The Comprehensive Sourcebook of Bacterial

Transcription

The Comprehensive
Sourcebook of Bacterial
Protein Toxins
Fourth Edition
Joseph Alouf †
Institut Pasteur, Paris, France
Daniel Ladant
Institut Pasteur, Unité de Biochimie des
Interactions Moléculaires, Paris, France
Michel R. Popoff
Institut Pasteur, Unité des Bactéries
anaérobies et Toxines, Paris, France
†
Deceased
AMSTERDAM • BOSTON • HEIDELBERG • LONDON • NEW YORK • OXFORD
PARIS • SAN DIEGO • SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO
Contents
List of Contributors
Introduction to the Fourth Edition
Section I Basic Genomic and Physiological Aspects of
Bacterial Protein Toxins
1
Evolutionary aspects of toxin-producing bacteria
Brenda A. Wilson and Mengfei Ho
2
Mobile genetic elements and pathogenicity islands encoding
bacterial toxins
Ulrich Dobrindt, Sarah Tjaden, Sadrick Shah and Jörg Hacker
3
News and views on protein secretion systems
Alain Filloux and Agnes Sagfors
Section II
Intracellularly Alive Bacterial Protein Toxins
ix
xvii
1
3
40
77
109
4
Diphtheria toxin
Daniel Gillet and Julien Barbier
111
5
Pseudomonas aeruginosa toxins
Stephanie Rolsma, Dara W. Frank and Joseph T. Barbieri
133
6
Bordetella protein toxins
161
Jiri Masin, Radim Osicka, Ladislav Bumba, Peter Sebo and Camille Locht
7
Vibrio cholerae and Escherichia coli heat-labile enterotoxins
and beyond
Julie E. Heggelund, Victoria A. Bjørnestad and Ute Krengel
8
Vibrio parahaemolyticus virulence determinants
Marcela de Souza Santos, Dor Salomon, Peng Li, Anne-Marie
Krachler and Kim Orth
195
230
vi
Contents
9
Typhoid toxin
Jorge E. Galan
261
10
Shiga toxins: properties and action on cells
Kirsten Sandvig, Anne Berit Dyve Lingelem, Tore Skotland and
Jonas Bergan
267
11
Clostridial neurotoxins: from the cellular and molecular mode
of action to their therapeutic use
Bernard Poulain, Jordi Molgó and Michel R. Popoff
287
12
Uptake and transport of clostridial neurotoxins
Nathalie Schmieg, Kinga Bercsenyi and Giampietro Schiavo
337
13
Bacillus anthracis toxins
Shihui Liu, Mahtab Moayeri, Andrei P. Pomerantsev
and Stephen H. Leppla
361
14
ADP-ribosylating toxins modifying the actin cytoskeleton
Holger Barth, Bradley G. Stiles and Michel R. Popoff
397
15
Large clostridial cytotoxins modifying small GTpases:
structural aspects
Klaus Aktories and Thomas Jank
426
Large clostridial glycosylating toxins modifying small GTPases:
cellular aspects
Harald Genth and Ingo Just
441
16
17
Pasteurella multocida toxin
463
Brenda A. Wilson, Stefan Bergmann, Mengfei Ho and Joachim H.C. Orth
18
Deamidase toxins
Emmanuel Lemichez, Patrick Munro and Laurent Boyer
499
19
Helicobacter pylori vacuolating toxin
Vittorio Ricci, Patrizia Sommi and Patrice Boquet
515
20
Bacterial genotoxins
Teresa Frisan
558
Section III Bacterial Protein Toxins Active on the
Surface of Target Cells
21
Basic mechanism of pore-forming toxins
Roland Benz
603
605
Contents
22
Membrane-damaging and cytotoxic sphingomyelinases
and phospholipases
Marietta Flores-Díaz, Laura Monturiol-Gross and Alberto Alape-Girón
23
Structure and function of RTX toxins
A. Chenal, A.C. Sotomayor-Perez and Daniel Ladant
24
Perfringolysin O and related cholesterol-dependent cytolysins:
mechanism of pore formation
Kristin R. Wade, Eileen M. Hotze and Rodney K. Tweten
vii
627
677
719
25
The staphylococcal alpha-toxin and leukotoxins
Gilles Prévost, Mira Y. Tawk, Gaëlle Zimmermann-Meisse
and Emmanuel Jover
739
26
Aerolysin and Related Aeromonas Toxins
Ioan Iacovache, Matteo Dal Peraro and F. Gisou van der Goot
773
27
Structural relationships between small β-pore-forming toxins from
Clostridium perfringens
Ajit K. Basak, Claire E. Naylor, Jessica Huyet, Christos Savva,
Michel R. Popoff and Richard Titball
794
28
Clostridium perfringens enterotoxin
Archana Shrestha and Bruce A. McClane
815
29
Bacillus cereus phospholipases, enterotoxins, and other hemolysins
Toril Lindbäck and Per Einar Granum
839
30
Mechanism of action of Bacillus thuringiensis insecticidal toxins
and their use in the control of insect pests
858
Alejandra Bravo, Diana L. Martínez de Castro, Jorge Sánchez,
Pablo Emiliano Cantón, Gretel Mendoza, Isabel Gómez, Sabino Pacheco,
Blanca I. García-Gómez, Janette Onofre, Josue Ocelotl and Mario Soberón
31
Escherichia coli heat-stable enterotoxins
J. Daniel Dubreuil
874
32
Bacterial superantigens and superantigen-like toxins
Ries J. Langley, John D. Fraser and Thomas Proft
911
Section IV Clinical Aspects, Applications of Bacterial
Protein Toxins in Cell Biology and Therapy, and Toxin
Inhibitors
33
Clostridial toxins in the pathogenesis of gas gangrene
Amy E. Bryant and Dennis L. Stevens
975
977
viii
Contents
34
Engineering of botulinum neurotoxins as novel therapeutic tools
J. Oliver Dolly and Jiafu Wang
35
Engineering of bacterial toxins for research and medicine
Julien Barbier and Daniel Gillet
1016
36
Toxins as tools
Klaus Aktories and Gudula Schmidt
1045
37
Exploiting endocytic pathways to prevent bacterial toxin infection
Callista B. Harper, Adam McCluskey, Phillip J. Robinson
and Frederic A. Meunier
1072
38
Inhibitors of pore-forming toxins
Sergey M. Bezrukov and Ekaterina M. Nestorovich
1095
39
Bacterial protein toxins as biological weapons
Virginia I. Roxas-Duncan and Leonard A. Smith
1135
Index
995
1151
Structure and function
of RTX toxins
23
Alexandre Chenal, A.C. Sotomayor-Perez, and Daniel Ladant
Institut Pasteur, CNRS UMR 3528, Unité de Biochimie des Interactions Macromoléculaires,
Département de Biologie Structurale et Chimie, Paris, France
Introduction: an overview of RTX proteins
Repeat-in-ToXin (RTX) toxins are important virulence factors produced by a variety
of Gram-negative bacteria, the prototype of which is the α-hemolysin, HlyA, produced by pathogenic Escherichia coli. These RTX toxins, like HlyA, are able to form
pores in the membranes of their eukaryotic target cells, and this membrane-damaging
capacity directly contributes to their cytotoxic activities [1–3]. Members of the RTX
toxin family share several common structural characteristics with HlyA, particularly
the presence of a series of glycine and aspartate-rich nonapeptide repeats of prototype
sequence GGXGXDXUX (i.e., a one-letter code for amino acids, with X standing for
any amino acid, and U standing for any large hydrophobic amino acid such as L, V, I,
F, or Y). This so-called RTX motif, which defines this class of cytotoxins, constitutes
a specific calcium-binding sequence [4,5]. Indeed, most cytotoxins of the HlyA family are calcium-dependent [1–3], and few of them have been shown to bind calcium
in solution [6,7].
In addition, RTX nonapeptide repeats are also found in a distinct group of bacterial
proteins that display various enzymatic activities (particularly proteolytic or lipolytic
activities), adhering capacities, or both [8–16]. These proteins are not usually referred
to as “toxins,” although they may contribute to the virulence of the bacteria that produce them [2,3,17].
A prominent feature of all these RTX-containing proteins is that they share the
common property of being secreted by a dedicated type I secretion system (T1SS), a
tripartite machinery that constitutes one of the major export systems used by Gramnegative bacteria to secrete proteins into their external medium [17–20] (see also
Chapter 3 of this book). The T1SS encoding genes (or at least some of them) are
generally in the same genetic locus as the RTX protein substrates [8–10,21–26]. The
secreted proteins are recognized through a noncleavable secretion signal, localized
at the C terminus of the polypeptide chain downstream to the RTX motifs and are
exported from the bacterial cytosol to the extracellular medium without any periplasmic intermediates [8–10,27–29]. The tight link between the presence of RTX motifs
in a protein and its secretion by a dedicated T1SS machinery strongly supports the
The Comprehensive Sourcebook of Bacterial Protein Toxins. DOI: http://dx.doi.org/10.1016/B978-0-12-800188-2.00023-9
© 2015 Elsevier Ltd. All rights reserved.
678
Bacterial Protein Toxins Active on the Surface of Target Cells
view that the RTX motifs are structural features that favor efficient secretion through
the T1SS pathway [29,30].
A third class of RTX-containing proteins, originally identified in Vibrio species,
regroups the so-called multifunctional-autoprocessing RTX (MARTX) toxins [31–
33]. Classified at first among the RTX toxins, the structural organization as well as the
particularities of the RTX repeated sequences of these virulent factors clearly specify
a distinct family. The MARTX toxins are secreted via an atypical four-component
T1SS and harbor several effector domains that contributes to the virulence of the
bacterial hosts [32,33].
The RTX protein family has been constantly expanding from the original few
members identified in the late 1980s, to over 1000 putative RTX proteins found in
more than 250 bacterial species as reviewed by Linhartova and coworkers in 2010
[3]. Bacteria harboring RTX protein genes are mostly found in Gammaproteobacteria,
Alphaproteobacteria, Betaproteobacteria, and Cyanobacteria. RTX proteins exhibit a
variety of biological activities, being pore-forming toxins, metalloproteases, lipases
(I.3 lipase family), iron-regulating proteins, nodulation-related proteins, or proteins
implicated in the formation of S-layer, bacterial adherence/motility or host-receptor
interaction [2,3,17]. The origin of this phylogenetically diverse bacteria family is
unknown, as it contains members that are closely related (such as the Pasteurellaceae
family) and others that are genetically more distant, such as the adenylate cyclase
(AC) toxin, CyaA, from the Bordetella species or the MARTX toxins from the Vibrio
species. The family may have originated from Pasteurellaceae and spread to the different species through multiple horizontal gene transfer events [34,35].
