V. harveyi

Concepto y estructura
de los biopeliculas
Iwona B. Beech
University of Portsmouth, UK
BACTERIAL GROWTH
IN NATURAL
AND
MAN MADE ENVIRONMENTS
PLANKTONIC CELLS
(PESENT IN THE LIQUID PHASE)
SESSILE CELLS
(ASSOCIATED WITH SURFACES)
MICROSCOPY
IN BIOFILM RESEARCH
• LIGHT
• CONFOCAL SCANNING LASER
• ATOMIC FORCE
• (ENVIRONMENTAL)
SCANNING
ELECTRON
CONCEPTO BIOPELÍCULA
Conjunto de microorganismos embebidos en una matriz
exopolimérica de origen microbiano que los mantiene unidos
junto a otras sustancias del medio en que se encuentran
(Characklis y Marshall, 1990)
FORMACIÓN Y DESARROLLO
1.- TRANSPORTE A LA
SUPERFICIE
2.- ADHESIÓN INICIAL
3.- CONSOLIDACIÓN DE LA
ADHESIÓN O “ATTACHMENT “
4.- COLONIZACIÓN
5.- DESORCIÓN
BIOFILM
MICROBIAL GROWTH
AT INTERFACES
BIOPELÍCULAS
Morning Glory Pool in
Yellowstone National Park
BIOPELÍCULAS
Intercambiadores
de calor
Tuberías
Placa dental
Lentes de contacto
Cateteres
Implantes
Characteristic
Characteristic features
features of
of aa multilayer
multilayer biofilm
biofilm
BIOFILMS ON HISTORIC STRUCTURES
FRACTURES
Fuente de Los Leones de
La Alhambra
Cracking of marble
Fuente de Los Leones de
La Alhambra
Catedral Santiago
Compostela
PIGMENTATION
BIODETERIORO
Cambio indeseable en las propiedades de un material
causado por la acción de un organismo vivo
(Hueck, 1965)
The Mary Rose
• The Mary Rose is the only 16th century warship on display
anywhere in the world. Built between 1509 and 1511, she
was a firm favourite of King Henry VIII.
• In 1545, while maneuvering to engage a French fleet
outside Portsmouth, she unexpectedly went down in 14 m
of water.
• The wreck was rediscovered in 1971, and salvaged, along
with some 19,000 objects in 1982.
• Approximately one-third of the original hull remains. Most
of the starboard side and parts of the decks had survived
deeply embedded in soft yielding clay.
• Currently the hull is still in a ‘wet’ state. It is in the final
stages of PEG spraying before drying and open public
exhibition in 2010/2011.
Biofouling
Biofilms in health
Three examples of possible points of entry
into the body for infectious biofilms:
catheter
hip replacement
periodontal disease
STAGES OF BIOFILM FORMATION
Biofilm
sloughing
Cell
association
Irreversible
adhesion
Reversible
adhesion
Conditioning
layer
Biofilm
formation
Cell
division
Microcolony
formation
SUBSTRATUM
Biofilm model based on CSLM imaging
EPIFLUORESCENCE MICROSCOPY
Titanium
62 days
174 days
90 days
254 days
254 days
Attachment of D. alaskensis and D. indonensiensis mixed population to the AISI 316 stainless
steel surface after 4 h (a) and 12 h (b) of exposure.
DNA-DAPI-staining shows in blue all attached cells.
D. indonensiensis cells (arrows) are identified by double staining in green (FITC) and blue
(DAPI). Superposed images of FITC and DAPI (insets) show that D. alaskensis colonises surface
more rapidly than D. indonesiensis.
Bar =1µ
µm.
SEM images of biofilms
Titanium
Smooth
26 days
90 days
285 days
Rough
Copper
AISI 316 stainless steel
BIOFILM MATRIX
BACTERIAL EXTRACELLULAR POLYMERIC
SUBSTANCES
EPS
facilitate irreversible cell adhesion to a substratum
form the biofilm matrix
EPS
Marine biofilm on the surface of carbon steel
ESEM image
PROPIEDADES DE LAS BIOPELÍCULAS
Agregación de células
Adhesion a superficies
Reconocimiento celular
Retención de agua
Diferencia entre las
condiciones externas y las
propias de la biopelícula:
MICROAMBIENTE
Transporte de sustancias
Concentracion de nutrientes
Protección frente a
condiciones externas
Gradiente de O2
Cambios de pH
Varios tipos de microorganismos
Quorum Sensing
in Bacteria
Bacteria prefer to live in communities
Multispecies biofilm
1 µm
Single species biofilm
Bacterial cells “talk” to each other …
Quorum sensing (QS) is the ability
of bacteria to communicate and
coordinate behavior
via signaling molecules.
