2017
Teilprojekt B04: Bilddatenanalyse
und Agenten-basierte Modellierung der Wechselwirkung zwischen Immunzellen und humanpathogenen Pilzen in Raum und Zeit
Figge MT
10/2013 – 06/2017
Teilprojekt C01: Charakterisierung
molekularer Mechanismen der
Translokation von Candida albicans durch Epithelgewebe
Hube B
10/2013 – 06/2017
Teilprojekt C04: Mechanismen der
Adhäsion und Modulation humaner Immunzellen durch Candida
albicans
Skerka C
10/2013 – 06/2017
Teilprojekt C05: Mechanismen der
gastrointestinalen Kolonisierung
und der Translokation von Candida
albicans durch die Darmschleimhaut
Jacobsen ID
10/2013 – 06/2017
Teilprojekt C06: Einfluss sekretierter Proteine von Candida albicans
bei der Immunevasion und der
Pathogenität
Zipfel PF
10/2013 – 06/2017
Teilprojekt INF: Integrierte Datenbank für Experimentaldaten (Data
Warehouse OmniFung)
Guthke R
10/2013 – 06/2017
Teilprojekt Z01: Projektmanagement
Brakhage AA
10/2013 – 06/2017
Teilprojekt Z01: Die Rolle der
Regulation putativer Pathogenitätsassoziierter Gene in der Virulenz
des humanpathogenen Mucormykose-Erregers Lichtheimia corymbifera: Vergleichende RNAseq-Expressionsanalyse von attenuiertem
versus virulentem Stamm
Voigt K
10/2013 – 12/2013
Teilprojekt Z02: Methoden zur
Analyse der zeitlichen und räumlichen Änderung der Proteome bei
der Interaktion pilzlicher Pathogene mit dem Wirt
Kniemeyer O
10/2013 – 06/2017
Schwerpunktprogramm 1580:
Intracellular Compartments
as Places of Pathogen-Host Interaction
Survival and proliferation of human pathogenic Candida species
within phagocytes
Hube B
10/2010 – 08/2014
Forschergruppe FOR 1334:
Determinants of Polarized
Growth and Development in
Filamentous Fungi
Redox regulation, development
and hyphal growth in Aspergillus
nidulans
Brakhage AA, Hortschansky P
07/2010 – 06/2013
Europäische Union,
7. Forschungsrahmenprogramm
Marie Curie International
Research Staff Exchange Scheme
KRAB-ZNF: KRAB zinc finger
gene biology in evolution and
disease
Guthke R
07/2011 – 06/2013
Marie Curie Initial Training
Network
ARIADNE – Signaling circuitry
controlling fungal virulence:
identification and characterization of conserved and specific
fungal virulence genes as common antifungal targets
Brakhage AA, Heinekamp T
01/2010 – 12/2013
Network
Quantfung – Quantitative Biology for Fungal Secondary Metabolite Producers
Brakhage AA
10/2013 – 09/2017
Network
FINSysB – Pathogenomics and
Systems Biology of Fungal Infections – An Integrative Approach
Hube B
11/2008 – 09/2012
Large-scale Integrating Project
EURenOmics – European Consortium for High-Throughput Research in Rare Kidney Diseases
WP 05: Complement Disorders
Zipfel PF
10/2012 – 10/2016
Bundesministerium für Bildung
und Forschung
DiNaMid: Genom-basierte Findung neuer antimikrobieller
Naturstoffe in mikrofluidischen
Chips
Teilprojekt: Identifizierung neuer
Stoffwechselwege und NaturstoffIsolierung
Nett M, Roth M
10/2009 – 12/2012
ERA-NET AspBIOmics
Biomarker zur Therapie von invasiver Aspergillosis
Kniemeyer O
05/2011 – 10/2014
ERA-Net – CandiCol
Grundlagen zur Kolonisation und
der Dissemination pathogener
Candida-Spezies: Entwicklung früher Diagnose- und Therapieansätze
Hube B
05/2011 – 04/2014
ERA-NET OXYstress
Humapathogene Pilze unter
Sauerstoff-Stress: Adaptation an
Sauerstoffmangel und reaktive
Sauerstoffspecies und die Folgen
für die Interaktion mit dem Wirt
und die Therapie
Heinekamp T
04/2011 – 09/2014
ERA-NET PathoGenoMics 2
Apergillosis – Die Zellwand als
Target zur Verbesserung der antifungalen Therapie der Aspergillose
Kniemeyer O
02/2009 – 05/2012
ERASysBio – LINCONET
Modellierung des genregulatorischen Netzwerks für die Linienspezifische Differenzierung in
menschlichen mesenchymalen
Stammzellen: Identifikation von
