Structure and mode of action of clostridial glucosylating toxins: the ABCD model
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Review
Structure and mode of action of
clostridial glucosylating toxins: the
ABCD model
Thomas Jank and Klaus Aktories
Institut für Experimentelle und Klinische Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg,
Otto-Krayer-Haus, Albertstrasse 25, D-79104 Freiburg, Germany
Toxins A and B, which are the major virulence factors of Toxins with multimodular structure
antibiotic-associated diarrhea and pseudomembranous Toxin A consists of 2710 amino acid residues with a
colitis caused by Clostridium difficile, are the prototypes molecular mass of 308 kDa and toxin B comprises 2366
of the family of clostridial glucosylating toxins. The residues with a mass of 269.6 kDa. These toxins are there-
toxins inactivate Rho and Ras proteins by glucosylation. fore also known as large clostridial cytotoxins [10]. On the
Recent findings on the autocatalytic processing of the basis of their amino acid sequences, a tripartite structure for
toxins and analysis of the crystal structures of their the toxins had been suggested [10,12,13], with a biologically
domains have made a revision of the current model of active N-terminal domain, a middle translocation section
their actions on the eukaryotic target cells necessary. characterized by a small hydrophobic stretch (prediction of
transmembrane regions), and a C-terminal receptor-bind-
Introduction ing domain. This prediction of the structure–function
Antibiotic-associated diarrhea and pseudomembranous relationship was in line with the model of AB-toxins such
colitis induced by Clostridium difficile have emerged as as diphtheria toxin, consisting of a biologically active
important nosocomial infections. During the past decade, domain and a binding or translocation domain [14]. The
these diseases seem to have become more serious and more binding domain can be separated into subdomains (e.g. for
frequently refractory to therapy [1]. The occurrence of receptor binding and translocation) resulting in a tripartite
hypervirulent strains of C. difficile such as PCR ribotype structure. However, recent studies indicate that a multi-
027/PFGE type NAP1 [2] with attributable mortality rates modular structure more accurately describes the structure–
of up to 16.7% is concerning [3]. The major virulence factors function relationship of the clostridial glucosylating toxins
of C. difficile are two protein toxins, named toxin A and toxin (Figure 1). These toxins have a biologically active domain
B (also designated TcdA and TcdB), which have been recog- and a binding and translocation domain but in addition they
nized for their important role in disease for the past 30 years. have an autocatalytic, self-cutting protease domain for toxin
In addition, some strains of C. difficile produce a binary processing. Thus, the AB-toxin model can be extended to an
ADP-ribosylating toxin (C. difficile transferase, CDT) that ABCD model (A, biological activity; B, binding; C, cutting; D,
modifies G-actin [4]. Notably, hypervirulent strains are delivery).
characterized by production of 10- to 20-fold larger amounts
of toxins A and B, the resistance to fluoroquinolones and The C terminus binds to target cell membranes
production of the actin-ADP-ribosylating toxin [2]. The receptor-binding domain located at the C terminus of
the clostridial glucosylating toxins consists of repeating
Structure–function relationships of clostridial polypeptide units [12,15–17]. Recently, two C-terminal
glucosylating toxins fragments (terminal 127 and 255 residues) of toxin A
C. difficile toxin A and toxin B are the prototypes of the (toxinotype VI) were crystallized [18,19]. These studies
family of clostridial glucosylating toxins [5,6]. Other mem- gave new insights into the overall structure of the C
bers of this toxin family are the hemorrhagic and the lethal terminus of the toxin (Figure 1). Toxin A folds in a sole-
toxins from Clostridium sordellii and the a-toxin from noid-like structure with 32 small repeats consisting of 15–
Clostridium novyi. Moreover, several isoforms of toxin A 21 residues and seven large repeats of 30 residues. Each
and B have been described, adding to this family of cyto- repeat forms a single b-hairpin that is rotated by 1208 to
toxins [7–9]. All of these toxins share sequence identities each other, thereby forming a screw-like superfold
ranging from 36% to 90% and have molecular masses (Figure 1). Solenoid structures are frequently found in