QS is regulated by population density
of the same species and the presence
of other species
Vibrio fischeri
Quorum sensing was first discovered in a marine
luminescent bacterium, Vibrio fischeri, which is a
facultative symbiont of marine animals.
The
The jelly-fish
jelly-fish lounge
lounge (Image:
(Image:
JJ Nicholson
and
K
Takayama)
Nicholson and K Takayama)
3-Oxohexanoyl
3-Oxohexanoyl homoserine
homoserine
lactone
lactone (AHL)
(AHL) is
is produced
produced by
by
LuxI
LuxI and
and recognized
recognized by
by LuxR
LuxR
in
in Vibrio
Vibrio fischeri
fischeri
http://www.che.caltech.edu/groups/fha/quorum.html
Bacteria are sensitive to the presence of
“neighbors”
It was discovered that when Vibrio fischeri cells
were solitary, they did not luminesce.
Only when many cells came together, in places
such as the gut of a fish, did the luminescence
“turned on”.
This is makes sense, as there is no advantage
for a single, isolated bacterium to produce
light.
The General Principle of Intra-Species QS
Each bacterium produces autoinducer molecules into its
environment. Different species typically produces
different compounds.
The autoinducers are sometimes referred to as
pheromones or AI-1.
Each bacterium has a receptor for its own AI-1.
When only a few other bacteria of the same kind are in
the vicinity, the concentration of the inducer in the
surrounding medium is very low.
Why intra-species quorum sensing?
The purpose of quorum sensing is to coordinate
certain behaviour or actions between bacteria,
based on their local density.
QS can occur within a single bacterial species
(as well as between disparate species)
and regulates a range of different processes,
essentially serving as a communication network.
When many bacteria of the same kind are present, the
concentration of the autoinducer increases above a critical
threshold.
In response, the bacteria start to synthesis more autoinducer.
This forms a positive feedback loop.
The receptor becomes fully activated, and this induces the upregulation of specific genes.
For example, activation of luciferase (lux gene) in V. fishcheri
causes light emission, and activation of genes in other bacteria
causes pathogenicity.
Quorum sensing makes cells able to
react to high cell densities
http://www.che.caltech.edu/groups/fha/quorum.html
Different species usually have different autoinducers
(Quorum Pheromones)
Gram-negative
Homoserine lactones (AHL)
Gram-positive
Peptides
Science (2006) 311: 1113-1116
Cell communication
in Gram-negative and Gram-positive bacteria
www.nottingham.ac.uk/quorum/
The model luminous bacterium Vibrio harveyi
produce two different autoinducers, called AI-1 and
AI-2,
•
each autoinducer is detected by its own sensor
protein.
•
Both sensors transmit information to a shared
integrator protein to control the output, light emission.
•
An analogous mechanism operate in V. cholerae to
control virulence.
The AI-2 autoinducer
•V. harveyi and V. cholerae use the AI-1 quorum
sensing circuit for intra-species communication and
the AI-2 quorum sensing circuit for inter-species
communication
The Al-2 autoinducer appears to serve as a
'universal' signal for inter-species communication.
The chemical identity of AI-2 remained unknown
until recently …
Inter-species communication
To investigate the mechanism of AI-2 signaling,
Bessler et al. constructed mutants and cloned the
gene responsible for AI-2 production from several
bacteria.
In each case the gene was highly homologous, and
they named it luxS. Homologues of luxS and AI-2
production are widespread in the bacterial world,
suggesting that communication via an AI-2 signal
response system is a common mechanism that
bacteria employ for inter-species interaction in
natural environments.
What is AI-2?
The chemical identity of AI-2 was obtained in
2002 by solving the crystal structure of the V.
harveyi sensor protein in complex with its AI-2
molecule1.