potenziellen Zielmolekülen für die
anabole Therapie von Gewebserkrankungen
Guthke R
03/2010 – 08/2013
GenBioCom: Genombasierte
Produktion bioaktiver Verbindungen aus Aktinomyceten
Komplexe Polyketide aus Aktinomyceten: Genom-Analyse, Gensynthese und Aktivitätsbestimmung
Hertweck C
03/2010 – 07/2013
GERONTOSYS II
Rolle von oxidativen Stress in
der Alterung und therapeutische
Implikationen
Guthke R
03/2011 – 05/2014
Integrierte Forschungs- und Behandlungszentrum (IFB) Sepsis
und Sepsisfolgen (CSCC)
In-house Professorship Molekulare
Mechanismen der Candida-Sepsis
Hube B, Jacobsen ID
Integriertes Forschungs- und Behandlungszentrum (IFB) Sepsis
Project: Inprot Identifizierung
von C. albicans und A. fumigatus
Proteinantigenen für die Pilzsepsisdiagnostik und Untersuchung
der A. fumigatus Stressantwort
Kniemeyer O
Integriertes Forschungs- und Behandlungszentrum (IFB) Sepsis
Project: Pathogen-Wirt-Interaktion
während der Infektion mit Candida
albicans und die Entwicklung zur
Sepsis
Hube B, Brunke S
Jenaer Centrum für die Systembiologie des Alterns – JenAge
Systembiologie von mildem Stress
beim gesunden Altern ein MultiSpezies-Ansatz
Guthke R
10/2009 – 09/2014
Kompetenznetz
Die Virtuelle Leber
Guthke R
04/2020 – 03/2015
PET-CT II
In-vivo-Quantifizierung von Inflammation und Destruktion in
murinen Arthritismodellen mittels
PET/CT als Beitrag zur Reduktion
der Zahl der in Forschung und
Entwicklung verwendeten Versuchstiere
Saluz HP
11/2011 – 10/2014
223
Resistom – Antibiotika-Resistom
in der Lebensmittelkette
Krügel H
04/2011 – 03/2012
SPICE III – Wissenschaftlichtechnische Zusammenarbeit mit
Indonesien
MABICO – Einfluss von Meeresverschmutzungauf Biodiversität und
den Lebensunterhalt von Küstenbewohnern; Mikrobielle Biodiversität
Saluz HP
03/2012 – 02/2015
Technologietransfer
Verstetigung der Verwertungskonzepte in den lebenswissenschaftlichen Instituten der WGL – LeibnizInstitut für Naturstoff-Forschung
und Infektionsbiologie e.V. – HansKnöll-Institut - (HKI)
Ramm M
01/2009 – 03/2012
Verbundvorhaben Medizinische
Infektionsgenomik:
Wirt-Pathogen-Interaktion: Wirkung sekretierter Proteine von Staphylococcus aureus auf Zellen und
Komponenten des Immunsystems
Zipfel PF
10/2010 – 12/2013
ZIK Septomics
Forschungsgruppe Fungal Septomics: Molekulare Mechanismen in
der Pathogenese der Sepsis durch
Pilze
Brakhage A, Kurzai O
11/2009 – 10/2014
Zoonotische Chlamydien
Teilprojekt 4: Wechselwirkung
von zoonotischen Chlamydien mit
ihren Wirtszellen
Saluz HP, Hänel F
0172011 – 04/2014
Zwanzig20 – Partnerschaft für
Innovation
InfectControl 2020 – Neue Antiinfektionsstrategien – Wissenschaft
• Gesellschaft • Wirtschaft; Strategievorhaben
Brakhage AA
Weitere Verbundprojekte
Integriertes Multiplex Genotyping
System basierend auf einem hyperspektralen Vielfarben-Scanner
zur schnellen qualitativen und
quantitativen Sequenzanalyse von
SNPs und DNA-Polymorphismen
auf Microarrayoberflächen
Saluz HP
10/2009 – 09/2012
Freistaat Thüringen,
ProExzellenz
NanoConSens – Nanocontainer
224
und nanostrukturierte Trägermaterialien für Sensorik und Wirkstofftransport
Brakhage AA, Hertweck C
10/2009 – 09/2012
Freistaat Thüringen,
ProExzellenz
MikroInter – Characterisation of
receptors of the human-pathogenic
fungus Aspergillus fumigatus - How
does the fungus communicate with
the environment and the host
Brakhage AA
04/2010 – 03/2013
German-Israeli Foundation
The hypoxic response in the pathogenic mold Aspergillus fumigatus
and its relevance to disease
Kniemeyer O
01/2010 – 06/2013
Leibniz-Gemeinschaft
Kryostress – Anpassungsmechanismen der Zelle an Tiefstemperaturen (KAIT)
Kniemeyer O
05/2013 – 05/2017
NIH
Collaborative Study of Membranoproliferative Glomerulonephritis
Type II
Zipfel PF
09/2007 – 04/2012