This chapter mainly focuses on the structure and mode of action of the RTX toxins.
We first introduce the overall structure of RTX toxins, the RTX structural fold, and
the T1SS machinery. Then we present an overview of the main RTX toxins. Finally,
we discuss some key issues about the molecular mode of action of the RTX toxins.
Common features of RTX toxins
The most typical traits that define the RTX toxin family are the following [1–3,36,37]:
●
●
RTX toxins are large, single-chain proteins with a molecular mass of about 100 kDa, or
up to 200 kDa for a few of them. Most RTX toxins are acidic proteins with a theoretical
pI around 4, a property related to the presence of tandem repetitions of Gly- and Asp-rich
nonapeptide RTX sequences [1–4]. These repeated motifs, which vary in number from 6 to
more than 50, are usually located in the C-terminal part of the toxin, just upstream of the
C-terminal secretion signal. Another feature is the lack of cysteine residues in the RTX toxin
primary sequence.
The RTX toxins display calcium-dependent cytolytic activities, which result from their ability to form short-lived cation-selective pores in lipid membranes [38–42]. The effective pore
diameter appears to be uniform for a given RTX toxin but varies widely between different
RTX toxins. It is thought that the pores result from the insertion within the membrane of
several amphipathic and hydrophobic α-helices located within the N-terminus part of the
proteins [43–50].
●
●
●
●
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The RTX toxins are calcium-binding proteins. Calcium binds to the RTX motifs and triggers their folding into a parallel β-roll structure (as discussed later in this chapter) that is
somehow required for the functional cytolytic activities of the toxins [28,45,51–56].
The RTX toxins are all secreted by a T1SS, a tripartite machine made of (i) an inner-membrane ATP-binding cassette (ABC) transporter that recognizes the protein substrate, (ii) a
membrane fusion protein (MFP) that is inserted in the inner membrane and extends mainly
into the periplasm, and (iii) an outer membrane protein (OMP). The RTX toxins have a
C-terminal secretion signal that is specifically recognized by the ABC transporter system
and that is not cleaved during or after the secretion process [17,19,57].
The RTX toxins are synthesized as inactive protoxins that require a posttranslational activation prior to their export from the toxin-producing bacteria [21,25,27,58–61]. This modification is carried out by an accessory protein, RtxC, which is a specific acyltransferase
that acylates one or two key lysine residues located in the middle region of the RTX toxin
polypeptide [62–66].
The structural gene coding for the RTX toxin, usually called rtxA, is part of an rtx operon
(Figure 23.1) that typically contains four (of five) genes in the following order: rtxC, which
Figure 23.1 Genetic organization of various rtx locus. The genetic organization of different
RTX toxin operons is schematized with arrows representing the different coding regions: the
rtxA genes coding for the RTX toxins are in orange (light gray in print) (and yellow (white
in print)), the rtxC genes coding for the acyltransferases are in black, the rtxB genes coding
for the inner membrane ABC transporters are in blue, the rtxD genes coding for the MFPs
are in green, and the genes encoding the OMPs are in red. The arrows indicate the different
transcripts. The lower part shows the genetic organization of the the locus encoding the
RTX proteases from P. aeruginosa and E. chrysantemi. The same color code applies for the
component of the T1SS, while the genes coding for the different proteases are in brown and
the genes coding for the specific protease inhibitors are in magenta [3,17].
680
Figure 23.2 Schematic organization of HlyA and CyaA RTX toxins. The different domains
of the proteins are indicated as follows: H: hydrophobic segments; RTX: RTX motifs;
SS: secretion signal; AC: CyaA catalytic domain; and T: CyaA translocation domain.
The acylated lysines are also indicated.
●
codes for the accessory acyltransferase; rtxA; and then rtxB and rtxD, which codes for the
inner-membrane ABC transporter and the membrane fusion protein (MFP), respectively
[1–3,17,57]. The third component of the T1SS machinery, the OMP, is in most cases TolC,
which is encoded elsewhere on the bacterial chromosome. Occasionally, the OMP is part of
the operon, as illustrated by the Bordetella pertussis cyaE gene. The transcriptional order is
typically rtxC, rtxA, rtxB, and rtxD, as for the hemolysin operon, while for the B. pertussis
cya operon, the rtxC (cyaC) gene is transcribed divergently from the cyaA, cyaB, cyaD, and
cyaE genes, that form an operon (Figure 23.1).
The RtxA polypeptides exhibit a common structural organization with typically four distinct
regions (Figure 23.2):
A N-terminal hydrophobic region (H), 200–300 residue long that contains several predicted hydrophobic and amphiphilic α-helical structures, which interact with and insert
into the target cell membrane to create cation-selective pores [43–50,67]
A central region harboring one or two lysine residues (K564 and K690 in HlyA) that are
fatty-acylated by the RtxC acyltransferase [62,64]
A RTX domain that contains a variable number of calcium-binding nonapeptide repeats
essential for RtxA recognition of target cells [28,44,51,53]
A C-terminal secretion signal that is recognized by the RtxB/RtxD/TolC(or RtxE) secretion machinery [24,51,68–70]
●
●
●
●
RTX-repeats: motifs and structures
The most salient characteristic of RTX proteins is the presence of nonapeptide motifs
that are repeated in tandem fashion, most commonly in the C-terminal moiety of the
polypeptide, and that constitute specific calcium-binding sequences [2–4,17]. The
prototypic, consensus sequence of these motifs is GGXGXDXUX (using one-letter
code for amino acids, with X standing for any amino acid, and U standing for a
hydrophobic residue, generally L, and occasionally V, I, F, or Y). However, in most
RTX toxins, many of the putative repeats are only partially matched to the consensus
681
Figure 23.3 Structure of an RTX domain and model for its calcium-induced folding.
Top: Two different views of the RTX domain (residues 326–377) of the alkaline protease
(pdb.1KAP) from P. aeruginosa showing the RTX motifs folded in the presence of calcium
(blue sphere) into a characteristic calcium-binding structure, the parallel β-roll or parallel
β-helix [4]. Alignment of the corresponding RTX motifs is shown on the right. The turn
involved in calcium binding is formed by the first six residues, GGXGXD, while the short
β-strand is made of the last three residues, XUX (underlined), of the nonapeptide RTX repeat.
Calcium is coordinated by aspartic acids, shown in red, and the backbone carbonyl groups.
Bottom: Model for calcium-induced folding of the RTX motifs. In the absence of calcium,
RTX motifs are natively disordered (left) and adopt various premolten globule conformations.
Calcium binding triggers compaction, dehydration, and folding of the RTX motifs into a
compact and stable β-roll conformation.
GGXGXDXUX sequence (see, for example, [71]). As a consequence, the number of
repeats in any particular toxin remains partly undefined, as it depends upon how much
deviation from the consensus is tolerated. The total number of repeats also varies
widely among members of the RTX family, from 6 to more than 50. The RTX motifs
have been rapidly recognized as being critical for the activity of the various RTX toxins and shown to be involved in calcium binding [6,7,28,51,53]. Calcium is indeed a
key cofactor for most RTX toxin hemolytic and cytolytic activities [28,51,52,54,55].
Three-dimensional (3D) structures of the RTX motifs in the presence of calcium
have only been obtained thus far for the protease and lipase subfamily of RTX proteins [4,5,72], but the overall fold is likely pertinent for the RTX toxin subfamily as
well. The X-ray crystal structure of the alkaline protease from Pseudomonas aeruginosa [4] showed that RTX motifs fold in the presence of calcium into a characteristic
calcium-binding structure (Figure 23.3), the parallel β-roll or parallel β-helix, which
is a highly regular super-secondary structure with calcium ions as an integral part
[4,5,72]. The first six residues (GGXGXD) form a turn involved in calcium binding;
meanwhile, the last three residues (XUX) form a short β-strand where the X residues
point outward and the U residue points inward (it is buried in the core), contributing
682
to the hydrophobic core of the β-helix, as shown in Figure 23.3. The conserved hydrophobic U residue stabilizes the hydrophobic core of the β-helix and is ideally a leucine
(which interdigitates nicely in the interior of the β-helix), but it can also be isoleucine,
valine, phenylalanine, or tyrosine. Although X can be any amino acid, X3 and X5 are
preferably small hydrophobic residues [4,5,73], while the conserved glycines within
the RTX motif are required for steric considerations [74]. The consecutive stacking
of turns and β-strands builds up a right-hand helix of parallel β strands, with calcium
bound in the loops at both sides of the structure separated by a narrow hydrophobic
core. One turn of the helix consists of two consecutive RTX motifs (therefore, each
GGXGXD motif forms two half-sites for calcium binding; see Figure 23.3) and calcium ions are hexacoordinated between two spatially adjacent turns by the conserved
aspartic acids and by the backbone carbonyl groups [4,72]. Similar structures have
been found in other RTX proteins, such as the metalloproteases from Serratia marescens and Serratia sp [5,75], the lipase LipA from S. marcescens (which possesses
two blocks of tandem repeats) [72], or the R-module of the Azotobacter vinelandii
calcium-dependent mannuronan C-5 epimerase [76].
Insights into the structure-function relationships of the RTX fold have been
obtained by biochemical and biophysical studies of particular RTX toxins, the E. coli
HlyA and the AC toxin, CyaA, from B. pertussis. CyaA contains about 40 RTX motifs
and binds, in solution, about 40 calcium ions with an affinity in the submillimolar
range (i.e., 0.1 to 1 mM [7,71,77–79]), while HlyA, which has 11–13 RTX repeats,
can bind up to 12 calcium ions [80]. This indicates a binding stoichiometry of one calcium ion per RTX motif. Calcium binding triggers important conformational changes
in the secondary and tertiary structures of the proteins. Sr2+ and Ba2+ are, to some
degree, able to substitute for Ca2+, while other divalent cations have no or little effect
[7,71,77,81]. The calcium-induced structural changes were further characterized on
the RTX-containing domain of CyaA, RD, which encompasses residues 1006–1706.
