Large Clostridial Glucosylating Toxins Research Articles

Clostridiumnovyi's Alpha-Toxin Changes Proteome and Phosphoproteome of HEp-2 Cells.

C. novyi type A produces the alpha-toxin (TcnA) that belongs to the large clostridial glucosylating toxins (LCGTs) and is able to modify small GTPases by N-acetylglucosamination on conserved threonine residues. In contrast, other LCGTs including Clostridioides difficile toxin A and toxin B (TcdA; TcdB) modify small GTPases by mono-o-glucosylation. Both modifications inactivate the GTPases and cause strong effects on GTPase-dependent signal transduction pathways and the consequent reorganization of the actin cytoskeleton leading to cell rounding and finally cell death. However, the effect of TcnA on target cells is largely unexplored. Therefore, we performed a comprehensive screening approach of TcnA treated HEp-2 cells and analyzed their proteome and their phosphoproteome using LC-MS-based methods. With this data-dependent acquisition (DDA) approach, 5086 proteins and 9427 phosphosites could be identified and quantified. Of these, 35 proteins were found to be significantly altered after toxin treatment, and 1832 phosphosites were responsive to TcnA treatment. By analyzing the TcnA-induced proteomic effects of HEp-2 cells, 23 common signaling pathways were identified to be altered, including Actin Cytoskeleton Signaling, Epithelial Adherens Junction Signaling, and Signaling by Rho Family GTPases. All these pathways are also regulated after application of TcdA or TcdB of C. difficile. After TcnA treatment the regulation on phosphorylation level was much stronger compared to the proteome level, in terms of both strength of regulation and the number of regulated phosphosites. Interestingly, various signaling pathways such as Signaling by Rho Family GTPases or Integrin Signaling were activated on proteome level while being inhibited on phosphorylation level or vice versa as observed for the Role of BRCA1 in DNA Damage Response. ZIP kinase, as well as Calmodulin-dependent protein kinases IV & II, were observed as activated while Aurora-A kinase and CDK kinases tended to be inhibited in cells treated with TcnA based on their substrate regulation pattern.

Another surprise in receptor binding of C. difficile toxins

Clostridioides difficile (formerly named Clostridium difficile) is the dominant causative agent of a spectrum of illnesses, which frequently occur as the consequence of antibiotic treatment of patients. C. difficile infections (CDIs) cause mild to severe diarrhea (so-called antibiotic-associated diarrhea) but also pseudomembranous enterocolitis with complications like toxic megacolon, bowel perforation, and death. Three protein toxins are involved in the pathology of CDIs, toxin A (TcdA) and toxin B (TcdB), which are the prototypes of large clostridial glucosylating toxins, and C. difficile ADP-ribosyltransferase CDT.

A Highly Specific Holin-Mediated Mechanism Facilitates the Secretion of Lethal Toxin TcsL in Paeniclostridiumsordellii.

Protein secretion is generally mediated by a series of distinct pathways in bacteria. Recently, evidence of a novel bacterial secretion pathway involving a bacteriophage-related protein has emerged. TcdE, a holin-like protein encoded by toxigenic isolates of Clostridioides difficile, mediates the release of the large clostridial glucosylating toxins (LCGTs), TcdA and TcdB, and TpeL from C. perfringens uses another holin-like protein, TpeE, for its secretion; however, it is not yet known if TcdE or TpeE secretion is specific to these proteins. It is also unknown if other members of the LCGT-producing clostridia, including Paeniclostridium sordellii (previously Clostridium sordellii), use a similar toxin-release mechanism. Here, we confirm that each of the LCGT-producing clostridia encode functional holin-like proteins in close proximity to the toxin genes. To characterise the respective roles of these holin-like proteins in the release of the LCGTs, P. sordellii and its lethal toxin, TcsL, were used as a model. Construction and analysis of mutants of the P. sordellii tcsE (holin-like) gene demonstrated that TcsE plays a significant role in TcsL release. Proteomic analysis of the secretome from the tcsE mutant confirmed that TcsE is required for efficient TcsL secretion. Unexpectedly, comparative sample analysis showed that TcsL was the only protein significantly altered in its release, suggesting that this holin-like protein has specifically evolved to function in the release of this important virulence factor. This specificity has, to our knowledge, not been previously shown and suggests that this protein may function as part of a specific mechanism for the release of all LCGTs.