Calls for Appointments
2012/2013
Rufe 2012/2013
Dr. habil. Guthke, Reinhard
apl. Professor für Systembiologie
an der Friedrich-SchillerUniversität Jena (2011)
Prof. Hoffmeister, Dirk
Professor für Pharmazeutische
Biologie an der Friedrich-SchillerUniversität Jena
Ruf an die Universität des
Saarlandes, Saarbrücken (2013)
– abgewendet –
PD Jacobsen, Ilse, PhD
Ruf an die Universität Leipzig
(2013)
– abgewendet –
Postdoctoral Lecture
Qualifications 2012/2013
Habilitationen 2012/2013
Ilse Jacobsen, PhD
Infektionsmodelle zur Untersuchung der Pathogenese von
Pilzinfektionen
2013
Graduations 2012
Promotionen 2012
Brendel, Nicole
Molekularbiologische Untersuchungen und Manipulation der
Rhizoxin-Biosynthese in Burkholderia rhizoxinica
Kusebauch, Björn
Biogenese und synthetische Modifizierung des antimitotischen
Makrolids Rhizoxin
Barthel, Diana
Die Rolle von Proteasen bei der
Immunevasion von pathogenen
Mikroorganismen
Braukmann, Maria
Wirt-Pathogen-Wechselwirkungen
in Chlamydia psittaci- und Chlamydia abortus-infizierten Hühnerembryonen
Linde, Jörg
Systems biology of human pathogenic fungi: Modelling transcriptional networks of virulence
Mayer, François
Indentification and characterization of novel infection-associated
genes in Candida albicans
Mech, Franziska
Image-based Systems Biology of
Human-Pathogenic Fungi
Müller, Sebastian
Application of Bayesian statistics
in natural product research and
infection biology
Nützmann, Hans-Wilhelm
Protein acetyltransferases of
Aspergillus nidulans – impact on
secondary metabolism and fungal
– bacterial interaction
Bruns, Sandra
Interaktion des human-pathogenen
Schimmelpilzes Aspergillus fumigatus mit humanen Neutrophil
Extracellular Traps: Induktion und
Evasion
Scharf, Daniel
Neue Mechanismen der Regulation
und Biochemie von Penicillin- und
Gliotoxin-Biosynthese in Aspergillus sp.
Heddergott, Christoph
Sekretierte Pathogenitätsfaktoren
des Dermatophyten Arthroderma
benhamiae
Seider, Katja
Immune evasion as a pathogenicity
strategy of Candida glabrata: survival within macrophages
Horn, Fabian
Integrative Analysis of Omics-data
for Aspergillus nidulans
Slesiona, Silvia
Etablierung von Infektionsmodellen zur Untersuchung invasiver
Aspergillosen durch Aspergillus
terreus
Freie Universität
Icke, Selina
Rolle von Akut-Phase-Proteinen
und Komplement in entzündlichen
Prozessen
Koch, Tina
Evasionsstrategien pathogener
Bakterien gegen die angeborene
Immunreaktion
Kopp, Anne
Crosstalk between the long pentraxin PTX3 and soluble complement inhibitors
Kuhnert, Anja
Die Rolle des multifunktionellen
Proteins TopBP1 bei der Initiation
von Replikation und DNA-Schadensantwort
Svobodová, Eliška
Interaction of Candida albicans and
Candida dubliniensis with cells of
the human innate immune system
Voigt, Anja
Comperative genomics of chlamydial pathogens
Wartenberg, Dirk
Identifizierung und Charakterisierung von Effektorproteinen bei
Aspergillus fumigatus und Aspergillus nidulans
Graduations 2013
Promotionen 2013
Behnken, Swantje
Genome mining of bacteria leads to
closthioamide, the first antibiotic
from the anaerobic world
Bereza, Magdalena
Assessment of structural diversity
of ß-amyloid with genetically engineered antibody fragments
Böhm, Sascha
Immune evasion proteins from
Staphylococcus aureus and therapeutic evaluation of staphylococcal
complement-controlling proteins
Boettger, Daniela
Exploring the biosynthetic potential of PKS and NRPS pathways in
filamentous fungi
Bretschneider, Tom
In-vitro-Charakterisierung nichtkanonischer Katosynthasen und
Imaging-Massenspektrometrie von
Naturstoffen
Döring, Nadia
Die Rolle der Plasmaproteine Kallikrein, Plasmin und beta2-Glykoprotein I (ß2GPI) bei der Regulation
des alternativen Komplementaktivierungsweges.