[1]
[1] Chen,
Chen, X.,
X., Schauder,
Schauder, S.,
S., Potier,
Potier, N.,
N., Van
Van Dorsselaer,
Dorsselaer, A.,
A., Pelczer,
Pelczer, I.,
I.,
Bassler,
Bassler, B.
B. L.,
L., and
and Hughson,
Hughson, F.
F. M.
M. (2002).
(2002). Nature
Nature 415,
415, 545-549
545-549
Chemical Identity of AI-2
The V. harveyi AI-2 was found to
be a furanosylborate diester.
Finding boron in the active
molecule was surprising because
boron, while widely available in
nature, has almost no known role
in biology.
AI-2 as recognized by V. harveyi
X-ray crystal structure of the V. harveyi
AI-2 / sensor protein complex
X-ray crystallographic electron density of AI-2 (blue), with the
boron atom shown in yellow. Protein side chains of the AI-2
sensor protein LuxP hydrogen bond (red dashed lines) to the
furanosyl borate diester ligand.
Quorum sensing makes cells able to
react to high cell densities
http://www.che.caltech.edu/groups/fha/quorum.html
Different species usually have different autoinducers
(Quorum Pheromones)
Gram-negative
Homoserine lactones (AHL)
Gram-positive
Peptides
Science (2006) 311: 1113-1116
Cell communication
in gram-negative and gram-positive bacteria
www.nottingham.ac.uk/quorum/
Modern work has shown that
there are two different autoinducers in
Vibrio harveyi
Bonnie Bassler et al. have shown that
•
the model luminous bacterium Vibrio harveyi produce
two different autoinducers, called AI-1 and AI-2,
•
each autoinducer is detected by its own sensor protein.
•
Both sensors transmit information to a shared integrator
protein to control the output, light emission.
•
An analogous mechanism operate in V. cholerae to
control virulence.
The AI-2 autoinducer
•V. harveyi and V. cholerae use the AI-1 quorum
sensing circuit for intra-species communication
and the AI-2 quorum sensing circuit for interspecies communication
The Al-2 autoinducer appears to serve as a
'universal' signal for inter-species
communication.
The chemical identity of AI-2 remained unknown
until recently …
Inter-species communication
To investigate the mechanism of AI-2 signaling,
Bessler et al. constructed mutants and cloned
the gene responsible for AI-2 production from
several bacteria. In each case the gene was
highly homologous, and they named it luxS.
Homologues of luxS and AI-2 production are
widespread in the bacterial world, suggesting
that communication via an AI-2 signal response
system is a common mechanism that bacteria
employ for inter-species interaction in natural
environments.
So what is AI-2?
The chemical identity of AI-2 was obtained in
2002 by solving the crystal structure of the V.
harveyi sensor protein in complex with its AI2 molecule11.
[1]
[1] Chen,
Chen, X.,
X., Schauder,
Schauder, S.,
S., Potier,
Potier, N.,
N., Van
Van Dorsselaer,
Dorsselaer, A.,
A., Pelczer,
Pelczer, I.,
I., Bassler,
Bassler, B.
B. L.,
L., and
and Hughson,
Hughson, F.
F. M.
M.
(2002).
(2002). Nature
Nature 415,
415, 545-549
545-549
X-ray crystal structure of the V. harveyi
AI-2 / sensor protein complex
X-ray
X-ray crystallographic
crystallographic electron
electron density
density of
of AI-2
AI-2 (blue),
(blue), with
with the
the boron
boron atom
atom
shown
shown in
in yellow.
yellow. Protein
Protein side
side chains
chains of
of the
the AI-2
AI-2 sensor
sensor protein
protein LuxP
LuxP
hydrogen
hydrogen bond
bond (red
(red dashed
dashed lines)
lines) to
to the
the furanosyl
furanosyl borate
borate diester
diester ligand.
ligand.
[1]
[1] Chen,
Chen, X.,
X., Schauder,
Schauder, S.,
S., Potier,
Potier, N.,
N., Van
Van Dorsselaer,
Dorsselaer, A.,
A., Pelczer,
Pelczer, I.,
I.,
Bassler,
Bassler, B.
B. L.,
L., and
and Hughson,
Hughson, F.
F. M.
M. (2002).
(2002). Nature
Nature 415,
415, 545-549
545-549
Chemical Identity of AI-2
The V. harveyi AI-2 was found
to be a furanosylborate
diester. Finding boron in the
active molecule was
surprising because boron,
while widely available in
nature has almost no known
role in biology.