In the absence of calcium, RD is natively disordered, weakly stable, and highly
hydrated [78,79]. Such premolten globule conformations are likely maintained in
these unstructured states by the electrostatic repulsion between the negatively charged
Asp residues of the RTX motifs and the low hydrophobicity of the sequence. Calcium
binding triggers a strong compaction and dehydration of RD, as well as the formation
of secondary and tertiary structures in a highly cooperative manner [78,79,82,83].
Calcium ions likely balance the repulsive charges of the aspartate residues in the RTX
repeats, allowing the polypeptide chain to collapse and fold into a compact and stable β-roll conformation [79] (Figure 23.3). Importantly, calcium binding to the RTX
motifs also induces conformational changes in the other functional domains of the
toxins [47,81,84,85] and affects the overall conformation of the full-length proteins
[7,86–88].
The RTX fold, therefore, appears as a structural module that is finely tuned for
allowing efficient protein secretion by the T1SS machinery (as discussed later in this
chapter). Indeed, in the low-calcium environment (<μM) of the bacterial cytosol, the
intrinsically disordered character of the RTX motifs should be favorable for polypeptide export through the narrow channel of the T1SS. In the extracellular medium
(enriched in calcium), calcium binding can trigger the folding of the RTX motifs into
the characteristic RTX β-roll fold, which may then serve as a nucleation site for the
683
folding of the whole toxin into its functional state, in the absence of any chaperone
(Figure 23.4). This likely explains the general occurrence of RTX motifs in proteins
secreted by the T1SS machinery [2,3,17,19] and the importance of this motif for an
efficient secretion [24,28,51,89–91].
As said previously, calcium is an essential cofactor of most RTX toxins hemolytic
and cytolytic activities [28,51,52,54–56], indicating that the calcium-bound conformations are functionally active ones. Indeed, deletions or mutations of the RTX
motifs result in toxins with elevated calcium requirements, displaying reduced activity, or both [92–94]. Besides the critical role in toxin folding, the RTX domains may
have additional functions, such as promoting toxin interaction with the membrane
surface [81] or with specific receptors at the cell surface [95,96].
T1SS
All known RTX toxins are secreted by a T1SS, a Sec-independent pathway that, in
Gram-negative bacteria, drives the secretion of proteins in a single step from the
cytoplasm to the extracellular medium, without a stable periplasmic intermediate (see
Chapter 3) [17,97]. The hemoprotein HasA from S. marcescens is the only known
protein to date that is exported by a dedicated T1SS system, although it has no RTX
motif [17,98].
Figure 23.4 Schematic model for the secretion pathway of RTX toxins. Proposed model for
the secretion of a RtxA polypeptide through the T1SS. The proRtxA polypeptide is acylated by
RtxC and exported by the T1SS, made of RtxB, RtxD, and RtxE/ TolC (in blue, green and red,
respectively, in e version). Upon reaching the extracellular environment, RtxA binds calcium
ions (dots, blue in e version) that trigger the folding of the toxin into its active conformation.
684
The T1SS secretion apparatus is composed of three proteins: an inner membrane
protein with an ABC domain (coded by the rtxB gene), a membrane fusion protein
(coded by the rtxD gene) and an OMP (coded by the rtxE or tolC genes for HlyA and
many other RTX toxins). The three components assemble to form a continuous channel
from the inner membrane, across the periplasm to the outer membrane [17,19,97], and
the protein substrates can be delivered through this channel directly from the cytosol to
the external medium. The assembly of the three components is transient and induced
by the recognition of the substrate. Once the RTX protein has been exported, the transport machinery disengages [97]. The protein substrates are assumed to be in an unfolded
state to pass across the narrow channel of the T1SS secretion machinery [99–101].
Inner membrane ABC transporter
This ATPase protein is encoded by the hlyB gene (generally rtxB). The ATPase activity is essential for transport, as mutants with reduced ATPase activity present reduced
secretion rates [29]. RtxB is dimeric and comprises two main modules, a transmembrane domain (TMD) made of six predicted hydrophobic helical segments [102] and a
cytoplasmic nucleotide-binding domain (NBD) [103]. The TMD is assumed to form a
channel in the inner membrane to allow passage of the protein substrate across the lipid
bilayer. The NBD is responsible for the recognition of the protein substrate and also
provides energy for the export process through the hydrolysis of adenosine triphosphate
(ATP) [103–105]. Detailed structural analysis have been reported for the isolated NBD
of HlyB, which exhibits the common overall architecture of ABC-transporter NBDs
[106–108], while no structural data are yet available for the TMD. Recently, Lecher
and colleagues [109] identified in the N-terminal cytosolic region of HlyB an additional domain that plays a critical role in the HlyA substrate recognition. This domain
is called CLD (which stands for “C39-like domain”), as it shows homology to the C39
peptidase domain found in ABC transporters that transport bacteriocins. CLD has no
proteolytic activity, as it lacks key catalytic residues, but it is able to interact with the
unfolded state of the RTX domain from HlyA and is essential for efficient secretion
[109]. This domain may thus help to maintain the large and unfolded HlyaA substrate
protected inside the cells in the vicinity of the TISS to facilitate its efficient secretion.
Membrane fusion protein (MFP)
The MFP HlyD (or related RtxD) is a connector between the ABC transporter and the
OMP, which is similar to the adaptor proteins in drug efflux pumps [103]. HlyD has
a small N-terminal cytosolic domain, a single transmembrane helix, and a large periplasmic domain [110]. No structural studies have yet been reported for HlyD, but the
periplasmic region is predicted to be a long helical domain followed by a C-terminal
β domain [19]. HlyD was recently shown to interact with TolC via the tip of the putative α helical hairpin region [110], while the C-terminal domain may interact with the
inner membrane transporter and the N-terminal part with the substrate [17,19,111,112].
HlyD is trimeric (or hexameric) and forms a stable complex with HlyB in the absence
of TolC or substrate [111]. Mutations in HlyD were shown to result in secretion of the
685
incorrectly folded HlyA toxin with low hemolytic activity [113], suggesting that HlyD
could help prevent the misfolding of secreted proteins [57].
Outer membrane protein (OMP)
Outer membrane proteins (OMPs) are encoded normally by the rtxE gene but lack
the α-hemolysin operon. The OMP required for HlyA secretion in E.coli is TolC
[114], an outer membrane channel, which functions in several efflux systems (e.g.,
AcrAB-TolC) [103]. TolC is a trimer that forms a continuous channel of over 140 Å in
length, consisting of a 100-Å-long α-helical channel that extends into the periplasm,
anchored via a long helical coil, to a 40-Å-long β-barrel integrated in the outer membrane [115]. The β-barrel spans the outer membrane and forms a pore that appears
wide open to the extracellular medium in the structure [57,97,103,115]. Koronakis
and coworkers have proposed that an irislike movement of the helices at the distal end
of the channel controls the opening and closing of the periplasmic entrance [116,117].
This generates a maximal opening of 30–35 Å, which has been proposed to be wide
enough to allow the passage of unfolded proteins [57,97,115].
Assembly of the T1SS apparatus
Assembly of the T1SS apparatus has been mainly explored for the secretion systems
of HlyA, Erwinia chrysanthemi proteases PrtB and PrtC, and S. marcescens HasA.
Letoffe and coworkers first showed (on PrtB, PrtC, and Has A) a sequential assembly
of the T1SS components triggered by protein substrate recognition by the ABC protein,
which interacts with the MFP, which in turn binds the outer membrane component
[118]. Thanabalu et al. [111] found that HlyD and HlyB form a stable complex in the
inner membrane in the absence of TolC and substrate. Binding of HlyA to HlyB/HlyD
promotes interaction with TolC and conformational changes in all three components,
potentially leading to the opening of the export channel. The HlyB ATPase activity is not
necessary for the T1SS assembly but is essential for export of the HlyA substrate [111].
Recognition of the protein substrate occurs partly via the C-terminal secretion
signal that interacts specifically with the NBD of the ABC protein [57,104,105]. The
secretion signals are located, in most cases, at the very C-terminus of the RTX proteins [88,119]. For example, the 60 C-terminal residues of HlyA, when fused to different passenger polypeptides, can promote the secretion of the chimeric proteins by
the HlyA T1SS [88,120,121]. In addition, the C-terminal secretion signal of HlyA can
be functionally replaced by secretion signals from other RTX toxins, displaying very
little primary sequence identity [17]. The HlyA T1SS can also export heterologous
RTX toxins [90,122,123]. Yet the precise motifs and structural features recognized in
the secretion signal are still poorly defined and may consist in few important residues
scattered throughout the sequence, as well as some potential secondary structures
(e.g., amphiphatic helix) [2,19,124–127].
The CLD of HlyB that binds the unfolded RTX domain of HlyA is likely involved
in tethering the protein substrate to prevent its aggregation and degradation during
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secretion, as proposed recently by Lecher et al. [109]. Once the machinery is assembled, HlyA is rapidly secreted, and then the T1SS apparatus disassembles [118].
The energy required for driving protein secretion has two sources:
1. The hydrolysis of ATP by RtxB is essential for secretion; it is possibly involved in the opening of the transperiplasmic channel formed by HlyD/TolC [102,105].
2. The proton motive force across the inner membrane is also required for RTX toxin export;
the protonophore CCCP (that disrupts the proton gradient across the bacterial membrane)
strongly inhibits the secretion of HlyA and CyaA [57,69,128].
Numerous studies have been carried out to characterize the secretion process
using a variety of heterologous secretion apparatus, chimeric proteins, or both [91].
For example, the E. coli T1SS machinery can export a variety of heterologous RTX
proteins, albeit with varying efficiencies [122,129,130]. Chimeric substrates have
also been widely analyzed in particular for biotechnological or vaccinal purposes
[99,131–134]. These studies revealed (i) the importance of the RTX motifs for
secretion, although this varies widely among the secretion apparatus and chimeric
proteins tested [24,28,91,135]; (ii) a prerequisite for efficient secretion of the protein
substrate is that it can unfold, likely to pass through the TolC channel, about 30 Å
wide [115,116]; and (iii) premature folding of the protein substrates or stabilization
of their 3D fold (e.g., via cytoplasmic disulfide bond formation) prevents secretion by
T1SS [99,101,133,136]. Chaperones, like SecB or GroEL are not needed for HlyA or
PtrB secretion [19,100], and it has been proposed that the RTX motifs could act as an
internal chaperone that can prevent the folding of the protein substrate [17].