Eberhardt, Hannes
The role of CFHR proteins in human autoimmune diseases
Friedrich, Lydia
Etablierung des Hühnerembryonenmodells zur Darstellung
von Infektionen mittels Positronenemissionstomographie/Computertomographie (PET/CT) am Beispiel
von Chlamydophila psittaci
Heidrich, Alexander
A glance through the shell: establishing the chick embryo as in vivo
model system for preclinical molecular imaging by PET and CT
Kupfer, Peter
Dynamic modeling of stimulated
cells involved in cartilage and bone
degenerative diseases
Lopez, Crisanto
Immunoevasion of pathogenic
microbes
Miramón, Pedro
Molecular dissection of the interactions between Candida albicans
and polymorphonuclear granulocytes
Priebe, Steffen
Transcriptome analysis of multiple
species using novel sequencing
technologies
Thywißen, Andreas
Molekulare Mechanismen der Interaktion von Aspergillus fumigatus
mit Makrophagen
Voigt, Jessica
The role of NK cells in immuneresponse against Candida albicans
Weber, Michael
Integrative Methods for reconstruction of dynamic networks in
chondrogenesis
Bachelor / Master / Diploma
Theses 2012
Bachelor-/ Master-/ Diplomarbeiten 2012
Adam, Jonathan
Approaches to detecting boundaries of biosynthetic gene clusters
(pathways) in bacterial genomes
Bartz, Christoph
Charakterisierung von Aspergillus
fumigatus Gendeletionsmutanten
unter hypoxischen Wachstumsbedingungen
Fachhochschule Jena
Bauer, Laura
Konstruktion und Verifizierung
von filamentösen und filamentdefizienten Candida albicans Stämmen
unter Einsatz des Tetrazyklinregulierbaren Expressionssystems
Baunach, Martin
Aufklärung der Indolterpenbiosynthese in Mangrovenendophyten
Bens, Martin
Analyse der transkriptionellen
Antwort von Rattenhepatozyten
auf Hepatotoxine
Bonkowski, Katharina
Die Rolle der DNasen von Aspergillus fumigatus beim Stickstoffmetabolismus und der Pathogenese
Bresan, Stefanie
Epidemiologie von Klebsiella ESBLStämmen
Brockmöller, Thomas
Metabolische Rekonstruktion des
Malonyl-CoA-Biosynthese-Weges
in Aspergillus nidulans
Buhlmann, Denise
Expression und Charakterisierung
des humanen löslichen Komplementrezeptors Typ 2 (CR2/CD21)
Conrad, Theresia
Die Bedeutung von Single Nucleotide Polymorphismen (SNPs) der
Alarmin-erkennenden Rezeptoren
MINCLE und RACE für die Suszeptibilität und den Krankheitsverlauf
bei Sepsis
Dettling, Anna
Hyperspektrale Analyse von Single-Nucleotide-Polymorphisms auf
Chipoberflächen am Beispiel von
Chlamydia psittaci
Fachhochschule Jena
Dix, Andreas
MicroRNA-Seq data analysis for
age-related comparison of mouse
and short-lived fish Nothobranchius
furzeri
Fischer, Juliane
Impact of selected histone H3
amino acid residues on Aspergillus
nidulans secondary metabolism and
TAP-tagging of SAGA/ADA complex
Hochschule Lausitz (FH) Senftenberg
Freitag, Janine
Vorhersage von MicroRNAs in
Candida albicans
Fricke, Markus
UV-Laser ChIP-Seq in vivo: Bioinformatische Analyse von genomweiten Protein-DNA-Interaktionen
Hoffmann, Bianca
Export dynamischer Modelle aus
NetGenerator und TILAR in SBML
scriptomics of secondary metabolite gene clusters in filamentous
fungi
Jacksch, Susanne
Complement evasion of Candida
albicans - Characterization of Pra1
Kämpfer, Philipp
Phylogeny of MADS-box transcription factors in fungi.