between 250 and 308 kDa [10,11]. Because toxins A and bacterial virulence proteins [20]. They increase the sur-
B are of major clinical importance and because most data face area of proteins and enable protein–protein or
obtained are from these toxins, this review focuses on protein–carbohydrate interactions. Some years ago, Kri-
recent progress in the understanding of the structure– van and Tucker showed binding of the trisaccharide
function relationship of these toxins. Gala1–3Galb1–4GlcNAc to toxin A [21,22]. This was con-
firmed and structurally explained by co-crystallization of
Corresponding author: Aktories, K. (klaus.aktories@pharmakol.uni-freiburg.de). toxin A with an artificial trisaccharide containing the
222 0966-842X/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2008.01.011
Review Trends in Microbiology Vol.16 No.5
Figure 1. Domain structure of clostridial glucosylating toxins. The ABCD model (labeled in bold at top of figure) of clostridial glucosylating toxins is shown with Clostridium
difficile toxin B as an example. The biologically active glucosyltransferase A-domain (bold) is located at the N terminus (amino acids 1–543) and has been crystallized
recently. The DXD motif, which is involved in Mn2+ coordination, is located in this domain. The C terminus of the clostridial glucosylating toxins, which consists of
polypeptide repeats, is involved in receptor binding (B-domain, bold). A fragment of the C-terminal repeats of toxin A has been crystallized, showing a solenoid-like
structure. From this rather small fragment, the structure of the whole C-terminal part of toxin A has been deduced, which consists of 39 repetitive elements [19]. According
to this model the repeats have a b-hairpin structure. Rotation of the hairpins by 1208 relative to the neighboring hairpin forms a screw-like structure. The cysteine protease
domain (residues 544–767), which is similar to Vibrio cholerae RTX toxin, is located adjacent to the glucosyltransferase domain. This C-domain (bold) is involved in
processing and cutting of the toxin. The cysteine protease C-domain can be characterized by the catalytic triad consisting of Asp587, His653 and Cys698 (DHC). Alternatively,
the DXG (D1665) motif was suggested to be part of an aspartate protease domain, possibly involved in processing of the toxins. In the middle part of the protein is a short
hydrophobic region (residues 956–1128), which might be involved in pore formation and delivery of the catalytic domain into the cytosol (D-domain, bold). Images were
created using PyMOL (www.pymol.org).
Gala1–3Galb1–4GlcNAc-glycan [19]. The carbohydrate- A. However, structural data are not available to date and
binding groove is formed between the junction of a large the nature of the receptor of C. difficile toxin B is even
repeat and the hairpin turn of the following small repeat further from being defined.
by several amino acids. These residues are not conserved
in the other clostridial glucosylating toxins and might Toxin uptake: a question of cutting and delivery
have a crucial role in carbohydrate receptor specificity. Following receptor binding, the clostridial glucosylating
However, human tissue generally does not produce a- toxins are endocytosed [25] (Figure 2) – the precise endo-
anomeric galactose bonds [23], indicating that the carbo- cytosis pathway is not known. After endocytosis, the toxins
hydrate structure Gala1–3Galb1–4GlcNAc cannot be part translocate through the early endosomal membrane into
of intestinal receptors in humans. Therefore the disac- the cytosol. This process depends on the acidification of
charide Galb1–4GlcNAc, which is present in humans, has endosomes by vesicular H+-ATPase. Bafilomycin, which
been suggested to be part of a possible glycan receptor. blocks the H+-ATPase, inhibits cytosolic entry of the toxin
Whether a glycoprotein or glycosphingolipid [24] (either and intoxication of cells [26]. Therefore, C. difficile toxins A
with or without an additional protein receptor) comprises and B belong to the short trip toxins group (which includes
the intestinal receptor remains to be clarified. The C- diphtheria toxin) that enter the cytosol of host cells from an
terminal part of toxin B is probably similar to that of toxin early endosomal compartment. These protein toxins can be
223
