Structural characterization of the cell wall binding domains of Clostridium difficile toxins A and B; evidence that Ca2+ plays a role in toxin A cell surface association

Clostridium difficile (C.difficile) is a nosocomially acquired intestinal bacillus which can cause chronic diarrhea and life-threatening colitis. The pathogenic effects of the bacillus are mediated by the release of two toxins, A and B. The C-terminal portions of both toxins are composed of 20 and 30 residue repeats known as cell wall binding (CWB) domains. We have cloned and expressed the CWB-domains of toxins A and B and several truncated CWB-domain constructs to investigate their structure and function. The smallest CWB-domain that folded in a cooperative manner was an 11 repeat construct of toxin A. This differentiates the C-terminal domains of toxins A and B from the CWB-domain of Streptococcus pneumoniae LytA, which only requires six repeats to fold. The 11 repeat toxin A construct bound Ca2+ directly with millimolar affinity and interacted with mammalian cell surfaces in a concentration and Ca2+-dependent fashion. Millimolar Ca2+ levels also accelerated toxin mediated CHO cell killing in an in vitro cell assay. Together, the data suggest a role for extracellular Ca2+ in the sensitization of toxin A/cell-surface interactions.     

Super toxins from a super bug: structure and function of Clostridium difficile toxins

Clostridium difficile, a highly infectious bacterium, is the leading cause of antibiotic-associated pseudomembranous colitis. In 2009, the number of death certificates mentioning C. difficile infection in the U.K. was estimated at 3933 with 44% of certificates recording infection as the underlying cause of death. A number of virulence factors facilitate its pathogenicity, among which are two potent exotoxins; Toxins A and B. Both are large monoglucosyltransferases that catalyse the glucosylation, and hence inactivation, of Rho-GTPases (small regulatory proteins of the eukaryote actin cell cytoskeleton), leading to disorganization of the cytoskeleton and cell death. The roles of Toxins A and B in the context of C. difficile infection is unknown. In addition to these exotoxins, some strains of C. difficile produce an unrelated ADP-ribosylating binary toxin. This toxin consists of two independently produced components: an enzymatic component (CDTa) and the other, the transport component (CDTb) which facilitates translocation of CDTa into target cells. CDTa irreversibly ADP-ribosylates G-actin in target cells, which disrupts the F-actin:G-actin equilibrium leading to cell rounding and cell death. In the present review we provide a summary of the current structural understanding of these toxins and discuss how it may be used to identify potential targets for specific drug design.     

Rho-glucosylating Clostridium difficile toxins A and B: new insights into structure and function

Clostridium difficile causes pseudomembranous colitis and is responsible for many cases of nosocomial antibiotic-associated diarrhea. Major virulence factors of C. difficile are the glucosylating exotoxins A and B. Both toxins enter target cells in a pH- dependent manner from endosomes by forming pores. They translocate the N-terminal catalytic domains into the cytosol of host cells and inactivate Rho guanosine triphosphatases by glucosylation. The crystal structure of the catalytic domain of toxin B was solved in a complex with uridine diphosphate, glucose, and manganese ion, exhibiting a folding of type A family glycosyltransferases. Crystallization of fragments of the C-terminus of toxin A, which is characterized by polypeptide repeats, revealed a solenoid-like structure often found in bacterial cell surface proteins. These studies, which provide new insights into structure, uptake, and function of the family of clostridial glucosylating toxins, are reviewed.     

Evidence that Clostridium difficile TcdC is a membrane-associated protein

Clostridium difficile produces two toxins, A and B, which act together to cause pseudomembraneous colitis. The genes encoding these toxins, tcdA and tcdB, are part of the pathogenicity locus, which also includes tcdC, a putative negative regulator of the toxin genes. In this study, we demonstrate that TcdC is a membrane-associated protein in C. difficile.     

Rho GTPases as targets of bacterial protein toxins

Several bacterial toxins target Rho GTPases, which constitute molecular switches in several signaling processes and master regulators of the actin cytoskeleton. The biological activities of Rho GTPases are blocked by C3-like transferases, which ADP-ribosylate Rho at Asn41, but not Rac or Cdc42. Large clostridial cytotoxins (e. g., Clostridium difficile toxin A and B) glucosylate Rho GTPases at Thr37 (Rho) or Thr35 (Rac/Cdc42), thereby inhibiting Rho functions by preventing effector coupling. The 'injected' toxins ExoS, YopE and SptP from Pseudomonas aeruginosa, Yersinia and Salmonella ssp., respectively, which are transferred into the eukaryotic target cells by the type-III secretion system, inhibit Rho functions by acting as Rho GAP proteins. Rho GTPases are activated by the cytotoxic necrotizing factors CNF1 and CNF2 from Escherichia coli and by the dermonecrotizing toxin DNT from B. bronchiseptica. These toxins deamidate/transglutaminate Gln63 of Rho to block the intrinsic and GAP-stimulated GTP hydrolysis, thereby constitutively activating the GTPases. Rho GTPases are also activated by SopE, a type-III system injected protein from Salmonella ssp., that acts as a GEF protein.     