Folding of the exported protein may be initiated within the external chamber of
TolC [133] or upon reaching the extracellular environment (Figure 23.4). As suggested previously, the high calcium concentration in the extracellular medium should
favor the folding of the RTX domain, which may serve as a nucleation site for the
folding of the remaining regions of the secreted protein [78,87,88]. It is thought that
the C-terminal extremity is released first into the extracellular medium, but this has
not been proven yet. Interestingly, mutations in either TolC or HlyD also result in
secreted HlyA with reduced hemolytic activity, suggesting a tight coupling between
the secretion process and the folding of the exported protein [113,137]. Other studies
have suggested a role of the outer membrane lipopolysaccharide (LPS) in the secretion, folding, and activity of several RTX toxins [138–141].
Finally, it appears that several RTX proteins remained attached to the bacterial cell
surface after export rather than being released into the environment [128,142,143]. In
numerous cases, the secreted RTX toxins (EHEC HlyA, and CyaA) are found to be
associated with outer membrane vesicles (OMVs) that may serve to deliver the toxins
to the target cells [144–147].
Main RTX toxins
RTX toxins are important virulence factors produced by many bacterial pathogens,
including Escherichia, Pasteurellaceae, Proteus, and Bordetella (Table 23.1). Many
Table 23.1
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Main RTX toxins
Toxin
Bacterial hosts
Size
(kDa)
Operon
Target
species
Target cells
HlyA
UPEC
110
>CABD/tolC
Erythrocytes,
leukocytes,
lymphocytes,
epithelia,
endothelia
EhxA
EHEC
107
>CABD/tolC
ApxIA
Actinobacillus sp
A.rossii
suis
lignieressi
A.pleuropneumoniae
110
>CABD/tolC
Human,
monkey,
pig, rat,
mouse,
horse,
sheep, cow
Human,
sheep, cow
Pig
Others ?
ApxIIA
105
>CA
ApxIIIA
120
>CABD/tolC
ApxIVA
170–200
>Orf1/A?
AqxA
A. equuli
110
>CABD/tolC
CyaA
B. pertussis
178
C<> ABDE
LktA
M. haemolytica
102
>CABD/tolC
Sheep,
goat, cow
LtxA
108
>CABD/tolC
MbxA
A. actinomycetemcomitans
M. bovis
110
>CABD/tolC
Human,
primate
Bovine
MmxA
M. morganii
110
>CABD/tolC
PaxA
108
>CABD/tolC
PlktA
P. aerogenes
P. mairi
M. varigena
Human,
animals
Pig
105
>CABD/tolC
Pig
PvxA
P. vulgaris
110
>CABD/tolC
Human,
animals
Horse,
pig
Human
Erythrocytes,
leukocytes
Erythrocytes,
leukocytes,
lymphocytes,
macrophages,
endothelia
Leukocytes,
macrophages
weakly
haemolytic
Leukocytes,
macrophages
nonhemolytic
Leukocytes,
erythrocytes?
Erythrocytes,
leukocytes,
Erythrocytes,
leukocytes,
macrophages,
neutrophils
Leukocytes,
lymphocytes,
macrophages,
platelets
Leukocytes
Erythrocytes,
leukocytes,
Erythrocytes,
leukocytes,
Erythrocytes
(cohaemolytic)
Erythrocytes,
leukocytes,
Erythrocytes,
leukocytes,
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of these toxins have been originally identified as hemolysins, as they could lyse
erythrocytes in vitro [1,148–151]. This hemolytic activity is due to the formation of
cation-selective pores in the cell membrane [38–40,152–154], and although it is likely
irrelevant from a physiological point of view, it has been highly useful for the genetic
and biochemical characterization of the corresponding RTX toxins, and it still serves
as a marker for bacteriological identification [21,148,150,155–157]. The physiologically relevant target cells of most RTX toxins are leukocytes that express β2 integrins,
which serve as specific receptors for many RTX toxins [158–163]. At high concentrations, the RTX toxins can cause direct lysis of the target leukocytes [38,39,153];
alternatively, when present at sublytic concentrations (which is most likely the case
during in vivo infection), they can alter the membrane permeability, resulting in calcium entry, potassium efflux, or both [154,164–170]. This can trigger various signaling cascades that may alter the cell physiology, leading eventually to cell apoptosis
[147,171–175]. Ultimately, these damages will result in inflammation and contribute
to tissue or organ lesions, and consequently, to the pathogenicity of the bacterial hosts.
Indeed, many of these RTX toxins are important—even essential—virulence factors.
Traditionally, RTX toxins have been subdivided in two categories (namely, hemolysins and leukotoxins) due to their range of activity against different cell types. The
so-called hemolysins were initially found to be active toward many different cell types
from a wide variety of species, while cytotoxins and leukotoxins display toxicity
toward a narrower range of cell types and species [1,2,176].
E. coli α-hemolysin (HlyA)
The archetypal RTX toxin is α-hemolysin (HlyA), which is produced by a variety of
uropathogenic E. coli (UPEC) strains [21,148], and occasionally by enterotoxigenic E.
coli (ETEC), enteropathogenic E. coli (EPEC), or Shiga toxin (Stx)–producing E. coli
(STEC) [1,150,177,178]. The hlyCABD operon that is necessary for the production and
secretion of HlyA is usually encoded in pathogenicity islands [177,179–182; see also
Chapter 2] or on plasmids [178]. UPEC strain 536 possesses two copies of the hemolysin operon, localized on distinct pathogenicity islands [179]. The coding regions of the
two hlyCABD operons are conserved, but not the upstream sequences. Consequently,
although the two hly paralogues are functionally identical, their expression pattern is
very different [183], and they likely participate at distinct stages of the infectious process, as both hemolysin determinants contribute to the full virulence of UPEC strain
536. Interestingly, the unrelated UPEC strain J96 also has the same two hly alleles,
supporting the view that the two paralogues provide a selective advantage [183].
A related hemolysin, EhxA (which is about 60% identical to HlyA at the amino
acid level), is produced by typical enterohemorrhagic E. coli (EHEC) strains, such
as O157:H7, the etiological agent of hemorrhagic colitis and hemolytic-uremic syndrome (HUS), a thrombotic microangiopathy affecting the renal glomeruli, the intestine, and the brain [184]. The ehxCABD operon is located on large EHEC virulence
plasmids [185–187]. Although Shiga toxins are the major virulence factors of EHEC,
the contribution of EhxA is likely clinically important, as it is found in all EHEC
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serogroups that are known to cause the most severe infections in humans, such the
O157:H7 serotype [37,184].
HlyA is by far the most widely studied RTX toxin and many fundamental aspects
on the structure and function of this family have been established on this model toxin
[1,2,37,176]. It consists of a single polypeptide of 1024 amino acids (110 kDa) that
is posttranslationally modified by the fatty acylation of two lysine residues, Lys-564
and Lys-690 [21,22,62,64,188,189]. This covalent modification, which is performed
by the HlyC acyltransferase (170 residues, 20 kDa), is essential to confer hemolytic
activity to the unmodified, nonhemolytic proHlyA polypeptide.
Once activated, the HlyA toxin is exported from E. coli by the means of the specific Type1 secretion machinery composed of the inner membrane ABC transporter
HlyB (707 residues, 80 kDa), the MFP HlyD (478 residues, 55 kDa) and the OMP
TolC (471 residues, 52 kDa) [27,57,111,114,190]. The three components assemble to
create a continuous channel across both bacterial membranes, and the HlyA protein
is delivered through this conduit directly from the cytosol to the external medium.
HlyA is then released in the medium without cleavage of a signal peptide occurring
[27,190]. The EHEC EhxA is also found to be associated with OMVs that are released
by EHEC during growth [145]. The hlyB and hlyD genes are organized in a single
operon with the structural gene hlyA and the hlyC gene that encodes the acytransferase [22,23,191], while tolC is encoded separately on the chromosome [114]. TolC
is also a major component of several efflux systems (e.g., AcrAB/TolC, AcrEF/TolC,
EmrAB/TolC, and MacAB/TolC) that are involved in export of antibiotics or other
toxic compounds from the cell [103].
Expression of the rtx operon is controlled by the nucleoid-associated H-NS and
Hha proteins [192,193]. HlyA is mainly expressed during the log phase [194], but
its expression is modulated by environmental conditions such as osmolarity of the
culture medium, temperature, and anaerobiosis [195]. Northern analysis revealed
full-length transcripts covering the rtxCABD genes and shorter rtxCA transcripts that
terminate downstream of the rtxA gene [196]. Inverted repeat sequences characteristic
of rho-independent transcription terminators have been described in the rtxA-rtxB
intergenic region of the hly operon (as well as in other rtx operons) [176,196,197].
The transcription antitermination protein RfaH has been shown to enhance the hlyCABD transcript elongation and the expression of the hlyB/D genes [198–201].
The HlyA polypeptide comprises four distinct regions (Figure 23.2):
●
●
●
●
The N-terminal domain contains a pronounced hydrophobic region (amino acids 237–409),
in which several putative amphiphilic and/or hydrophobic α-helical structures can be predicted [21,157]. This region is thought to interact with and insert into the target cell membrane to create cation-selective pores that will trigger cell lysis [44,48,50].
The central region harbors the two lysine residues, K564 and K690, which are fatty-acylated
by HlyC [62,64,189]. This region also contributes to define the cell selectivity [202].
The RTX-containing region (residues 724–852) is made of about 11–13 nonapeptide repeats
that are involved in Ca2+-binding. Calcium binding triggers conformational changes that are
essential for HlyA recognition of the target cells [28,44,51,53].
The C-terminal extremity contains the secretion signal (about 60 residues long) that is recognized by the HlyB/D/TolC secretion machinery [24,28,68–70,119].
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HlyA is able to attack a variety of mammalian cells in addition to erythrocytes, particularly epithelial cells, lymphocytes, and leukocytes [37,129,164–166,170,203,204].