Kupis, Sabine
Das embryonierte Hühnerei als
Modellsystem für die molekulare
Bildgebung bei Infektion mit Chlamydia psittaci
Fachhochschule Jena
Lauterbach, Tom
Genom-basierte Suche nach Naturstoffen aus Rhizobium radiobacter
Manig, Steffi
Molekularbiologische Untersuchungen zur Funktion von Cys3
und Cys4 in Candida albicans
Fachhochschule Jena
Meyer, Florian
Synthese von Hydrazidomycinen
und Strukturanaloga
Molina Rodríguez, Juan Camilo
Investigation of iron acquisition
genes predicted by a Candida albicans regulatory network model
Pflieger, Astrid
Untersuchung und Optimierung
der Netropsinbildung durch Streptomyces netropsis
Fachhochschule Jena
Raguse, Marina
Identification of complement regulator binding sites and functional
domains of Lipoamide dehydrogenase (Lpd) of Pseudomonas
aeruginosa
Schaarschmidt, Barbara
Charakterisierung monoklonaler
Mausantikörper hinsichtlich ihrer
Spezifität gegenüber verschiedener
humanpathogener Pilze
Sehnert, Philipp
Funktionelle Analyse der Genexpression in humanen B-Lymphozyten
Hornung, Bastian
Comparative genomics and tran-
225
Shabuer, Gülmire
Strategie zur Aktivierung stiller
Gencluster in Clostridium acetobutylicum und Clostridium cellulolyticum
Sheikh MD, Rajuiddin
The role of methylthioadenosine
phosphorylase in virulence and immune evasion of Candida albicans
Tennstedt, Sebastian
Untersuchungen zur Einstellung
von Temperaturregelkreisen an
Bioreaktoren im pilottechnischen
Maßstab
Fachhochschule Jena
Tovar Ballen, Miguel Angel
Establishment of appropriate conditions for culturing Actinobacteria
in picolitre droplets: a microfluidic approach for novel antibiotic
screening
Technische Universität Dresden
Ulbricht, Andrea
Biosynthese von Indol-3-Essigsäure in Basidiomyceten
Üzüm, Zerrim
The role of chitinolysis and exopolysaccharide in Rhizopus microsporus and Burkholderia rhizoxinica endosymbiosis
Wagner, Lysett
Light microscopical and molecular
identification of terrestrial fungi
from taxonomic groups in Mortierella (Zygomycetes)
Walther, Elisabeth
Pathobiology of chlamydial epitheliocystis agents
Fachhochschule Jena
Wang, Ding
Analyse und Visualisierung genomweiter DNA-Polymorphismusdaten in R
Weist, Corinna
Immunevasion von Tumorzellen
durch lösliche und membrangebundene Komplementregulatoren
Wolf, Thomas
Analyse der transkriptionellen
Regulation von SekundärmetabolitGen-Clustern
Ziereisen, Jana
Charakterisierung der Komplementaktivierung und der Autoantikör-
226
per in den Seren von Patienten mit
den Nierenerkrankungen EHECHUS und MPGN
Bachelor / Master / Diploma
Theses 2013
Bachelor-/ Master-/ Diplomarbeiten 2013
Albán Proaño, María Cristina
Role of Candida albicans Eed1
protein in morphogenesis and
pathogenicity
Allert, Stefanie
Der Einfluss spezifischer Gene der
humanpathpogenen Hefe Candida
glabrata auf das Überleben in
Makrophagen
Baumeister, Tim
Charakterisierung Anthranilatbasierter Naturstoffe aus Amycolatopsis nigrescens
Bick, Jochen
Mapping non-coding RNAs in the
transcriptome of a bovine Chlamydia psittaci strain DC15
Böttger, Marco
Biochemische Untersuchung der
6-Thioguanin-Biosynthese in Erwinia amylovora
Fachhochschule Jena
Bredy, Florian
Funktionelle Charakterisierung
von Complement Factor H related
Protein 4 (CFHR4) A, B und C im
alternativen Weg der Komplementaktivierung
Dunker, Christine
Role of glutathione peroxidases
in the oxidative stress resistance
of the fungal pathogen Candida
albicans
Fichtner, Maximilian
Ermittlung spezies-spezifischer
Virulenzfaktoren in pathogenen
Candida-Spezies
Fischer, Daniel
Mikroevolution von Candida
glabrata: Untersuchungen zu genetischen Veränderungen eines
hypervirulenten Stamms
Gröning, Stephanie
RhiG, an unusual acyltransferase
involved in rhizoxin biosynthesis
Hartmann, Anja
Potential kryptischer BiosyntheseGencluster von Clostridium cellulolyticum
Helfrich, Eric
MALDI imaging and genome mining for natural product discovery
in Clostridia and Cyanobacteria