Analysis of synaptic neurotransmitter release mechanisms using bacterial toxins

Several bacterial toxins are powerful and highly specific tools for studying basic mechanisms involved in cell biology. Whereas the clostridial neurotoxins are widely used by neurobiologists, many other toxins (i.e. toxins acting on small G-proteins or actin) are still overlooked. Botulinum neurotoxins (BoNT, serotypes A-G) and tetanus neurotoxin (TeNT), known under the generic term of clostridial neurotoxins, are characterized by their unique ability to selectively block neurotransmitter release. These proteins are formed of a light (Mr approximately 50) and a heavy (Mr approximately 100) chain which are disulfide linked. The cellular action of BoNT and TeNT involves several steps: heavy chain-mediated binding to the nerve ending membrane, endocytosis, and translocation of the light chain (their catalytic moiety) into the cytosol. The light chains each cleaves one of three, highly conserved, proteins (VAMP/synaptobrevin, syntaxin, and SNAP-25 also termed SNAREs) implicated in fusion of synaptic vesicles with plasma membrane at the release site. Hence, when these neurotoxins are applied extracellularly, they can be used as specific tools to inhibit evoked and spontaneous transmitter release from certain neurones whereas, when the membrane limiting steps are bypassed by the mean of intracellular applications, BoNTs orTeNT can be used to affect regulated secretion in various cell types. Several members of the Rho GTPase family have been involved in intracellular trafficking of synaptic vesicles and secretory organelles. As they are natural targets for several bacterial exoenzymes or cytotoxins, their role in neurotransmitter release can be probed by examining the action of these toxins on neurotransmission. Such toxins include: i) the non permeant C3 exoenzymes from C. botulinum or C. limosum which ADP-ribosylate and thereby inactivate Rho, ii) exoenzyme S from Pseudomonas aeruginosa which ADP-ribosylates different members of the Ras, Rab, Ral and Rap families, iii) toxin B from C. difficile which glucosylates Rho, Rac and CDC42, iv) lethal toxin from C. sordellii which glucosylates Rac, Ras and to a lesser extent, Rap and Ral, but not on Rho or CDC42, and v) CNF deamidases secreted by pathogenic strains of E. coli which activate Rho and, to a lesser extent, CDC42. Since these toxins or exoenzymes have no or little ability to enter into the neurones, they must be applied intraneuronally to bypass the membrane limiting steps. Injection of several of these toxins into Aplysia neurones allowed us to reveal a new role for Rac in the control of exocytosis. ADP-ribosylating enzymes, which specifically act on monomeric actin (C2 binary toxin from C. botulinum and iota toxin from C. perfringens), are potential tools to probe the role of actin filaments during secretion.     

Analysis of the physicochemical interactions between Clostridium difficile toxins and cholestyramine using liquid chromatography with post-column derivatization

A potential therapy for antibiotic-associated pseudomembranous colitis is to bind Clostridium difficile toxins A and B using cholestyramine, a hydrophobic anion exchange medium. Frontal analysis in isotonic phosphate buffer was studied using post-column derivatization with o-phthalaldehyde, which gave a highly sensitive (> or =30 ng) flow-through analysis. Following load (1.5-3.0 microg toxin/3.6 mg), toxin A was bound at a slightly higher capacity than B, due to slower kinetics. A salt gradient eluted roughly 20% of bound toxin A with 0.6 M NaCl and toxin B with 1.1 M NaCl, hence toxin A showed weaker electrostatic affinity. The remainder of toxin A (65%) and some of toxin B (10% out of 50%) were eluted using a subsequent gradient to 60% acetonitrile in normal saline, which measured predominantly hydrophobic binding. Low and high affinity populations of both toxins were observed. Glycocholic acid or amino acids were competitive binders, although these components had little effect on the toxin A population bound primarily through ionic interactions. Competitive protein constituents in hamster cecal contents were also profiled. These results help to explain the variable clinical response in using cholestyramine to treat colitis. Using quaternary amine-polyhydroxymethacrylate (PHM) ion exchange chromatography, a trend for increased binding at higher pH was observed, especially for toxin A. Binding to strong cation exchange resins (sulfonate-PHM) was not observed. A range of reversed phase media retained both toxins, although recovery was very poor relative to protein standards. Size exclusion chromatography with light scattering detection showed that toxin B exists in different aggregation states, while toxin A remains monomeric.     