Bacterial intracellularly active toxins: Membrane localisation of the active domain.

Numerous bacterial toxins exert their activity by inactivating or modulating a specific intracellular host target. For this purpose, these toxins have developed efficient strategies to overcome the different host cell defences including specific binding to cell surface, internalisation, passage through the endosome or plasma membrane, exploiting intracellular trafficking and addressing to intracellular targets. Several intracellularly active toxins deliver an active domain into the cytosol that interacts with a target localised to the inner face of the plasma membrane. Thus, the large clostridial glucosylating toxins (LCGTs) target Rho/Ras-GTPases, certain virulence factors of Gram negative bacteria, Rho-GTPases, while Pasteurella multocida toxin (PMT) targets trimeric G-proteins. Others such as botulinum neurotoxins and tetanus neurotoxin have their substrate on synaptic vesicle membrane. LCGTs, PMT, and certain virulence factors from Vibrio sp. show a particular structure constituted of a four-helix bundle membrane (4HBM) protruding from the catalytic site that specifically binds to the membrane phospholipids and then trap the catalytic domain at the proximity of the membrane anchored substrate. Structural and functional analysis indicate that the 4HBM tip of the Clostridium sordellii lethal toxin (TcsL) from the LCGT family contain two loops forming a cavity that mediates the binding to phospholipids and more specifically to phosphatidylserine.

Quantitative Phosphoproteome Analysis of Clostridioides difficile Toxin B Treated Human Epithelial Cells

The large clostridial glucosylating toxin B (TcdB) is a major virulence factor of the nosocomial pathogen Clostridioides difficile. TcdB inhibits small GTPases by glucosylation leading to impaired downstream signaling. TcdB also possesses a glucosyltransferase independent effect described as pyknosis. To elucidate the impact of TcdB and its glucosylation-inactive mutant TcdBNXN on the kinome of human cells, SILAC labeled HEp-2 cells were treated with 2 nM TcdB for 8 h. Phosphopeptides were enriched using SCX chromatography, IMAC and TiO2 followed shotgun mass spectrometry analysis. Overall 4,197 phosphopeptides were identified; more than 1,200 phosphosites responded to treatment with TcdB or TcdBNXN. The data suggested that predominantly stress-activated MAPK-dependent signaling pathways were triggered by toxin B treatment.

A neutralizing antibody that blocks delivery of the enzymatic cargo of Clostridium difficile toxin TcdB into host cells

Clostridium difficile infection is the leading cause of hospital-acquired diarrhea and is mediated by the actions of two toxins, TcdA and TcdB. The toxins perturb host cell function through a multistep process of receptor binding, endocytosis, low pH-induced pore formation, and the translocation and delivery of an N-terminal glucosyltransferase domain that inactivates host GTPases. Infection studies with isogenic strains having defined toxin deletions have established TcdB as an important target for therapeutic development. Monoclonal antibodies that neutralize TcdB function have been shown to protect against C. difficile infection in animal models and reduce recurrence in humans. Here, we report the mechanism of TcdB neutralization by PA41, a humanized monoclonal antibody capable of neutralizing TcdB from a diverse array of C. difficile strains. Through a combination of structural, biochemical, and cell functional studies, involving X-ray crystallography and EM, we show that PA41 recognizes a single, highly conserved epitope on the TcdB glucosyltransferase domain and blocks productive translocation and delivery of the enzymatic cargo into the host cell. Our study reveals a unique mechanism of C. difficile toxin neutralization by a monoclonal antibody, which involves targeting a process that is conserved across the large clostridial glucosylating toxins. The PA41 antibody described here provides a valuable tool for dissecting the mechanism of toxin pore formation and translocation across the endosomal membrane.