Review Trends in Microbiology Vol.16 No.5 Figure 2. Processing of clostridial glucosylating toxins. Clostridial glucosylating toxins bind to the cell membrane through the C terminus. The receptors for the toxins are not yet defined. For C. difficile toxin A carbohydrates are proposed as receptors. After binding, the toxins are endocytosed to reach an acidic endosomal compartment. Here, conformational changes occur enabling insertion of the toxin into the endosomal membrane and subsequent pore-formation. The toxins are processed by autocatalytic cleavage, which in the case of toxin B depends on the presence of inositol hexakisphosphate (InsP6). Only the glucosyltransferase domain of the N terminus of the toxins is released into the cytosol. In the cytosol, Rho GTPases are glucosylated at Thr37 (e.g. RhoA) or at Thr35 (e.g. Rac or Cdc42). distinguished from the long trip toxins (which includes pore formation is directly involved in delivery of the toxin cholera toxin) that travel retrograde from endosomes to the into the cytosol remains unclear, but it is thought that the Golgi and from there to the ER, where they eventually hydrophobic region (residues 956–1128 of toxin B) is part of enter the cytosol [27]. The low pH of early endosomes the delivery domain. induces conformational changes of the clostridial toxins, The precise mechanism of the translocation process resulting in exposure of a hydrophobic region of the protein remains one of the most enigmatic puzzles of the action toxins, enabling membrane insertion [28]. It has been of these toxins. Recent studies indicate that the toxins suggested that residues 956–1128 of toxin B are part of must be processed to reach the cytosol. Only the catalytic the hydrophobic region, which is involved in membrane domain of the toxins, including the N-terminal 543 amino insertion [13]. A short-term decrease in extracellular pH of acids, is delivered into the cytosol [30,31]. The search for a the medium of cell cultures mimics the low pH of endo- host protease possibly involved in toxin processing resulted somes and enables the toxins to enter host cells directly in the unexpected finding that the clostridial glucosylating through the cell membrane [26,28]. Membrane insertion is toxins are auto-proteolytically processed [32]. Even more paralleled by formation of pores. This can be shown by surprising, inositol hexakisphosphate (InsP6, phytic acid) 86 Rb+ ion release from 86Rb+-loaded host cells when the pH was found to be an essential factor for activation of proteol- of the cell medium is reduced to pH 5 [26]. Toxin B mutants ysis [32], however its functional role is not clear. Polypho- and N-terminal truncated toxins consisting of the C termi- sphorylated inositol, a common inositol metabolite in nus and the middle part of the protein (including the hydro- mammalian cells, is highly charged and has diverse bio- phobic residues 956–1128) are sufficient for pore formation logical functions, including mRNA traffic king, binding to [26]. It has been suggested that the C terminus, which is clathrin-assembly protein AP-2, inhibition of protein phos- involved in binding, is not essential for pore formation but phatases and stimulation of protein kinases [33,34]. InsP6 enhances toxin–membrane interaction. The efficacy of pore might be involved in stabilization of toxin protein confor- formation induced by toxin A is largely cholesterol-depend- mation, which is essential for protease activity and/or ent. Cholesterol depletion of membranes with methyl-b- proper cleavage. cyclodextrin inhibits 86Rb+ efflux and cholesterol repletion Recently it was suggested that toxin B possesses reconstituted pore-forming activity of toxin A [29]. Whether aspartate protease activity, which is responsible for toxin 224
Review Trends in Microbiology Vol.16 No.5
cleavage because the aspartate protease inhibitor EPNP The N-terminal enzyme domain
(1,2-epoxy-3-p-nitrophenoxypropane) blocked the proces- The N terminus harbors the glucosyltransferase activity of
sing of toxin B and labeled aspartate 1665 as part of a the toxins and is the biological activity domain [37]. The 543
short Asp-Xaa-Gly (DXG) motif observed in many aspar- amino acid residues, which are delivered into the cytosol of
tate proteases [32]. Another hypothesis describes a host cells, form the glucosyltransferase domain. Recently,
cysteine protease activity as being responsible for the the 3D-structure of the catalytic domain (residues 1–543) of
processing of clostridial glucosylating toxins. There are toxin B has been solved [38]. The catalytic core is formed by a
several lines of evidence supporting this hypothesis. First, mixed a/b-fold with mostly parallel b-strands (Figure 3a).