Signal transduction pathways and cellular intoxication with Clostridium difficile toxins

In cultured cells the cytopathic effects (CPE) of Clostridium difficile toxins A and B are superficially similar. The irreversible CPEs involve a reorganization of the cytoskeleton, but the molecular details of the mechanism(s) of action are unknown. As part of the work to elucidate the events leading to the CPE, cultured cells were preincubated with agents known to either stimulate or inhibit some major signal transduction pathways, whereupon toxin was added and the development of the CPE was followed. Both toxin-induced CPEs were enhanced by phorbol esters and mezerein, which stimulate protein kinase C, while they were inhibited by the phospholipase A2 inhibitors quinacrine and 4-bromophenacylbromide. Agents affecting certain G-proteins, cGMP and cAMP levels, phosphatases, prostacyclin, lipoxygenase, and phospholipase C did not affect the development of the CPE of either toxin. Thus, the cytoskeletal effect induced by toxins A or B appears to require PLA2 activity and involves at least part of a protein kinase C-dependent pathway, but not pertussis toxin-sensitive G-proteins, cyclic nucleotides, eicosanoid metabolites, or phospholipase C activity. In addition, both toxins were shown to activate phospholipase A2.     

Evaluation of different methods for detection of Clostridium difficile toxins in Poland

The aim of this study was to compare different methods for C. difficile toxins detection. Fifty three stool samples taken from patients with antibiotic-associated diarrhoea were studied. TCD toxin A EIA (Becton Dickinson, USA), Tox A/B ELISA test (TechLab, USA), cytotoxicity and neutralization assay on McCoy cells and PCR for detection of both toxin A and B genes were performed in vivo (in stool samples) and in vitro (in isolated strains). Reference toxigenic and nontoxigenic and two Japanese toxin A-negative and toxin B-positive C. difficile strains were used as a controls. TCD toxin A EIA detected in vivo only 19 positive samples. Tox A/B test detected 52 positive samples out of 53 studied. All 53 stool samples were C. difficile culture positive (53 strains were cultured). Toxin B was detected in 52 strain-supernatants and in all controls (except the nontoxigenic one). Both toxin A and B genes were detected by PCR in all 53 isolated strains, Japanese and reference strain (except the nontoxigenic one). In vitro toxin A was detected by TCD toxin A EIA in 42 strains. These results were compared with those obtained in Tox A/B ELISA test. We observed 52 positive strains. Toxigenic reference strain and two Japanese toxA(-)/toxB(+) strains were also positive. Only 2 negative results were obtained with the nontoxigenic reference strain and unique nontoxigenic isolated strain. Tox A/B ELISA test seems to be the best for detection of C. difficile toxins in vivo and in vitro. Test avoids the false-negative results in the case of presence of toxin A-negative and toxin B-positive strain.     

ADP-ribosylation in Clostridium difficile toxin-treated cells is not related to cytopathogenicity of toxin B

ADP-ribosylation of a protein in human fibroblasts treated with partially purified Clostridium difficile toxin B was previously reported. Here we show that the same protein was ADP-ribosylated also in human fibroblasts exposed to supernatant from a C. difficile strain producing neither toxin A nor toxin B. Furthermore, in Chinese hamster ovary and in Vero cells, showing toxin B-induced cytopathogenic effect, the protein was not significantly ADP-ribosylated. The results indicate that the ADP-ribosylation is unrelated to the cytopathogenic effect of toxin B. It appears to be caused by another unidentified factor from C. difficile, and the substrate may correspond to a protein modified endogenously in cells exposed to stressful situations. Cellular actin was not ADP-ribosylated by toxin B.     