Clostridium difficile and Clostridium sordellii toxins, proinflammatory versus anti-inflammatory response

Clostridium difficile and Clostridium sordellii produce related potent toxins (C. difficile toxin A (TcdA) and toxin B (TcdB), C. sordellii lethal toxin (TcsL) and hemorrhagic toxin (TcsH)) which belong to the large clostridial glucosylating toxin (LCGT) family. TcsL is the main C. sordellii toxin as most of toxigenic C. sordellii strains only synthesize this toxin. Intestinal colonization by C. difficile subsequently to unbalanced microbiota is accompanied by release of toxins which induce local tissue destruction and severe inflammatory response. TcdA and TcdB inactivate Rho-GTPases. Notably inactivation of RhoA results in the stimulation of the pyrin/ASC inflammasome, which is one of the main signaling pathways used by these toxins to trigger the inflammatory response. In contrast, TcsL induces an anti-inflammatory effect, mainly by inactivating Ras proteins which results in blockage of the cell cycle and killing of immune cells. The absence or moderate local inflammatory response allows C. sordellii spreading in deep tissues, production of toxin which is released in the general circulation and causes a toxic shock syndrome. The toxin mechanisms of pro-versus anti-inflammatory responses are discussed.

The role of toxins in Clostridium difficile infection.

Clostridium difficile is a bacterial pathogen that is the leading cause of nosocomial antibiotic-associated diarrhea and pseudomembranous colitis worldwide. The incidence, severity, mortality and healthcare costs associated with C. difficile infection (CDI) are rising, making C. difficile a major threat to public health. Traditional treatments for CDI involve use of antibiotics such as metronidazole and vancomycin, but disease recurrence occurs in about 30% of patients, highlighting the need for new therapies. The pathogenesis of C. difficile is primarily mediated by the actions of two large clostridial glucosylating toxins, toxin A (TcdA) and toxin B (TcdB). Some strains produce a third toxin, the binary toxin C. difficile transferase, which can also contribute to C. difficile virulence and disease. These toxins act on the colonic epithelium and immune cells and induce a complex cascade of cellular events that result in fluid secretion, inflammation and tissue damage, which are the hallmark features of the disease. In this review, we summarize our current understanding of the structure and mechanism of action of the C. difficile toxins and their role in disease.

The Tip of the Four N-Terminal α-Helices of Clostridium sordellii Lethal Toxin Contains the Interaction Site with Membrane Phosphatidylserine Facilitating Small GTPases Glucosylation.

Clostridium sordellii lethal toxin (TcsL) is a powerful virulence factor responsible for severe toxic shock in man and animals. TcsL belongs to the large clostridial glucosylating toxin (LCGT) family which inactivates small GTPases by glucosylation with uridine-diphosphate (UDP)-glucose as a cofactor. Notably, TcsL modifies Rac and Ras GTPases, leading to drastic alteration of the actin cytoskeleton and cell viability. TcsL enters cells via receptor-mediated endocytosis and delivers the N-terminal glucosylating domain (TcsL-cat) into the cytosol. TcsL-cat was found to preferentially bind to phosphatidylserine (PS)-containing membranes and to increase the glucosylation of Rac anchored to the lipid membrane. We have previously reported that the N-terminal four helical bundle structure (1–93 domain) recognizes a broad range of lipids, but that TcsL-cat specifically binds to PS and phosphatidic acid. Here, we show using mutagenesis that the PS binding site is localized on the tip of the four-helix bundle which is rich in positively-charged amino acids. Residues Y14, V15, F17, and R18 on loop 1, between helices 1 and 2, in coordination with R68 from loop 3, between helices 3 and 4, form a pocket which accommodates L-serine. The functional PS-binding site is required for TcsL-cat binding to the plasma membrane and subsequent cytotoxicity. TcsL-cat binding to PS facilitates a high enzymatic activity towards membrane-anchored Ras by about three orders of magnitude as compared to Ras in solution. The PS-binding site is conserved in LCGTs, which likely retain a common mechanism of binding to the membrane for their full activity towards membrane-bound GTPases.

The catalytic domains of Clostridium sordellii lethal toxin and related large clostridial glucosylating toxins specifically recognize the negatively charged phospholipids phosphatidylserine and phosphatidic acid.