Hess, Carolin
Charakterisierung der immunmodulatorischen Proteine Pra1 und
Gpd2 von Candida albicans
Hofmann , Susann
Herstellung und phänotypische
Charakterisierung von Mutanten
im Pentose-Phosphat-Weg des
pathogenen Schimmelpilzes Aspergillus fumgiatus
Fachhochschule Jena Jena
Kaulfuß, Toni
Untersuchungen zur Interaktion
von humanen natürlichen Killerzellen mit dem pathologischen
Hefepilz Candida albicans
Fachhochschule Jena
Koske, Iris
Dihydrolipoamide dehydrogenase
(Lpd) of Pseudomonas aeruginosa binds to extracellular matrix
(ECM) proteins and mediates adherence to human cells
Kretzschmar, Tom
Einfluss von löslichem Komplementrezeptor 2 auf die Aktivierung von Immunzellen
Manzke, Melanie
Deregulation der Komplement
C3 Konvertase durch ein CFHRHybridprotein
Meinel, Christian
Characterization of the immune
evasion proteins PspC and Tuf of
two clinical Streptococcus pneumoniae isolates
Mohamed, Nizar Mustafa Yousif
Immune signals activated in mammalian host cells during interaction with Chlamydiales
Universitätsklinikum Freiburg und
Neuwirth, Toni
Totalsynthese von Heterocyclen
aus Aspergillus spp.
Opel, Viktoria
Komplexe Polyketide aus Burkholderia gladioli
Pann, Patrick
Interaction of the human-pathogenic fungus Aspergillus fumigatus
with neutrophil granulocytes – the
role of DNases
Universität Rostock
Piegsa, Judith
Role of NF-kB in epithelial barrier
maintenance during the interaction with C. albicans
Säuberlich, Marco
Konstruktion und Charakterisierung von rekombinanten VHHAntikörpern auf der Basis des
kameliden Antikörpers B10 zur
Produktion in E. coli mittels Hochzelldichtefermentation
Hochschule Zittau / Görlitz
Schmidt, Ann-Katrin
Participation of different innate
immune reseptors in recognition of
C. albicans in a whole blood model
of infection
Schmidt, Franziska
Untersuchung der intrazellulären
Prozessierung von Aspergillus
fumigatus Konidien durch Makrophagen
Schnell, Bastien
Genome mining for new natural
products in the bacterium Cupriavidus basilensis
Université Paris-Sud XI
Schreiner, Maria
Genetische Ursachen für die Wiedererlangung der Filamentbildung
einer evolvierten nicht filamentösen Candida albicans-Mutante
Schult, Kristin
Expression und Lokalisation des altersabhängigen Makulardegeneration assoziierten Proteins ARMS2
in humanen Zellen
Schulze, Sylvie
Prediction of a gene regulatory
network of MAPK- and NFƙBsignaling pathway components
under Candida albicans infection
Seibel, Elena
Analyse und Manipulation der
Indolosesquiterpen-Biosynthese
aus einem Mangroven-Endophyten
Shopova, Iordana
The effects of extracellular DNA
on Aspergillus fumigatus biofilm
formation
Thamm, Martin
Versuche zur Messung des intrazellulären pH-Wertes in einzelnen
Mono Mac 6-Zellen während der
Infektion mit Aspergillus-Sporen
Timme, Sandra
Automated estimation of motility parameters from agent-based
simulations of host-pathogen interactions
Wallstabe, Lars
Untersuchungen zur Regulation
des Gliotoxin-Genclusters aus
Fachhochschule Jena
Weiß, Elisabeth
The effects of metal depletion on
Candida albicans physiology: a
fatty solution to high salt
Wichmann, Christina
Bacterial Diversity in Indonesian
Epinephelus fuscoguttatus faeces
by 16S analysis
Zhang, Qi
Respiration vs. Fermentation in
dimorphen Pilzen
Zitzmann, Carolin
Mathematical Modelling of Infections in XLA Patients under IgG
Substitution Therapy
227
Inventions and Patents 2012/2013
Erfindungen und Schutzrechte 2012/2013
In addition to publications in peer reviewed journals, intellectual property rights are the main performance parameters reflecting the quality
of research at the HKI. In 2012/2013 a remarkable number of inventions
were filed both by departments focusing on natural product research and
by technology-oriented groups, thus broadening the patent-portfolio of the
institute. HKI patents resulted in a number of fruitful co-operations with
industry and affected the institute’s budget advantageously.