AI-2 as recognized by V. harveyi
Different QS strategies …
V. fischeri, a symbiont, glows when signal molecules
from its own kind reach critical levels.
In contrast, free-living V. harveyi require sufficient
amounts of two autoinducers — both the speciesspecific AI-1 and the universal AI-2, to activate their
luminescence genes.
V. harveyi mutant strains respond to only one signal or
the other. Mutants were used to show that one system
tells the bacteria how many of its own species are in the
area; the other tells how many other types of bacteria
are around.
In a petri dish, the arrow contains a mutant
form of V. harveyi.
On the left is a patch of E. coli that causes
intestinal infections; on the right is
Salmonella; in the middle, above and below
the stem of the arrow, is a lab strain of nonpathogenic E. coli.
In the dark (bottom photo), V. harveyi
glows in the presence of the two
pathogenic bacteria but not the harmless
one.
http://www.princeton.edu/pr/pwb/99/0329/
bacterial.htm
Variability of AI-2 among species
Different species of
bacteria recognize
different forms of AI-2.
S. typhimurim recognize a
chemically distinct adduct
of DPD as AI-2.
The reason for this
complexity is not known. It
may allow different
species to “interpret” the
signal in different ways.
Crystal
Crystal structure
structure of
of the
the S.
S. typhimurim
typhimurim AI-2
AI-2
receptor,
receptor, LsrB
LsrB (Miller,
(Miller, S.T.
S.T. et
et al.)
al.)
Quorum sensing and biofilms
In
In the
the cartoon
cartoon above,
above, various
various species
species of
of bacteria
bacteria are
are represented
represented by
by different
different colors.
colors.
Bacteria
Bacteria can
can produce
produce chemical
chemical signals
signals ("talk")
("talk") and
and other
other bacteria
bacteria can
can respond
respond to
to them
them
("listen")
("listen") in
in aa process
process commonly
commonly known
known as
as cell-cell
cell-cell communication
communication or
or cell-cell
cell-cell
signaling.
signaling. This
This communication
communication can
can result
result in
in coordinated
coordinated behavior
behavior of
of microbial
microbial
populations.
populations. Courtesy,
Courtesy, MSU-CBE.
MSU-CBE.
Although planktonic cells secrete chemical signals (HSLs, for
homoserine lactones), the low concentration of signal molecules
does not change genetic expression. Biofilm cells are held together
in dense populations, so the secreted HSLs attain higher
concentrations. HSL molecules then re-cross the cell membranes
and trigger changes in genetic activity.
Diagram of the P. aeruginosa biofilm-maturation pathway.
Acyl-homoserine lactone QS are required to form mature
biofilms of Gram-negative bacteria
• Unattached cells that approach a surface may attach. Attachment
involves specific functions.
• Attached cells will proliferate on a surface and use specific
functions to actively move into microcolonies.
• The high-density microcolonies differentiate into mature biofilms
by a 3OC12-HSL-dependent mechanism.
Scanning
Scanning confocal
confocal microscope
microscope images
images of
of aa mature
mature
P.
P. aeruginosa
aeruginosa wild-type
wild-type biofilm
biofilm (Upper)
(Upper) and
and aa
quorum-sensing
quorum-sensing mutant
mutant biofilm
biofilm (Lower).
(Lower). In
In this
this case
case
the
the quorum-sensing
quorum-sensing mutant
mutant was
was aa lasR,
lasR, rhlR
rhlR
double
double mutant.
mutant. The
The perspective
perspective is
is from
from above
above the
the
biofilm
biofilm on
on aa glass
glass surface.
surface. The
The glass
glass surface
surface is
is red,
red,
and
and the
the green
green is
is from
from the
the green
green fluorescent
fluorescent protein
protein
encoded
encoded by
by the
the gfp
gfp gene
gene in
in the
the recombinant
recombinant P.
P.
aeruginosa.
aeruginosa. The
The wild-type
wild-type biofilm
biofilm consists
consists of
of thick
thick
microcolonies.
microcolonies. The
The immature
immature mutant
mutant biofilm
biofilm
appears
appears thinner,
thinner, and
and more
more of
of the
the glass
glass surface
surface is
is
exposed.
exposed. With
With the
the lasR,
lasR, rhlR
rhlR mutant
mutant shown
shown here
here
(but
(but not
not with
with lasI,
lasI, rhlI
rhlI mutants)
mutants) zones
zones of
of clearing
clearing
around
around microcolony
microcolony towers
towers are
are often
often observed.
observed.