Clostridium sordellii lethal toxin (TcsL) is a potent virulence factor belonging to the large clostridial glucosylating toxin family. TcsL enters target cells via receptor-mediated endocytosis and delivers the N-terminal catalytic domain (TcsL-cat) into the cytosol upon an autoproteolytic process. TcsL-cat inactivates small GTPases including Rac and Ras by glucosylation with uridine-diphosphate (UDP)-glucose as cofactor leading to drastic changes in cytoskeleton and cell viability. TcsL-cat was found to preferentially bind to phosphatidylserine (PS)-containing membranes and to increase the glucosylation of Rac anchored to lipid membrane. We here report binding affinity measurements of TcsL-cat for brain PS-containing membranes by surface plasmon resonance and enzyme-linked immunosorbent assay (ELISA). In addition, TcsL-cat bound to phosphatidic acid (PA) and, to a lesser extent, to other anionic lipids, but not to neutral lipids, sphingolipids or sterol. We further show that the lipid unsaturation status influenced TcsL-cat binding to phospholipids, PS with unsaturated acyl chains and PA with saturated acyl chains being the preferred bindingsubstrates. Phospholipid binding site is localized at the N-terminal four helical bundle structure (1-93 domain). However, TcsL-1-93 bound to a broad range of substrates, whereas TcsL-cat, which is the active domain physiologically delivered into the cytosol, selectively bound to PS and PA. Similar findings were observed with the other large clostridial glucosylating toxins from C. difficile, C. novyi and C. perfringens.

Haemorrhagic toxin and lethal toxin from Clostridium sordellii strain vpi9048: molecular characterization and comparative analysis of substrate specificity of the large clostridial glucosylating toxins.

Large clostridial glucosylating toxins (LCGTs) are produced by toxigenic strains of Clostridium difficile, Clostridium perfringens, Clostridium novyi and Clostridium sordellii. While most C. sordellii strains solely produce lethal toxin (TcsL), C. sordellii strain VPI9048 co-produces both hemorrhagic toxin (TcsH) and TcsL. Here, the sequences of TcsH-9048 and TcsL-9048 are provided, showing that both toxins retain conserved LCGT features and that TcsL and TcsH are highly related to Toxin A (TcdA) and Toxin B (TcdB) from C. difficile strain VPI10463. The substrate profile of the toxins was investigated with recombinant LCGT transferase domains (rN) and a wide panel of small GTPases. rN-TcsH-9048 and rN-TcdA-10463 glucosylated preferably Rho-GTPases but also Ras-GTPases to some extent. In this respect, rN-TcsH-9048 and rN-TcdA-10463 differ from the respective full-length TcsH-9048 and TcdA-10463, which exclusively glucosylate Rho-GTPases. rN-TcsL-9048 and full length TcsL-9048 glucosylate both Rho- and Ras-GTPases, whereas rN-TcdB-10463 and full length TcdB-10463 exclusively glucosylate Rho-GTPases. Vero cells treated with full length TcsH-9048 or TcdA-10463 also showed glucosylation of Ras, albeit to a lower extent than of Rho-GTPases. Thus, in vitro analysis of substrate spectra using recombinant transferase domains corresponding to the auto-proteolytically cleaved domains, predicts more precisely the in vivo substrates than the full length toxins. Except for TcdB-1470, all LCGTs evoked increased expression of the small GTPase RhoB, which exhibited cytoprotective activity in cells treated with TcsL isoforms, but pro-apoptotic activity in cells treated with TcdA, TcdB, and TcsH. All LCGTs induced a rapid dephosphorylation of pY118-paxillin and of pS144/141-PAK1/2 prior to actin filament depolymerization indicating that disassembly of focal adhesions is an early event leading to the disorganization of the actin cytoskeleton.

Identification and characterization of Clostridium sordellii toxin gene regulator.