Schutzrechte sind neben Originalpublikationen in referierten Fachjournalen ein wesentlicher Leistungsparameter für die Forschungsarbeit am HKI.
Sowohl die naturstoffchemisch arbeitenden Abteilungen als auch Technologie-orientierte Gruppen trugen im Zeitraum 2012/2013 mit einer Reihe
von Erfindungen zum Schutzrechts-Portfolio des Instituts bei. Vom HKI
angemeldete Patente führten zu einer Reihe fruchtbarer Industriekooperationen und wirkten sich positiv auf das Budget des Instituts aus.
Headed by Prof. Christian Hertweck, a group of scientists at the HKI has
discovered a new antifungal substance. It is formed by a bacterium that
triggers decay in cultivated mushrooms. The challenge was to search for
an unknown substance that bacteria do not produce on standard cultivation conditions but only when they affect a mushroom. Hertweck and his
colleagues sequenced the genome of the bacteria, scanned it for relevant
biosynthesis genes and predicted anticipatory metabolite structures using
bioinformatic methods. By means of this technique called genome mining,
the researchers found a gene cluster called jag which could code for the
biosynthesis of a new compound. In order to trigger its production, they
injected slides of mushrooms with the bacterium and examined the emerging rotted spots using high-resolution mass spectrometry as an imaging
technique. The researchers identified a mass peak that only appears at the
affected spots. By adding pieces of mushroom and a special culture medium, they were able to extract and isolate the corresponding compound
even in liquid culture in a larger quantity. Jagaricin – as the compound
was called – is a novel lipopeptide consisting of a peculiar structure. Pure
jagaricin induced symptoms of soft rot in mushrooms while bacteria with
disabled jag genes are unable to induce soft rot.
Eine Gruppe von Wissenschaftlern aus dem HKI entdeckte unter Leitung
von Prof. Christian Hertweck einen neuen antifungalen Wirkstoff. Er wird
von einem Bakterium gebildet, das beim Kulturchampignon Fäulnis auslöst. Die Herausforderung bestand darin, nach einer unbekannten Substanz zu suchen, die die Bakterien nicht unter Standard-Kulturbedingungen produzieren, sondern erst, wenn sie einen Pilz befallen. Hertweck und
seine Kollegen sequenzierten das Genom der Bakterien, durchsuchten es
nach einschlägigen Biosynthesegenen und machten mithilfe bioinformatischer Methoden Voraussagen über die zu erwartenden Metabolitstrukturen. Die Forscher fanden durch ein solches genome mining einen als jag
bezeichneten Genabschnitt, der für den Biosyntheseapparat des gesuchten
Stoffes codieren könnte. Um dessen Bildung anzuregen, impften sie Scheiben von Champignons mit dem Bakterium und untersuchten die entstehenden fauligen Stellen mithilfe bildgebender Massenspektrometrie. Die
Forscher identifizierten einen Massenpeak, der nur auf befallenen Stellen
auftritt. Durch Zugabe von Pilzstückchen und ein spezielles Nährmedium
gelang es dann, die zugehörige Verbindung auch in flüssiger Kultur in größerer Menge zu gewinnen und zu isolieren. Bei Jagaricin – so wurde die
Verbindung getauft – handelt es sich um ein neuartiges Lipopeptid mit
ungewöhnlicher Struktur. Reines Jagaricin rief bei Pilzen die Symptome
der Nassfäule hervor während Bakterien mit nicht funktionstüchtigen jagGenen keine Nassfäule auslösen können.
Jagaricin is effective against Candida albicans, Aspergillus fumigatus and
Aspergillus terreus and might be a starting point in developing a novel antifungal agent.