The hemolysin can bind to the plasma membrane of target cells, supposedly by
inserting part of its polypeptide chain into the bilayer and then create a hydrophilic
transmembrane pore of about 2–3 nm diameter [39,40]. The membrane thus loses its
barrier function, solutes such as ions, amino acids, and nucleotides can leak out of the
cell, and upon extensive formation of pores, an osmotic swelling of the cell occurs,
followed by cell lysis (see Chapter 21). This cytolytic action occurs at high concentrations of HlyA and likely results from a direct association of the toxin with the cell
membrane surface in a receptor-independent manner. Initial binding studies indicated
that erythrocytes and granulocytes bind the toxin in a nonsaturable manner [152,205].
In addition, in vitro experiments showed that HlyA can bind to and permeabilize lipid
vesicles and form cation-selective pores in planar lipid membranes consisting solely
of phospholipids, indicating that a protein receptor is not needed [39,40,45,206–208].
Yet HlyA also binds to specific receptors on certain cell types. Glycophorin, a
major membrane protein in erythrocytes was shown to act as a specific receptor
for HlyA [209]. More important from a physiological point of view, the β2 integrin LFA-1 (lymphocyte function-associated antigen, also known as αLβ2 integrin
or CD11a/CD18) has been shown to serve as the cell surface receptor for HlyA on
polymorphonuclear neutrophils (PMNs) [158]. LFA-1 is widely expressed on cells
of hematopoietic origin (T- and B-cells, macrophages, and neutrophils), mediating
leukocyte adhesion and acting as a major receptor of T-cells.
Expression of LFA-1 markedly increases the sensitivity of the cells to the cytotoxic
action of HlyA (as well as many other RTX toxins, as discussed later in this chapter),
and therefore, the LFA-1-expressing leukocytes are likely the main physiological targets of HlyA in vivo during the course of infection, although this was questioned by
Valeva et al. [210]. Interestingly, Wiles et al. [211] have shown in a zebrafish model
of infection that HlyA plays a key role in the evasion of phagocyte-mediated killing
by extraintestinal pathogenic E. coli (ExPEC) at local sites of infection.
At high concentrations, HlyA can cause direct osmotic lysis of the target leukocytes [152]. When present at sublytic concentrations, HlyA can alter the membrane
permeability, triggering calcium entry or potassium efflux [164,165,167,170,204] and
interfere with cell-signaling cascades [166]. Bhakdi et al. [152] reported a potent leukocidal action on PMN of HlyA at low doses due to membrane permeabilization and
efflux of cellular ATP. Low doses of HlyA were also shown to stimulate superoxide
anion production in polymorphonuclear leukocytes [165], to stimulate the production
of IL-1 in monocytes [164], and to elicit leukotriene and hydroxyeicosatetraenoic
acid generation and cause thromboxane-mediated hypertension and vascular leakage
in blood-free perfused rabbit lungs [212,213]. HlyA also induces neutrophil apoptosis and necrosis/lysis in vitro and necrosis/lysis and lung injury in a rat pneumonia
model [214].
In addition, HlyA affects nonimmune cells: it has been shown to induce calcium
oscillations in a renal epithelial cell line and stimulate production of IL-6 and IL-8
[170]. HlyA is also a potent inductor of inflammatory and vasodilatory mediators
(such as platelet-activating factor, nitric oxide, and prostaglandin I2) in endothelial
691
cells [215,216]. HlyA thus may contribute to vasoregulatory and inflammatory disturbances observed in severe infections.
Other RTX toxins
Many RTX toxins closely related to HlyA are produced by bacteria belonging to the
Pasteurellaceae family, which comprises a large and diverse group of Gram-negative
bacteria that are obligate parasites or commensals of vertebrates, colonizing mainly
the mucosal surfaces of the upper respiratory tract, oropharynx, and reproductive
tracts and intestinal tract. RTX toxins are produced by several major pathogens:
Aggregatibacter (Actinobacillus) actinomycetemcomitans, Actinobacillus pleuropneumoniae, Pasteurella multocida, and Mannheimia (Pasteurella) haemolytica [35,36].
A. actinomycetemcomitans leukotoxin (LtxA)
A. actinomycetemcomitans is the causative organism of localized aggressive periodontitis, and possibly of subacute infective endocarditis in humans. It produces
various virulence factors, including a leukotoxin, LtxA, that is about 50% identical
to HlyA [217]. LtxA is unique due to its high selectivity toward leukocytes of human
and Old World monkeys [218], although it was recently shown to also kill rodent leukocytes [219]. This 114-kDa protein (1055 aa) is heterogeneously acylated by LtxC
on Lys562 and Lys687 [220,221]. An lktCABD operon is present in all strains and a
TolC-like protein, TdeA, is required for toxin secretion [222]. Balashova et al. [223]
found that secretion of LtxA is inhibited by free iron, while the expression of LtxA
and other associated genes is not. The production of leukotoxin varies greatly among
different strains of this species and under different culture conditions, and a catabolite-repression-like system may be involved in the regulation of leukotoxin production
[224]. LtxA is differently released in the culture medium by adherent (rough) and
nonadherent (smooth) strains of A. actinomycetemcomitans: LtxA remains associated
with adherent bacteria but can be abundantly secreted by smooth strains. Mutations in
genes required for tight nonspecific adherence (tad) of A. actinomycetemcomitans to
surfaces also cause leukotoxin to be released from the bacterial cell [142]. LtxA can
also be released in outer membrane-like vesicles that are secreted by A. actinomycetemcomitans [144] and that can deliver the leukotoxin to target cells [225].
LtxA binds to the β2 integrin, LFA-1, although it is still unclear which subunits of
LFA-1 is conferring specificity [163,226]. Interestingly, Fong and colleagues [227]
have shown that LtxA, via a cytosolic Ca2+ increase, can mobilize LFA-1 to lipid
microdomain (rafts), where it associates with LtxA. They suggested that LtxA utilizes
the raft to stimulate an integrin-signaling pathway to trigger the apoptosis of target
cells.
LtxA induces cell death of different leukocyte populations in a variety of ways
[171]. The leukotoxin can causes apoptosis in lymphocytes [228]. Macrophages are
highly sensitive to LtxA and their lysis depends on caspase 1 activation [172]. LtxA
also triggers an abundant secretion of IL-1 beta by human macrophages, in a caspase
692
1–dependent manner, and this proinflammatory cell death appears to also involve the
purinergic receptor P2X(7) [173]. LtxA triggers a potent release and activation of
matrix metalloproteinase 8 from human neutrophils [229] and can also cause shrinkage lysis of human erythrocytes in a P2X receptor-dependent manner [230]. Finally,
LtxA was found to induce proinflammatory effects on human brain endothelial cells
and to trigger apoptosis in microvascular endothelial cells [231]. Altogether, the
cytotoxicity of LtxA toward human leukocytes appears to be closely related to the
pathogenic mechanisms of periodontitis inflammatory disease [232].
M. haemolytica leukotoxin (LktA)
M. haemolytica is one of the most important respiratory pathogens of domestic ruminants [233]. It causes serious outbreaks of acute pneumonia, also known as shipping
fever, a leading cause of loss to the sheep and cattle industry throughout the world.
M. haemolytica secretes a leukotoxin, LktA, that is a primary determinant of the bacterial pathogenicity in septicemia and pneumonia [234]. LktA exhibits a narrow cell
type and species specificity as it affects only ruminant leukocytes and platelets. LktA,
is a 953 residue-long protein (105 kDa), with 62% similarity to HlyA [59,235]. It is
encoded by the lktA structural gene, which is part of the lktCABD operon [236]. LktA
is activated by LktC [202], but surprisingly, LktaA has only one classical acylation
site (K554, equivalent to K563 in HlyA), as an asparagine N684 is present instead of a
lysine at the homologous second acylation site (which is equivalent to K689 in HlyA).
LktA kills bovine immune cells by the induction of apoptosis [237], although it
also exhibits weak hemolytic activity [60]. LktA binds to different β2 integrins on the
surface of bovine leukocytes by interacting selectively with the CD18 subunit [162].
Yet, postbinding signaling events (e.g., elevation of intracellular calcium and CD18
tail phosphorylation) and cytotoxicity are only observed in the presence of the CD11a
subunit, indicating that LFA-1 is likely the functional leukotoxin receptor on bovine
alveolar macrophages [238]. Interestingly, Shanthalingam and Srikumaran [239] have
shown that the LktA-binding site on CD18 includes the intact signal peptide that is not
cleaved in ruminant CD18 because of the presence of a cleavage-inhibiting residue at
position − 5. These results thus explain the species selectivity of the M. haemolytica
leukotoxin. Thumbikat et al. [240] also reported that neither acylation nor the amino
terminal 344 amino acids are required for LktA binding, but they are essential for
LktA cytolysis of target cells.
At low concentrations, LktA can activate bovine leukocytes to undergo oxidative
bursting and release proinflammatory cytokines (IL-1, IL-6, and IL-8 and tumor
necrosis factor α), while at higher concentration, LktA induces the formation of
transmembrane pores and cell death [234]. The proinflammatory cytokines contribute
to the accumulation of leukocytes in the lung and also upregulate the expression of
β2-integrins at the surface, thus enhancing the biological effects of LktA on bovine
neutrophils [160]. LktA was also shown to induce extracellular trap (ET) formation
by bovine neutrophils or monocyte-derived macrophages [241,242].
Related leukotoxins are also produced by other Mannheimia species such
Mannheimia glucosida, Mannheimia varigena (originally described as Pasteurella
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haemolytica–like leukotoxin, PllktA) or Pasteurella trehalosi [243–245] but they are
less characterized.
A. pleuropneumoniae toxins, ApxI, ApxII, ApxIII, and ApxIV
A. pleuropneumoniae (previously Haemophilus pleuropneumoniae) is a prominent pathogen that causes severe, contagious pulmonary disease in pigs. The main disease is porcine
pleuropneumonia, a highly contagious respiratory disease that affects primarily young
pigs. A. pleuropneumoniae produces four different RTX toxins, ApxI, ApxII, ApxIII, and
ApxIV, that appear to contribute significantly to the pathogenesis of porcine pleuropneumonia [36,243,246–249]. These toxins display the following distinct properties:
●
●
●
ApxI (110 kDa) is strongly hemolytic and cytotoxic to leukocytes. It is encoded by the
apxIA gene (1023 codons) that is part of the apxI-CABD operon [246,247,250].