as observed frequently for cysteine proteases, dithiothrei- The overall structure of the catalytic core is similar to other
tol activates the auto-catalytic cleavage of the toxin. bacterial glycosyltransferases, such as Neisseria meningiti-
Second, the autocatalytic activity is blocked by N-ethylma- dis galactosyltransferase LgtC (lipooligosaccharide glycosyl
leimide, a typical inhibitor of cysteine proteases. Third, the transferase C) [39] and Bacillus subtilis glycosyltransferase
clostridial glucosylating toxins share sequence similarity SpsA [40], but also to eukaryotic glycosyltransferases, such
with a novel, recently identified autocatalytic cysteine as bovine galactosyltransferase a3GalT [41]. All of these
protease domain within the structure of the RTX (repeats transferases belong to the glycosyltransferase A (GT-A)
in toxins) toxin of Vibrio cholerae. Several essential cata- family [42]. Mainly based on the sequence homology
lytic residues, including the putative catalytic triad D587, described here, the family of clostridial glucosylating toxins
H653 and C698, are conserved [35,36] (Figure 1). Accord- has been designated glycosyltransferase family 44 as
ing to this model, a cysteine protease cutting domain, defined by Henrissat et al. (http://www.cazy.org/). This
which is adjacent to the glucosyltransferase domain, is family also includes genes from Escherichia coli and Chla-
responsible for processing of the toxin. mydia trachomatis coding for putative glycosyltransferases.
Figure 3. The glucosyltransferase domain of Clostridium difficile toxin B. (a) Structural model of the glycosyltransferase domain of C. difficile toxin B, where UDP-glucose
(blue) is attached to the catalytic cleft via Mn2+ (brown) and to the residues of the DXD motif (ball-and-stick model); water molecules are shown as small blue spheres. The
GT-A type fold catalytic core is presented in white and the additional subdomains are shown in brown. The putative ‘flexible loop’ region is shown in red with Trp520
hydrogen bonding to the b-phosphate of UDP-glucose. (b) The catalytic cleft of toxin B, as in (a) but rotated 458. Several amino acids involved in co-substrate binding are
shown. (c) Schematic representation of the catalytic cleft shown in (b). Images were created using PyMOL (www.pymol.org).
225
Review Trends in Microbiology Vol.16 No.5
The catalytic core of toxin B consists of 234 residues [54,55]. A SNi (internal return) mechanism has been
plus >300 additional residues. These additional residues suggested for the transferase reaction. This mechanism
are mainly helices. The four N-terminal helices are is characterized by an oxocarbenium-like transition state
particularly prominent and seem to be an independent [38].
subdomain, possibly involved in membrane interaction. The clostridial glucosylating toxins exhibit high co-sub-
Mesmin and coworker showed for lethal toxin from C. strate specificity. Whereas toxin A and B and also the
sordellii (the closest homolog of toxin B) that the first related lethal toxin from C. sordellii use UDP-glucose as
18 amino acids (correlating to the first helix of the N- a donor substrate, the co-substrate of a-toxin from Clos-
terminal subdomain) are crucial for lipid bilayer attach- tridium novyi is UDP-N-acetylglucosamine (UDP-GlcNAc)
ment [43]. Also, truncations beyond amino acid Lys65 [56]. Biochemical studies revealed that Ile383 and Glu385
[44] lead to inactivation of the glucosyltransferase and are responsible for co-substrate specificity. These residues
glucohydrolase activity, demonstrating the importance of limit the space of the catalytic cleft for binding of the co-
the N-terminal helical bundle for catalytic activity. substrate in toxin B. Exchange of these residues changes
Recently, it was shown that domain C1 of Pasteurella the co-substrate specificities of the toxins. Thus, the toxin
multocida toxin (PMT) is structurally similar (41% sim- B double mutant Ile383Ser, Gln385Ala accepts UDP-
ilarity with toxin B) to this subdomain of toxin B. A role GlcNAc as a co-substrate [57] and changing Ser385 and
in membrane interaction has also been proposed for PMT Ala387 to Ile and Glu, respectively, turns a-toxin into a
[45]. UDP-glucose-accepting transferase.
The crystal structure of the catalytic domain of toxin B
revealed a set of essential amino acid residues involved in Interaction of toxin B with Rho GTPases
glucosyltransferase reaction or in substrate binding. The Glycosyltransferases are region-selective enzymes. In
Asp-Xaa-Asp (DXD) motif is characteristic of GT-A family the case of the clostridial glucosylating toxins a
members (Asp286 and Asp288 in toxin B) [46,47]. This specific threonine residue in the substrate proteins
motif is involved in Mn2+, UDP and glucose binding. (small GTP-binding proteins) is monoglucosylated
Whereas Asp288 complexes directly with Mn2+, interaction [54,58]. This threonine senses the integrity of the nucleo-
of Asp286 with Mn2+ occurs via a water molecule. Asp286 tide GTP, which is bound to the GTPase in the active
also interacts with the 30 hydroxyl group of the UDP-ribose conformation and to the hydrolyzed GDP form in the
and with the 30 hydroxyl group of glucose, so it is of major inactive conformation. Sensing occurs via binding to a
importance for proper positioning of UDP-glucose in the Mg2+ ion, which is also complexed with the phosphates of
catalytic cleft of the enzyme. Trp102, which is also essen- the nucleotide. Glucosylation prevents the sensor func-
tial for enzyme activity, stabilizes the uracil ring of UDP by tion and thereby prevents the fundamental confor-
aromatic stacking. In addition to the DXD motif and mational switch of the GTP-binding proteins necessary
Trp102, Asp270, Arg273, Tyr284, Asn384 and Trp520 were for interaction with diverse effectors or regulator
identified by alanine scanning as being essential for proteins [59] (Box 1).