The application for new patents is stringently evaluated within the HKI and
focuses on novel biologically active natural products and their (bio-)synthetic derivatives. Since 2006, the HKI co-operates with Ascenion GmbH,
Munich, to further optimize technology transfer and commercialization
activities. This project is funded by the Federal Ministry of Education and
Research.
Jagaricin ist gegen Candida albicans, Aspergillus fumigatus und Aspergillus
terreus wirksam und könnte ein Ansatzpunkt für ein neues Antimykotikum sein.
Die Anmeldung neuer Schutzrechte unterliegt einer strengen hausinternen
Evaluation und konzentriert sich auf neue, biologisch aktive Naturstoffe
und deren (bio-)synthetische Derivate. Das HKI arbeitet zur effektiven Verwertung der Schutzrechte seit 2006 erfolgreich mit der Ascenion GmbH,
München zusammen.
Bretschneider T, Graupner K,
Hertweck C, Lackner G, Scherlach K (2012)
Jagaricin, derivatives and uses thereof
EP 12 006 113.0
Dahse HM, Hertweck C, Kirchner H, Kusebauch B,
Scherlach K, Fiebig HH, Kelter G, Maier A (2012)
Neue Rhizoxinderivate
DE 10 2012 000 956.9
Ding L, Hertweck C (2012)
Bixiamycins
US 61/684,941
Hertweck C, Ueberschaar N, Scherlach K,
Dahse HM, Bretschneider T, Metsä-Ketelä M (2012)
Novel chartreusin analogues
EP 12 005 528.0
Höfs S, Hube B, Wilson D, Naglik J, Moyes D,
Richardson J, Tang S (2013)
Peptides and binding partners therefor
GB 1306588.3
228
The antifungal lipopeptide jagaricin is formed by
Janthinobacterium agaricidamnosum, the pathogen
of soft rot in cultivated mushrooms.
External Funding 2012/2013
Drittmittel 2012/2013
2012
T€
1,400
Total receipts: 4,849 T €
1,200
1,000
800
600
400
200
0
DFG
BMBF/BMWi
EU
Industry
Leibniz
Competition
ZIK
Septomics
Free State of
Thuringia
Others
2013
T€
1,400
Total receipts: 4,426 T €
1,200
1,000
800
600
400
200
0
DFG
BMBF/BMWi
EU
Industry
Leibniz
Competition
ZIK
Septomics
Free State of
Thuringia
Third-party funds during the period reported: composition of the sources of income
Drittmittelausgaben im Berichtszeitraum: Verteilung über die Drittmittelgeber
229
Others
Maps | Lagepläne
230
HKI Research Report 2012 / 2013 Maps
1 HKI Leibniz-Institut für Naturstoff-Forschung
und Infektionsbiologie – Hans-Knöll-Institut
2 Leibniz-Institut für Altersforschung –
Fritz-Lipmann-Institut
3 Abbe-Zentrum Beutenberg
4
Fraunhofer-Institut für Angewandte Optik und Feinmechanik
5 Leibniz-Institut für Photonische Technologien
6
Institut für Angewandte Physik
(Friedrich-Schiller-Universität Jena)
7 Max-Planck-Institut für Biogeochemie
8 Max-Planck-Institut für chemische Ökologie
9 Technologie- und Innovationspark
10 BioInstrumentezentrum
11 Zentrum für Molekulare Biomedizin
(Friedrich-Schiller-Universität Jena)
Maps HKI Research Report 2012 / 2013
231
232
Imprint | Impressum
HKI
Leibniz Institute for Natural Product Research and Infection Biology e. V.
– Hans Knöll Institute –
Beutenbergstraße 11a
07745 Jena, Germany
fon +49 (0)36 41-532 1011
fax +49 (0)36 41-532 0801
[email protected]
www.hki-jena.de
Editorial Board
Prof. Dr. Axel A. Brakhage
Dr. Michael Ramm
Design and Layout
Bernd Adam
Copyright
© 2014 HKI Jena
The copyright for any material created by the HKI is reserved.
Any duplication or use of objects such as texts, diagrams, photos or
other artwork in electronic or printed publications is not permitted
without the HKI’s prior written agreement.
233
HKI
Leibniz Institute for Natural Product Research and Infection Biology e. V. – Hans Knöll Institute –
Beutenbergstraße 11 a | 07745 Jena, Germany | Fon +49 (0)36 41 532-1011 | Fax +49 (0)36 41 532-0801
[email protected] | www.hki-jena.de
234

Research Report 2012 | 2013

Transcription

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