ApxII (105 kDa) is weakly hemolytic and moderately cytotoxic. The apxII operon contains
only the structural gene apxIIA (956 codons) and the apxIIC gene, but no genes for the
secretion components [246,251,252]. Secretion of ApxII seems to occur via the products of
the secretion genes of the apxI operon.
ApxIII (120 kDa), encoded by the apxIIIA gene (1052 codons), is nonhemolytic but strongly
cytotoxic toward porcine lung macrophage [247].
All these three Apx toxins are about 50% identical to HlyA. They all exhibit a transient channel-forming activity in planar membrane, albeit with different efficiency
and with different channel conductances [253].
The fourth Rtx toxin, ApxIV, is encoded by the apxIVA gene, which varied in length
from 1382 to 1805 codons in different serotypes [249]. ApxIV presents significant
sequence similarity with the iron-regulated RTX proteins of Neisseria meningitidis,
FrpA and FrpC [254]. Indeed, a recombinant ApxIV expressed in E. coli was shown
to exhibit a calcium-dependent autoprocessing and crosslinking activity, similar to
the “clip-and-link” activity described for the FrpC protein [255]. Recombinant ApxIV
also displays a weak hemolytic activity in the presence of an additional gene, ORF1
(157 codons), located immediately upstream of apxIVA, but which has no homology
to rtxC genes. ApxIVA is not be detected in bacteria grown in vitro, but it must be
expressed in vivo during the infection, as antibodies directed against ApxIVA are found
in pigs infected with A. pleuropneumoniae [249]
All serotypes of A. pleuropneumoniae secrete ApxIV during infection of pigs
[249,256], but the different serotypes expressed variable combinations of the other
three Apx toxins [252,257]. The highly pathogenic serotypes secrete ApxI, ApxII,
and ApxIV, or ApxII, ApxIII, and ApxIV, while the other serotypes secrete only one
of the RTX toxins in addition to ApxIV [248,258]. The contribution of the Apx toxins
to the virulence of A. pleuropneumoniae has been established by experimental infection of pigs with genetically defined mutants of A. pleuropneumoniae, in which one
or several apx genes were inactivated [259,260]. It was thus demonstrated that both
ApxI and ApxII of A. pleuropneumoniae serotype 1 are necessary for full virulence
[259,261]. A direct role of these toxins in the pathogenesis was also established by
endobronchial inoculation of A. pleuropneumoniae [262].
694
RTX toxins have also been identified in the phylogenetically related species
Actinobacillus lignieresii, a bovine pathogen; Actinobacillus equuli, an equine
pathogen; and Actinobacillus suis, which is pathogenic for pigs [263]. Berthoud and
colleagues [264] have characterized the aqx operon from A. equuli and showed that
all A. equuli isolates contained the aqxCABD operon and expressed a hemolytically
active AqxA. The host cell specificity of the hemolytic and cytotoxic activity of AqxA
and of ApxI and ApxII from A. suis has been reported by Kuhnert et al. [265], who
showed that the cytotoxic activity of these RTX toxins is strongly species specific,
while the hemolytic activity is not.
Proteus, Morganella, and Moraxella spp toxins
Hemolysins genetically related to HlyA have been identified very early on in Proteus
mirabilis, Proteus vulgaris, and Morganella morganii [25,266]. Proteus mirabilis and
Proteus vulgaris are commensals of the normal flora of the human gastrointestinal
tract, but they also can be found in water and soil. There are opportunistic pathogens
that can infect the lungs, or wounds, and frequently cause urinary tract infections.
Morganella (previously Proteus) morganii is also a commensal of the intestinal tracts
of humans or animals that can be implicated in postoperative and other nosocomial
infections. The functional properties and pore-forming properties of the RTX determinants have been investigated in lipid-bilayer membranes and erythrocytes and were
showed to be similar to that of HlyA [42,267–269].
Moraxella bovis is the etiologic agent of infectious bovine keratoconjunctivitis.
Gray et al. [270] first identified in the culture supernatant of M. bovis, a possible
HlyA-related toxin of 110 kDa with hemolytic and cytotoxic activities that might
be involved in the pathogenesis of infectious bovine keratoconjunctivitis. Angelos
et al. [271] cloned the structural gene MbxA and characterized the mbx operon [272]
that harbors the putative acyltransferase MbxC and the secretory proteins MbxB and
MbxD, as well as a TolC homologue. This operon is absent in nonhemolytic M. bovis
strains. Closely related operons, designated mbvCABD/tolC and movCABD/tolC,
have been identified in Moraxella bovoculi and M. ovis, respectively [273].
The AC toxin, CyaA, from Bordetella species
A distinct member of the RTX toxin family is the AC toxin, CyaA (or ACT), produced by various Bordetella species, including Bordetella pertussis, the causative
agent of whooping cough, a highly contagious, acute respiratory illness in humans
(see Chapter 6). B. pertussis is a strict human pathogen with no known animal or
environmental reservoir, while other species, Bordetella bronchiseptica, Bordetella
parapertussis, and Bordetella holmesii, are associated with respiratory infections in
humans and other mammals [274]. CyaA is unique among other RTX toxins, as it is
a bifunctional protein that has an AC domain in addition to the core structural features
common to other RTX hemolysins [46,55,89,275]. CyaA exhibits a hemolytic activity
695
as other RTX toxins [46,89] and is able to deliver its catalytic AC domain into the
cytosol of target cells, where it is activated by calmodulin (CaM) [55,276,277]. The
CaM-activated AC has a very high catalytic rate and produces supra-physiological
levels of cyclic adenosine monophosphate (cAMP), which in turn alter major physiological processes in the intoxicated cells, eventually leading to cell death [278,279].
The cytotoxicity of CyaA stems primarily from this massive production of cAMP in
target cells [46,55,276,280–282], although its pore-forming/hemolytic activity may
also enhance the overall cytotoxicity [283–285].
CyaA is an essential virulent factor of B. pertussis [274,279]. In an infant mouse
model of infection, mutants deficient in CyaA were shown to be avirulent [286–288].
Furthermore, both passive and active immunization with CyaA significantly shortened the period of bacterial colonization of the mouse respiratory tract by B. pertussis [289,290]. The AC toxin plays an important role in the early stages of respiratory
tract colonization by B. pertussis [281,288,289]. Harvill and colleagues [291], by
using a murine model of infection, have identified neutrophils and macrophages
(that express CD11b/CD18) as the primary targets of the CyaA toxin from B. bronchiseptica, a related animal pathogen. The CyaA toxin, therefore, may represent an
essential mechanism of defense against the early steps of innate immune responses
[278,292]. CyaA inhibits the phagocytic functions of neutrophils and macrophages by
impairing chemotaxis and oxidative response, and eventually triggers cell apoptosis
[161,276,284,293–298]. These effects are primarily due to the elevation of cAMP
in target cells and activation of cAMP-signaling cascades that paralyze phagocytic
processes and modulate gene transcription (particularly of many inflammatory genes)
[299,300]. CyaA also induces K+ efflux and Ca2+ influx that contribute to the overall
cytotoxicity of the toxin [283,284]. In addition, CyaA has been shown to affect the
adaptive immune responses by altering the maturation of dendritic cells [301–304]
and the activation (and chemotaxis) of T lymphocytes [305,306]. In the more general
context of an infection, CyaA will not act alone, but more likely in synergy with the
various pathogenic factors produced by B. pertussis [274,307,308].
CyaA is encoded by the cyaA gene, which forms an operon with the cyaB, cyaD,
and cyaE genes that encode a dedicated T1SS apparatus [89,197] (Figure 23.1).
The cyaC gene, which codes for the acyltransferase involved in CyaA acylation, is
located upstream of this operon but is transcribed divergently [61,309]. The expression of the cyaA/B/D/E operon is coordinately regulated by environmental signals via
a two-component system, like other virulence genes of B. pertussis, [274,308,310].
The 1706-residue-long CyaA is a bifunctional protein endowed with both AC (CyaA)
and hemolytic activities [46,55,89,280,311]. The catalytic domain, AC, is located
in the 400 amino-proximal residues, while the 1306 carboxy-terminal residues are
responsible for the hemolytic phenotype of B. pertussis (Figure 23.2). Both activities
can function independently as AC and hemolysin, respectively [275,312–314]. The
catalytic domain exhibits high enzymatic activity (kcat>2000 s−1) upon activation by
CaM, which binds with a high affinity (KD<0.1 nM) to the enzyme and stimulates its
activity by more than 1000-fold [312,315,316]. The C-terminal part of the molecule
(residues 400–1706) is endowed with an intrinsic (albeit low) hemolytic activity that
696
results from its ability to form cation-selective channels in membranes [41,317–319].
More important, this domain mediates the binding and internalization of the toxin into
eukaryotic cells [46,55,280]. Several subregions can be identified in this hemolysinlike moiety (Figure 23.2):
●
●
●
●
●
The region located between residues 400–500 (translocation domain, T), is crucial for the
translocation of AC domain across the plasma membrane and exhibits properties related to
membrane-active peptides [320,321].
The region (H) located from residues 500–700 contains several hydrophobic segments with
potentially amphiphilic and hydrophobic α-helical structures that are similar to the poreforming region of E. coli HlyA. Mutations and deletions in this region affect the cytotoxicity
and the hemolytic activity of CyaA [46,67,322], indicating that this portion is involved in
both the CyaA delivery and pore formation.
A region that contains two lysine residues (K860 and K983) that are specifically acylated
in the presence of CyaC [61,63,309,323,324]. The acylation of these lysines is essential for
converting the proCyaA into an active toxin endowed with cytoxic and hemolytic activities.
The RTX domain (residues 913–1613) that contains about 40 copies of RTX motifs that
are organized in five separated blocks of about seven to nine repeats each, separated by
non-RTX flanking regions of variable lengths [275]. Calcium binding to the RTX domain
is essential for both the hemolytic activity and the translocation of the AC catalytic domain
into the cytosol of target cells [7,46,54,55,280]. The RTX domain is also involved in binding
to a β2 integrin receptor on target cells [96,161].