Toxigenic Clostridium sordellii causes uncommon but highly lethal infections in humans and animals. Recently, an increased incidence of C. sordellii infections has been reported in women undergoing obstetric interventions. Pathogenic strains of C. sordellii produce numerous virulence factors, including sordellilysin, phospholipase, neuraminidase, and two large clostridial glucosylating toxins, TcsL and TcsH. Recent studies have demonstrated that TcsL toxin is an essential virulence factor for the pathogenicity of C. sordellii. In this study, we identified and characterized TcsR as the toxin gene (tcsL) regulator in C. sordellii. High-throughput sequencing of two C. sordellii strains revealed that tcsR lies within a genomic region that encodes TcsL, TcsH, and TcsE, a putative holin. By using ClosTron technology, we inactivated the tcsR gene in strain ATCC 9714. Toxin production and tcsL transcription were decreased in the tcsR mutant strain. However, the complemented tcsR mutant produced large amounts of toxins, similar to the parental strain. Expression of the Clostridium difficile toxin gene regulator tcdR also restored toxin production to the C. sordellii tcsR mutant, showing that these sigma factors are functionally interchangeable.

Characterization of the enzymatic activity of Clostridium perfringens TpeL

TpeL is a toxin produced by Clostridium perfringens which belongs to the large clostridial glucosylating toxin family. It was shown that TpeL modifies Ras using UDP-glucose or UDP-N-acetylglucosamine as cosubstrates (Guttenberg et al., 2012; Nagahama et al., 2011). We confirmed that TpeL preferentially glucosaminates the three isoforms of Ras (cH-Ras, N-Ras, and K-Ras) from UDP-N-acetylglucosamine and to a lower extent Rap1a and R-Ras3, and very weakly Rac1. In contrast to previous report, we observed that Ral was not a substrate of TpeL. In addition, we confirmed by in vitro glucosylation and mass spectrometry that TpeL modifies cH-Ras at Thr35.

TcsL Is an Essential Virulence Factor inClostridium sordelliiATCC 9714

Clostridium sordellii is an important pathogen of humans and animals, causing a range of diseases, including myonecrosis, sepsis, and shock. Although relatively rare in humans, the incidence of disease is increasing, and it is associated with high mortality rates, approaching 70%. Currently, very little is known about the pathogenesis of C. sordellii infections or disease. Previous work suggested that the lethal large clostridial glucosylating toxin TcsL is the major virulence factor, but a lack of genetic tools has hindered our ability to conclusively assign a role for TcsL or, indeed, any of the other putative virulence factors produced by this organism. In this study, we have developed methods for the introduction of plasmids into C. sordellii using RP4-mediated conjugation from Escherichia coli and have successfully used these techniques to insertionally inactivate the tcsL gene in the reference strain ATCC 9714, using targetron technology. Virulence testing revealed that the production of TcsL is essential for the development of lethal infections by C. sordellii ATCC 9714 and also contributes significantly to edema seen during uterine infection. This study represents the first definitive identification of a virulence factor in C. sordellii and opens the way for in-depth studies of this important human pathogen at the molecular level.

Functional implications of lethal toxin-catalysed glucosylation of (H/K/N)Ras and Rac1 in Clostridium sordellii-associated disease

Clostridium sordellii-based diseases in humans and livestock rely on the activity of the major virulence factors, the single-chain protein toxins TcsL and TcsH, both belonging to the large clostridial glucosylating toxins. TcsL exclusively glucosylates Rho and Ras low molecular weight GTP-binding proteins. TcsL-induced loss of barrier function in epithelial (diarrhoea) and endothelial cells (extravasation of blood fluid) is based on Rac glucosylation whereas induction of apoptosis results from glucosylation of Ras. Intracellular glucosylation of Rac and Ras can be tracked by immunoblot applying the glucosylation-sensitive antibodies Rac1(Mab 102) and Ras(Mab 27H5). Induction of apoptosis especially of phagocytotic cells is crucial for the severity of C. sordellii-associated disease. The inhibition of TcsL-induced apoptosis by tauroursodeoxycholic acid (TUDCA) may be a promising therapeutic option.