enzyme activity [48]. Asp270 and Arg273 are conserved Substrate specificity of the GTP-binding proteins for the
in several GT-A type glycosyltransferases and form with toxins has not been structurally defined to date. Never-
the help of Asp286, a highly defined hydrogen-bond net- theless, distinct amino acids or regions on Rho GTPases
work for proper adjustment of the glucose moiety of the have been shown to have a role in defining specificity. The
donor substrate [41,49]. A common structural feature of all substrate properties of RhoA versus RhoD are defined by
GT-A type glycosyltransferase is the ‘flexible loop’, which the residues Ser73 and Phe85, respectively, located on the
switches from an open, disordered conformation (apo- switch II region [60]. The differential recognition of Rac
enzyme) to a closed, ordered conformation on UDP-sugar and RhoA by lethal toxin, toxin B1470 and toxin B8864, is
binding. Co-substrate binding creates a deep pocket that attributed to the N-terminal region around amino acids
serves as a binding site for the acceptor substrate [50] 22–27 of the GTPases where Lys27 of Rho has a major role
(Figure 3a). The deep burial of the UDP-sugar could pre- [61].
vent water molecules from acting as acceptors and hydro- Earlier studies showed that the C-terminal part of the
lyzing the nucleotide sugar. It was proposed that the catalytic domain of the toxins (residues 364–516) confers
‘flexible loop’ helped release the reaction products, leading substrate specificity [62]. Recent data indicate that amino
to suitable turnover of the enzyme [42,51]. Trp520 is well acids Arg455, Asp461, Lys463 and Glu472, and residues of
conserved in all of the clostridial glucosylating toxins and helix a17 (e.g. Glu449) of toxin B are essential for enzyme–
resides on the putative ‘flexible loop’ formed by amino acids protein substrate recognition [48]. Introduction of helix
510 to 523 and interacts with the scissile bond of the donor a17 of toxin B into C. sordellii lethal toxin prevents the
substrate (b-phosphate oxygen of UDP). It was shown for modification of Ras subfamily proteins but enables gluco-
other glycosyltransferases that the corresponding trypto- sylation of RhoA. Crystallographic and biochemical results
phan residue swings out thereby opening the conformation led to a docking model in which the GTPase consensus
while releasing UDP [52,53]. binding region (suggested for effector or regulator binding
Glycosyltransferases are strictly stereospecific [63]) interacts in a similar manner with the glucosyltrans-
enzymes. The known clostridial glucosylating toxins ferase toxins (Figure 4). In this model, the membrane-
are retaining glucosyltransferases, because the sugar associated regions of the GTPase and the region of the
is attached to the target protein in the same a- toxin, which is supposed to interact with the membrane,
anomeric configuration as in the substrate UDP-glucose are located on the same side [48].