Other
Other experiments
experiments have
have shown
shown that
that these
these zones
zones
are
are filled
filled with
with extracellular
extracellular polysaccharide
polysaccharide (M.R.P.,
(M.R.P.,
unpublished
unpublished data).
data).
Acyl-homoserine
Acyl-homoserine lactone
lactone quorum
quorum sensing
sensing in
in Gram-negative
Gram-negative bacteria:
bacteria: AA signaling
signaling mechanism
mechanism involved
involved in
in associations
associations with
with
higher
higher organisms;
organisms; Matthew
Matthew R.
R. Parsek*
Parsek* and
and E.
E. Peter
Peter Greenberg
Greenberg
Proposed roles of quorum
sensing and biofilm formation in
the life cycle of Vibrio cholerae.
Upon ingestion, the biofilm structure
protects V. cholerae cells from acid
shock in the gastric environment. After
passing through the stomach, individual
cells that escape the biofilm experience
conditions of low cell density.
Virulence gene expression is induced in these cells, which then colonize the
intestinal epithelium. Subsequent growth to high cell density represses
virulence factor expression, and induces the expression of factors aiding
detachment, such as Hap protease. Bacteria are shed from the host, possibly
as biofilms, and the biofilm structure may enhance V. cholerae persistence in
the environment, or infectivity for new hosts.
http://www.asm.org/news/index.asp?bid=24596
First glimpses of the complexity of bacterial
communications
“It is a jungle out there”
Bacteria manipulate AI-2 molecules for their own benefit:
Some hide their own signals to deceive competing species
Others cleave their neighbors' AHLs
Pseudomonas aeruginosa listens in on other microbes and
turns on its own virulence programs only when in a large,
protective group (Bassler).
From the games bacteria play …
… to the games people play
Enzymes involved in AI-2 production and
detection are potential targets for novel
antimicrobial drugs.
In particular, molecules that are structurally
related to AI-2 have many potential uses.
Furanone is an anti-biofilm
compound from the seaweed
Delisea pulchra that does not
affect the growth of Gramnegative strains, inhibits AI-2
quorum sensing in Gram-negative
strains.
Structure
Structure of
of (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone
(5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone
Inhibition of E.
coli biofilm
swarming
(quorum sensing
phenomenon)
using furanone
http://cheweb.tamu.edu/orgs/groups/wood/research.html
FORMACIÓN Y DESARROLLO
4.- COLONIZACIÓN
- Desarrollo de la arquitectura
intrínseca de la biopelícula;
formación de canales y poros y
redistribución de las bacterias
- Comunicación célula-célula,
que favorece la maduración:
“Quorum sensing”
(Homoserina Lactonas)
5.- DESORCIÓN
- Desprendimiento de células o de porciones de la biopelícula
- Existe regulación:
· Incremento en la concentración de una molécula inductora responsable
de la liberación de enzimas que degradan la matriz polimérica
· Densidad celular también puede ser responsable de la liberación de
estas enzimas
Who is talking?
PROCESAMIENTO
PROCESAMIENTO DE
DE MUESTRAS
MUESTRAS POR
POR MICROBIOLOGÍA
MICROBIOLOGÍA
MOLECULAR
MOLECULAR
(5'-CCT ACG GGA GGC AGC AG-3') y con cola –GC (Muyzer cols, 1993)
Toma de muestras de biopelicula 341F
907R (5’CCG TCA ATT CCT TTG AGT TT-3’) (Muyzer y cols, 1995)
531R (5’-TAC CGC GGC TGC TGG CAC-3’) (Muyzer y cols, 1995)
Extracción de ADN
Amplificación por PCR del ADN
Análisis por DGGE
Clonación de fragmento de ADN
amplificado
Aislamiento de clones y extracción
del ADN insertado en el plásmido
Inserto
Análisis de los clones por DGGE
Secuenciación de ADN
Análisis de secuencias y
relaciones filogenéticas
Bases de datos:
NCBI y EMBL