Clostridial Toxins

Clostridia produce the highest number of toxins of any type of bacteria and are involved in severe diseases in humans and other animals. Most of the clostridial toxins are pore-forming toxins responsible for gangrenes and gastrointestinal diseases. Among them, perfringolysin has been extensively studied and it is the paradigm of the cholesterol-dependent cytolysins, whereas Clostridium perfringens epsilon-toxin and Clostridium septicum alpha-toxin, which are related to aerolysin, are the prototypes of clostridial toxins that form small pores. Other toxins active on the cell surface possess an enzymatic activity, such as phospholipase C and collagenase, and are involved in the degradation of specific cell-membrane or extracellular-matrix components. Three groups of clostridial toxins have the ability to enter cells: large clostridial glucosylating toxins, binary toxins and neurotoxins. The binary and large clostridial glucosylating toxins alter the actin cytoskeleton by enzymatically modifying the actin monomers and the regulatory proteins from the Rho family, respectively. Clostridial neurotoxins proteolyse key components of neuroexocytosis. Botulinum neurotoxins inhibit neurotransmission at neuromuscular junctions, whereas tetanus toxin targets the inhibitory interneurons of the CNS. The high potency of clostridial toxins results from their specific targets, which have an essential cellular function, and from the type of modification that they induce. In addition, clostridial toxins are useful pharmacological and biological tools.

Toxin B is essential for virulence of Clostridium difficile

Clostridium difficile is the leading cause of infectious diarrhea in hospitals worldwide, because of its virulence, spore-forming ability and persistence1,2. C. difficile-associated diseases (CDAD) are induced by antibiotic treatment or disruption of the normal gastrointestinal flora3,4. Recently, morbidity and mortality resulting from CDAD have increased significantly due to changes in the virulence of the causative strains and antibiotic usage patterns1,2,5,6. Since 2002, epidemic toxinotype III NAP1/027 strains1,2, which produce high levels of the major virulence factors, toxin A and toxin B, have emerged. These toxins have 63% amino acid sequence similarity7 and are members of the large clostridial glucosylating toxin family, which are monoglucosyltransferases that are proinflammatory, cytotoxic and enterotoxic in the human colon8–10. Inside host cells, both toxins catalyze the transfer of glucose onto the Rho family of GTPases, leading to cell death8, 11. However, the role of these toxins in the context of a C. difficile infection is unknown. Here we describe the construction of isogenic tcdA and tcdB mutants of a virulent C. difficile strain and their use in the hamster disease model to show that toxin B is a key virulence determinant. Previous studies showed that purified toxin A alone can induce most of the pathology observed following infection of hamsters with C. difficile8,9, 12 and that toxin B is not toxic in animals unless it is co-administered with toxin A, suggesting that the toxins act synergistically12. Our work provides evidence that toxin B, not toxin A, is essential for virulence, which represents a major paradigm shift. Furthermore, it is clear that the importance of these toxins in the context of infection cannot be predicted exclusively from studies using purified toxins, reinforcing the importance of using the natural infection process to dissect the role of toxins in disease.

Processing of Clostridium difficile toxins

The pathogenicity of Clostridium difficile depends on the large clostridial glucosylating toxins A and B (TcdA and TcdB). The proteins accomplish their own uptake by a modular structure comprising a catalytic and a binding/translocation domain. Based on a proteolytic processing step solely the catalytic domain reaches the cytosol. Within the cells, the glucosyltransferases inactivate small GTPases by mono-O-glucosylation. Here, a short overview is given regarding latest insights into the intramolecular processing, which is mediated by an intrinsic protease activity.

Autoprocessing of the Vibrio cholerae RTX toxin by the cysteine protease domain

Vibrio cholerae RTX is a large multifunctional bacterial toxin that causes actin crosslinking. Due to its size, it was predicted to undergo proteolytic cleavage during translocation into host cells to deliver activity domains to the cytosol. In this study, we identified a domain within the RTX toxin that is conserved in large clostridial glucosylating toxins TcdB, TcdA, TcnA, and TcsL; putative toxins from V. vulnificus, Yersinia sp., Photorhabdus sp., and Xenorhabdus sp.; and a filamentous/hemagglutinin-like protein FhaL from Bordetella sp. In vivo transfection studies and in vitro characterization of purified recombinant protein revealed that this domain from the V. cholerae RTX toxin is an autoprocessing cysteine protease whose activity is stimulated by the intracellular environment. A cysteine point mutation within the RTX holotoxin attenuated actin crosslinking activity suggesting that processing of the toxin is an important step in toxin translocation. Overall, we have uncovered a new mechanism by which large bacterial toxins and proteins deliver catalytic activities to the eukaryotic cell cytosol by autoprocessing after translocation.