The C-terminal end of CyaA harbors a secretion signal that is recognized by the CyaB/
CyaD/CyaE secretion machinery [89,122]. Depending on the strain, CyaA can be released
in the culture medium or remain adsorbed on the bacterial surface, or even be released with
OMVs [46,143,146].
CyaA can intoxicate a variety of cell types by a process that is independent of receptor-mediated endocytosis [54,55,325,326]. CyaA is thought to bind to the target cells by
inserting its hydrophobic region (aa 500–750) into their plasma membrane [327]. Then
the catalytic domain is translocated across the cell membrane into the cytosol, where
it is activated by CaM to produce cAMP [54,55,276,277,280]. How CyaA crosses the
plasma membrane of the target cells remains largely unknown. The translocation is
dependent on calcium, temperature, and negative potential across the target cell membrane [54,55,276,280,328–330], and may involve a transient destabilization of the lipid
bilayer by the translocation domain located between AC and the hydrophobic region
[320,321]. Interestingly, an atypical entry of CyaA into polarized T84 epithelial cells via
their basolateral membranes has been reported [331]. CyaA insertion into the membrane
also leads to the formation of cation selective pores of small diameter (0.6–0.8 nm), in a
process that likely involves the oligomerization of the protein [41,317,318,332].
In addition, CyaA binds in a calcium-dependent manner and with high affinity to
the CD11b/CD18 β2 integrin [96,161], that is expressed by a subset of leukocyte,
including neutrophils, macrophages, and dendritic cells (DCs), and that is thus the
primary target of the toxin in vivo [291,294,295]. Bumba et al. [333] showed recently
that after binding to the receptor via its RTX domain, CyaA mobilizes CD11b/CD18
into membrane microdomains, where the translocation of the AC domain occurs in a
cholesterol dependent manner.
697
MARTX toxins
The first MARTX toxin was identified in El Tor Vibrio cholerae by Lin et al. [31], as a
gene cluster physically linked to the cholera toxin element in the V. cholerae genome
and resembling that of the classical RTX toxins. The MARTX toxin was shown to
contribute to the virulence of the bacterial host [31]. Similar toxins have been identified in other Vibrio species, as well as in various bacterial genus such as Aeromonas,
Yersinia, Proteus, and Photorhabdus [3,32,33]. Although these proteins were initially
grouped with the RTX toxins, their structural organization and their mode of action
are completely different than that of the classical RTX toxins and clearly specify a
distinct family. This section briefly summarizes the main features of these toxins; for
further details, see the recent and comprehensive review of K. Satchell [33].
The MARTX gene cluster contains two divergent operons: one encoding proteins
RtxH, RtxC, and RtxA, the other encoding RtxB, RtxD, and RtxE (Figure 23.1):
●
●
●
RtxH is a protein of unknown function, RtxC is a putative acyltransferase, and RtxA is a
large protein with glycine and aspartic-rich repeated motifs [32].
RtxB and RtxE are two putative ABC transporters, homologous to HlyB, while RtxD is a
putative membrane fusion homologous to HlyD. RtxB, RtxD, RtxE, and the outer membrane TolC constitute an atypical four-component type I secretion system (TISS) required
for the MARTX toxin secretion [33,334,335].
The RtxA proteins are gigantic polypeptides of 3000–5000 amino acids (depending on the
strain) and contain two different types of repeated sequences [33]:
At the C-terminus, there are about 15 atypical RTX motifs, 18 residues in length.
The first 9 residues of these repeats are similar to a classical nonapeptide RTX motif.
Whether these motifs bind calcium and how they might fold are still unknown.
At the N-terminus, there are 14 repeats of 20 residues in length, followed by 38 repeats
of 19 residues in length. Again, the structure and properties of these repeats are unknown.
●
●
The central part of the MARTX RtxA toxins contains a series of distinct modules
or effector domains [33]. These domains are delivered to the cytosol of eukaryotic
target cells by a still-poorly-characterized pathway [336]. The last C-terminal domain
(CPD) is always a cysteine protease that is required for autoprocessing of the different
modules [337]. The CPD is allosterically activated by inositol phosphate (preferentially inositol hexakisphophate) and cleaves the RtxA polypeptide at Leu-Xaa bonds
to liberate the different effector domains [338].
The activities of several of these effector domains have been characterized. They
include:
●
●
●
●
An actin-cross-linking domain (ACD) that links the side chain of Glu270 of a first actin
monomer to Lys50 of a second actin monomer [339–341].
A Rho GTPase-inactivating domain (RID) [342,343].
An AC domain similar to the ExoY toxin of P. aeruginosa [344]. Its enzymatic activity is
activated by an unknown eukaryotic cofactor.
An alpha-beta hydrolase domain (ABH) has been recently shown to activate CDC42 [336].
Some effector domains are predicted through homology with various toxins and
protein folds, while many others are still of unknown function [33].
698
Molecular mode of action of RTX toxins: some key issues
Acylation of RTX toxins
RTX toxins are produced as inactive protoxins (proRtxAs) that have to be modified by
a specific posttranslational acylation to become cytolytic [2,65,66]. The proRtxA activation is carried out by a specific acyltransferase, RtxC, that catalyzes the transfer of acyl
chains from the acyl-ACP (acyl carrier protein) to the ε-amino group of internal lysine
residues [2,3,62]. RtxC proteins constitute a distinct subclass of acyltransferases [66].
They are relatively well conserved among different species and exhibit a certain degree
of redundancy in their capacity to acylate heterologous proRtxA toxins [60,129,202,345–
347], indicating that the reaction mechanism of these various acyltransferases is similar.
The biochemistry of RTX toxin activation has been mainly characterized for the
HlyC acylation of proHlyA. In 1991, Issartel et al. [62] set up an in vitro assay to
demonstrate that activation of prohemolysin to mature toxin is achieved by the transfer of a fatty acyl group from acyl-ACP to proHlyA. Various fatty acid chains (C12,
C14, C16, C16:1, C18, and C18:1) can be added, but acyl-ACP is the only accepted
acyl donor. In vivo, HlyA is predominantly acylated with myristic acid (68%), but also
with the rare C15 and C17 saturated fatty acids [189]. Stanley et al. [64] identified
lysine 564 and lysine 690 as the residues that are modified by acylation. Resistance
of the acylation to hydroxylamine indicated that the fatty acid is amide-linked, and
mutagenesis of the two lysines confirmed that they are the only modified residues
in vivo [64,188]. Both lysine residues are independently fatty-acylated [64,188]. The
amino acid sequences flanking the two acylation sites are partly conserved among
the various proRtxA proteins [64,65], but relatively large sequences around the
corresponding acylation sites are required for the acylation reaction [65,188,348].
Presently, the molecular determinants of the highly specific acylation of lysine 564
and 690 of proHlyA still remain elusive [348].
The enzymology of the HlyC-catalyzed reaction has been further characterized
by Ernst-Fonberg and colleagues. The acyltransferase reaction proceeds through
two partial reactions (a so-called ping pong mechanism) and involves the formation
of an acyl-enzyme intermediate in which His23 serves as a transient acyl acceptor
[349,350]. His23 is highly conserved among RtxC enzymes and is absolutely essential for activity, while Ser20 also appears to be critical for catalysis [349,350]. The
equivalent residues, Ser30 and His33, are also essential for CyaC activity [351]. The
proposed model is that in a first reaction, the acyl chain is transferred from acylACP to the His23 of HlyC via an acyl-imidazole intermediate. In a second step, the
acyl chain is transferred from acyl-HlyC to the ε-amino nucleophiles of Lys564 and
Lys690 of proHlyA. Other mutagenesis studies revealed additional residues that are
important for HlyC activity and or selectivity [352–354].
The acylation mechanism of other RtxC acyltransferases is likely very similar, given
the protein sequence conservation and the fact that the various RtxC enzymes are able
to acylate different proRtxA substrates [60,129,202,345–347]. Yet the nature and number of the acyl chain covalently attached to the protoxins are distinct [63,188,221,323].
For example, HlyA is preferentially acylated by myristic acid (68%), while CyaA is
exclusively palmitoylated on Lys983 (corresponding to Lys690 of HlyA) in native B.
699
pertussis. Furthermore, a second lysine (Lys860 corresponding to Lys564 of HlyA)
is acylated in recombinant proteins obtained by overexpression of CyaC and CyaA
either in E. coli or in B. pertussis [323,355], and this modification differently affects
the hemolytic and cytotoxic activities of the toxin. The A. actinomycetemcomitans
leukotoxin was even found to contain hydroxylated fatty acyl chains [221].
The precise role of the acyl groups attached to the RTX toxins in the cytolytic
activities of these proteins is still unclear. If the nonacylated proteins are unable
to lyse target cells, they are still able to form pores in planar lipid bilayer, albeit
with reduced efficiency [41,42,188,356]. The nonacylated proHlyA is able to bind
lipid vesicles in vitro but cannot permeabilize liposome [357] nor insert into a lipid
monolayer [358]. In contrast, the proCyaA was shown to permeabilize liposome as
efficiently as the acylated toxin [356]. Acylation also increases the affinity of the RTX
toxins for their β2-integrin receptors, as reported for CyaA and LtxA [96,220,240].
Regarding the potential mechanism of action, it was initially thought that the fatty
acids attached to the RtxA polypeptide might partition into the membrane, thus initiating the toxin interaction with the target cells [2,359]. Other studies have suggested
that acylation may play a structural role, affecting the folding and oligomerization
of the protein [2]. Herlax et al. [360] reported that acylation of HlyA is essential
for the oligomerization of the toxin in erythrocyte membranes, and it is thought that
oligomerization is involved in pore formation [361]. Conversely, Lee et al. [362],
reported that the CyaA toxin can oligomerize in solution, while the nonacylated
proCyA cannot. It is unclear, however, if these oligomeric species are physiologically relevant, as CyaA, like other RTX toxins, is naturally prone to aggregation. At
variance, Karst et al. [87] recently reported that the monomeric form of CyaA is the
physiologically active species of the toxin in solution. They further showed that CyaA
folding into a monomeric form is critically dependent upon the presence of both
calcium and acylation and is favored by molecular confinement, which may partly
mimic the steric constrains imposed by secretion thru the T1SS channel. The acyl
groups may also play a structural role in maintaining the toxins in partially folded or
molten globule–like conformations, which would be favorable for membrane insertion [2,363]. However, no strong experimental support for this hypothesis has been
obtained thus far. Future studies may help to clarify this central question.