226Review Trends in Microbiology Vol.16 No.5
Box 1. Functional consequences of glucosylation of Rho GTPases by clostridial toxins
The clostridial glucosylating toxins modify Rho proteins (Figure I). oxide production, cytokine secretion and immune cell signaling. The
These are low molecular mass GTPases, which control numerous clostridial glucosylating toxins modify Rho GTPases at threonine 35
signaling pathways [64,65] (Figure Ia). Approximately 20 Rho or 37, which is located in the switch-I region of the GTPases [58,66]
GTPases have been described, including RhoA, Rac1 and Cdc42, the (Figure Ib). The toxin-catalyzed glucosylation of Rho proteins results
best studied targets of the toxins. Rho GTPases are inactive in the in inhibition of effector coupling and subsequent blocking of signal
GDP-bound form and associated with guanine nucleotide dissociation transduction pathways [67]. It also blocks nucleotide exchange by
inhibitors (GDI), which keep the GTPases in the cytosol. Guanine GEFs [67] and inhibits intrinsic and GAP-stimulated GTPase activity
nucleotide exchange factors (GEFs) activate Rho GTPases. This [67]. Glucosylated Rho is no longer able to interact with GDI and is
enables interaction with different effectors to control numerous therefore found at the plasma membrane [68]. However, crystal-
signaling processes. The active state of Rho GTPases is terminated lographic [54] and NMR [55] data obtained from the Rho-related Ras
by hydrolysis of bound GTP facilitated by GTPase-activating proteins protein, which has been glucosylated by the lethal toxin of C. sordellii,
(GAP). Rho proteins regulate the actin cytoskeleton, enzyme activa- indicate that glucosylation prevents formation of the active ‘GTP
tion (e.g. protein kinases, phospholipases), cell polarity, gene conformation’ of the GTPases, whereas binding to GTP itself is
transcription and cell proliferation (for review see [65]). However, possible. Surprisingly, the glucosylating toxins cause up-regulation of
considering host–pathogen interactions and immune cell activities, it Rho B, which is an immediate-early gene product [69]. Some Rho B
is of note that Rho proteins are essential for epithelial barrier seems to be able to escape modification by toxins A or B and might
functions, immune cell migration, adhesion, phagocytosis, super- have important signaling functions [70].
Figure I. Regulation and signaling of Rho proteins (a) and functional consequences of the modification of Rho proteins by clostridial glucosylating toxins (b).
227Review Trends in Microbiology Vol.16 No.5
Figure 4. Model of toxin B interaction with RhoA – docking model of the glycosyltransferase domain toxin B (white) with its substrate RhoA (green) according to reference
[48]. RhoA is shown as a surface representation with the acceptor amino acid threonine 37 (T37) shown in white. Ser73 and Lys27 of RhoA defining the specificity towards
the toxins are marked and shown in red. The positions of amino acids in toxin B necessary for protein substrate recognition and helix a17 are circled in black. The different
locations of amino acids important for enzyme activity are circled in yellow. The N-terminal 18 amino acids of toxin B involved in membrane attachment are shown in blue.
UDP-glucose in toxin B and GDP in RhoA are marked and shown in stick representation (black). The C-terminal linker peptide of RhoA with its isoprenyl membrane anchor is
schematically represented. Images were created using PyMOL (www.pymol.org).
Concluding remarks 5 Voth, D.E. and Ballard, J.D. (2005) Clostridium difficile toxins:
mechanism of action and role in disease. Clin. Microbiol. Rev. 18,
Studies from recent years have yielded important progress
247–263
in our knowledge of the structure and mode of action of 6 Just, I. and Gerhard, R. (2004) Large clostridial cytotoxins. Rev.
clostridial glucosylating toxins. These results indicate the Physiol. Biochem. Pharmacol. 152, 23–47
need for a revision of the current AB-model of clostridial 7 Mehlig, M. et al. (2001) Variant toxin B and a functional toxin A
glucosylating toxins. Characterization of clostridial gluco- produced by Clostridium difficile C34. FEMS Microbiol. Lett. 198,
171–176
sylating toxins according to this model seems to be a scien-
8 Chaves-Olarte, E. et al. (1999) A novel cytotoxin from Clostridium
tific Procrustes’ bed. Thus, we suggest that an ABCD model difficile serogroup F is a functional hybrid between two other large
is more appropriate for describing the structure and func- clostridial cytotoxins. J. Biol. Chem. 274, 11046–11052
tion of these toxins. However, clostridial toxins are large and 9 Rupnik, M. et al. (1998) A novel toxinotyping scheme and correlation of
their full 3D-structure remains unknown, we also have yet toxinotypes with serogroups of Clostridium difficile isolates. J. Clin.
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to understand the precise functions of all toxin domains. 10 Von Eichel-Streiber, C. et al. (1996) Large clostridial cytotoxins - a
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future. Nevertheless, recent results on the structural Trends Microbiol. 4, 375–382
analysis of the toxins have increased our knowledge about 11 Busch, C. and Aktories, K. (2000) Microbial toxins and the
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