Membrane-pore structures
Another important question that remains to be addressed is the structure of the putative membrane pores formed by RtxA toxins [2,3,37]. In the absence of structural
data, hypothetical models about the potential organization of the RtxA proteins in
the membrane of target cells have been elaborated from a large set of biochemical and biophysical data. It is generally assumed that the hydrophobic domain
located in the N-terminal part of the RtxA polypeptide is directly implicated in
the permeabilization of the cell membrane. This region contains several predicted
amphiphilic/hydrophobic α-helical segments. In a commonly accepted model, these
sequences are inserted into the membrane to form transmembrane α-helices and create the membrane pores [2,42,44,318,364,365]. Indeed, selective deletions within
the N-terminal region of HlyA or CyaA or point mutations that alter the predicted
700
amphipathicity/hydrophobicity of these segments have been shown to abolish the
hemolytic activity toward erythrocytes and pore formation in artificial lipid bilayers
[44,46,67,176,322]. In addition, Hyland et al. [48] have shown that these hydrophobic
sequences (encompassing residues 177–411 of HlyA), are the major determinant of
membrane binding and insertion, as revealed by liposome-binding experiments and
labeling with a photoactivatable probe incorporated into the target lipid bilayer.
Possible topological arrangements of these hydrophobic structures within the
putative pore have been further explored by Schindel and coworkers [47] by using
fluorescently labeled HlyA variants. HlyA variants harboring single cysteine residue
introduced at various positions along the putative membrane-inserting region (i.e.,
residues 170–400) were produced and labeled with a polarity-sensitive fluorescent
probe. Protein insertion into lipid bilayer was monitored by fluorescent changes.
These studies revealed several amino acids that possibly become inserted into the
lipid bilayer during pore formation [47] and identified an amphipathic alpha-helix
(residues 272–298) that may line the aqueous pore [49]. Disruption of this putative
helical structure by introducing prolines at positions 284 and 287 did not affect binding properties, but it totally abolished the hemolytic activity of the molecule. These
data demonstrate the key role of these segments, but how they are organized in the
membrane remains unknown.
Several lines of evidence support the view that oligomerization of the RtxA polypeptides in the membrane is required to form a conducting pore:
●
●
●
●
Different nonfunctional RtxA variants can complement each other to create functional pores
[361,366,367].
The hemolytic and channel forming activities show concentration dependence with a cooperative character [40,41,317,368].
The limited lifetime of the channels suggests that the channel-forming unit is a dynamic
structure [40,42].
Toxin oligomers are formed in the cell membrane as detected by fluorescence resonance
energy transfer (FRET) or blue native polyacrylamide gel electrophoresis [332,360]. Yet
the exact stoechiometry of HlyA or CyaA in these putative channel-forming, oligomeric
structures is still unknown.
At variance with this oligomer model, Menestrina [369] showed that the permeabilization of small unilamellar lipid vesicles by HlyA follows a pseudo first-order reaction. This suggested that the toxin is active as a monomer and Menestrina proposed
a “single-hit” mechanism [369]. Soloaga and colleagues [370] proposed a model in
which HlyA is not inserted as an authentic transmembrane protein, but rather occupies only the external leaflet of the phospholipid membrane bilayer. They speculated
that the amphipathic helices of HlyA could anchor the protein to the external leaflet
of the membrane. Insertion of HlyA molecules in the outer leaflet of the membrane
may induce an increase in the lateral pressure of the monolayer lipids and trigger
membrane leakage [370]. This nontransmembrane arrangement of HlyA in the target
membrane cannot be easily reconciled with the model of a classical pore-forming
toxin. Structural characterization of the membrane-associated RtxA proteins will be
needed to clarify this critical issue.
701
Interaction with cell receptors and traffic
Several RTX toxins can interact with target cells in the absence of specific receptors by directly binding to the membrane bilayer. This process may involved two
distinct steps: namely, a reversible partition of the toxin to the membrane surface
(e.g., adsorption, driven in part by electrostatic interactions) followed an irreversible
membrane insertion that may lead to formation of pores [208,371]. Different protein
regions may be implicated in each step; e.g., the RTX domain and the toxin acylation
in initial cell binding and the hydrophobic region in subsequent membrane insertion
[2,28,37,81,129,176,202,346]. This direct membrane binding may explain the broad
specificity of several RTX hemolysins that are able to attack a variety of cell types,
including erythrocytes, although these latter cells are no longer considered real physiological targets of these toxins [2,371]. Specific receptors can also be involved in the
binding of RTX toxins to erythrocytes, such as glycophorin, that act as receptors for
HlyA on red blood cells [95,209]. The initial adsorption of the RTX toxins may also
occur through the recognition of glycosylated membrane components, such as glycoproteins and gangliosides [372,373], and recently, Munksgaard et al. [374] showed
that sialic acid residues are important for LtxA-induced cell lysis.
More important, many RTX toxins are using β2 integrins as specific cellular receptors. β2 integrins are leukocyte-specific, heterodimeric cell surface glycoproteins
sharing the same β2 subunit, CD18, but containing different α-subunits [375]. Lally
and coworkers were the first to identify the CD11a/CD18 β2 integrin (αL/β2 or LFA-1,
leukocyte function-associated antigen-1) as a receptor for A. actinomycetemcomitans
LtxA and E. coli HlyA on the surface of human leukocytes [158]. LFA-1 also serves
as a functional receptor for M. haemolytica LktA on bovine alveolar macrophages
[159,160]. Which subunit of LFA-1 is important for interaction with these toxins is
still debated: some studies indicated that the β-propeller domain of CD11a is involved
in LtxA recognition [226], while others suggested that the β2-chain (CD18) of the
integrin is responsible for selective binding of LtxA and LktA [163,238,376–378].
Another β2 integrin, MAC-1, or complement receptor 3 (CR3), made of the
CD11b (αM) and CD18 (β2) subunits, is the receptor of B. pertussis CyaA at the surface of neutrophils and macrophages [161,379]. El-Azami-El-Idrissi et al. [96] further
mapped the putative site of interaction with CD11b within the RTX domain of CyaA.
Interestingly, Morova et al. [372] later showed that binding and cytotoxic action of
different toxins, CyaA, LtxA, or HlyA depend on the presence of N-linked oligosaccharide chains on the β2 integrin receptors.
Several recent studies indicate that the RTX toxins are not using the β2 integrins as passive recruiting structures; rather, these toxins are actively mobilizing the receptors to membrane microdomains. This was first reported by Fong and colleagues [227], who showed
that A. actinomycetemcomitans LtxA can mobilize LFA-1 to lipid rafts, where they
associate to stimulate an integrin signaling pathway that leads to apoptosis of target cells.
LFA-1 clustering to lipid rafts is initiated by a LtxA-induced increase in cytosolic calcium
that triggers the activation of calpain, cleavage of talin, and mobilization of LFA-1 into
cholesterol and sphingolipid-rich microdomains of the plasma membrane. More recently,
Brown et al. [380] demonstrated that LtxA contains two cholesterol recognition/amino
702
acid consensus (CRAC) sites, one (CRAC336) highly conserved among RTX toxins,
while the second (CRAC503) is unique to LtxA. They further showed that a peptide corresponding to CRAC336 inhibited the LtxA cytotoxic activity and demonstrated that a point
mutation in the CRAC336 site abolished LtxA toxicity. The conservation of CRAC336
among RTX toxins suggests that this mechanism may be conserved among RTX toxins,
and indeed a specific interaction between cholesterol and E. coli HlyA has recently been
reported [381]. DiFranco et al. [382] reported that binding of LtxA to LFA-1 results in the
internalization of both LtxA and LFA-1. LtxA then specifically relocalizes to the lysosomal compartment, where it causes the disruption of the lysosomal membrane and release
of lysosomal contents into the cytosol, leading to cell death.
Atapattu and Czuprynski [383] suggested that M. haemolytica leukotoxin LktA,
after binding to LFA-1 on bovine lymphoblastoid cells (BL-3), also moves to lipid
rafts and clathrin-coated pits, where it is internalized. Further studies showed that
LktA is able to enter into and traffic to the mitochondria of BL-3 cells, where it
interacts with the translocase of the outer membrane of mitochondria (TOM) and
cyclophilin D, a member of the mitochondria permeability transition pore [384,385].
B. pertussis CyaA also mobilizes its CD11b/CD18 receptor into lipid rafts to accomplish translocation across target cell membrane in two steps. Bumba et al. [333] showed
that upon binding to the receptor, CyaA inserts into the membrane to form a translocation intermediate that allows the influx of extracellular calcium. Calcium triggers a
calpain-mediated cleavage of talin that enables the β2 integrin-CyaA complex to cluster into lipid rafts, where the membrane translocation of the catalytic domain occurs.
Interestingly, in T cells, CyaA induces a premature disengagement of LFA-1 from the
immunological synapses in a cAMP-dependent manner [306]. These studies indicate
that the cell biology of RTX toxins is likely to be more elaborate than initially thought.
Concluding remarks
Many issues regarding the structure and mode of action of the RTX toxins are under
active investigation. A better understanding of the biology of this important class of virulence factors will hopefully suggest novel strategies to prevent their pathogenic actions
and cure the diseases caused by their bacterial hosts. The rich biological resources of
this family of toxins are also exploited for various biotechnological and therapeutic
purposes. For example, the T1SS is used to export antigens or other proteins of biotechnological interest from bacterial cells [132,134,386]. In addition, some RTX toxins
can be engineered for therapeutic applications. The A. actinomycetemcomitans LtxA
leukotoxin is being developed as a targeting agent (Leukothera®) for the treatment of
hematological malignancies or autoimmune/inflammatory diseases, characterized by
chronic activation of leukocytes expressing high levels of LFA-1 [387–390]. The B. pertussis CyaA toxin is used as an antigen delivery vehicle to trigger cell-mediated immune
responses against a variety of antigens [379,391,392]. Two recombinant CyaA-based
immunotherapeutic vaccines [393–395] are currently in phase I and II clinical trials. It
can be anticipated that other members of the RTX toxin family will also be exploited for
specific applications and the development of new tools for cell biology.
703
Acknowledgments
The authors have been supported by Institut Pasteur and CNRS (UMR3528).
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