Cholesterol-dependent pore formation of Clostridium difficile toxin A

The large clostridial cytotoxins toxin A and toxin B from Clostridium difficile are major virulence factors known to cause antibiotic-associated diarrhea and pseudomembranous colitis. Both toxins mono-glucosylate and thereby inactivate small GTPases of the Rho family. Recently, it was reported that toxin B, but not toxin A, induces pore formation in membranes of target cells under acidic conditions. Here, we reassessed data on pore formation of toxin A in cells derived from human colon carcinoma. Treatment of 86Rb+-loaded cells with native or recombinant toxin A resulted in an increased efflux of radioactive cations induced by an acidic pulse. The efficacy of pore formation was dependent on membrane cholesterol, since cholesterol depletion of membranes with methyl-beta-cyclodextrin inhibited 86Rb+ efflux, and cholesterol repletion reconstituted pore-forming activity of toxin A. Similar results were obtained with toxin B. Consistently, methyl-beta-cyclodextrin treatment delayed intoxication of cells in a concentration-dependent manner. In black lipid membranes, toxin A induced ion-permeable pores only in cholesterol containing bilayers and at low pH. In contrast, release of glycosylphosphatidylinositol-anchored structures by phosphatidylinositol specific phospholipase C treatment did not reduce cell sensitivity toward toxins A and B. These data indicate that in colonic cells toxin A induces pore formation in an acidic environment (e.g. endosomes) similar to that reported for toxin B and suggest that pore formation by clostridial glucosylating toxins depends on the presence of cholesterol.     

Typing of toxic strains of Clostridium difficile using DNA fingerprints generated with arbitrary polymerase chain reaction primers

Clostridium difficile is the causative agent for pseudomembranous colitis in humans. Toxic strains of C. difficile produce two toxins, toxin A and toxin B. A reliable and definitive method of typing the toxic strains of C. difficile is needed since nosocomial cross infection is a primary concern in hospitals and other health care facilities. A method for typing toxic strains of Clostridium difficile using arbitrary polymerase chain reaction (PCR) primers is presented in this study. The C. difficile strains were initially characterized for the toxin A genetic determinant using specific PCR primers which differentiate toxin positive from toxin negative strains. These toxic strains were then PCR typed using six arbitrary primers which generated DNA patterns that were unique for all toxic strains examined. The use of this typing scheme in clinical applications is discussed.     

Bacterial toxins modifying the actin cytoskeleton.

Numerous bacterial toxins recognize the actin cytoskeleton as a target. The clostridial binary toxins (Iota and C2 families) ADP-ribosylate the actin monomers causing the dissociation of the actin filaments. The large clostridial toxins from Clostridium difficile, Clostridium sordellii and Clostridium novyi inactivate, by glucosylation, proteins from the Rho family that regulate actin polymerization. In contrast, the cytotoxic necrotic factor from Escherichia coli activates Rho by deamidation and increases the formation of actin filaments. The enterotoxin of Bacteroides fragilis is a protease specific for E-cadherin and it promotes the reorganization of the actin cytoskeleton. The bacterial toxins that modify the actin cytoskeleton induce various cell disfunctions including changes in cell barrier permeability and disruption of intercellular junctions.     

Structure and mode of action of clostridial glucosylating toxins: the ABCD model

Toxins A and B, which are the major virulence factors of antibiotic-associated diarrhea and pseudomembranous colitis caused by Clostridium difficile, are the prototypes of the family of clostridial glucosylating toxins. The toxins inactivate Rho and Ras proteins by glucosylation. Recent findings on the autocatalytic processing of the toxins and analysis of the crystal structures of their domains have made a revision of the current model of their actions on the eukaryotic target cells necessary.     

N-acetylcysteine protects epithelial cells against the oxidative imbalance due to Clostridium difficile toxins.

Toxins A and B from the anaerobic bacterium Clostridium difficile are the causative agents of the antibiotic-associated pseudomembraneous colitis. At the subcellular level, they inhibit the Rho family GTPases, thus causing alterations of the actin cytoskeleton. The cytoskeletal integrity is also controlled by the redox state of cells. Therefore, we have evaluated whether an oxidative imbalance could be involved in the toxin-induced cytopathic effects. Our results indicate that both toxins induce oxidative stress with a significant depletion of protein SH-groups. These responses and the cytoskeleton-dependent cell retraction and rounding are significantly counteracted by N-acetylcysteine but not by alpha-tocopherol. Our study provides the first evidence that the thiol supplier N-acetylcysteine impairs the cellular intoxication by acting on the cytoskeleton integrity. This also suggests a possible beneficial role for this drug during therapeutic intervention.     

Binary bacterial toxins: biochemistry, biology, and applications of common Clostridium and Bacillus proteins.

Certain pathogenic species of Bacillus and Clostridium have developed unique methods for intoxicating cells that employ the classic enzymatic "A-B" paradigm for protein toxins. The binary toxins produced by B. anthracis, B. cereus, C. botulinum, C. difficile, C. perfringens, and C. spiroforme consist of components not physically associated in solution that are linked to various diseases in humans, animals, or insects. The "B" components are synthesized as precursors that are subsequently activated by serine-type proteases on the targeted cell surface and/or in solution. Following release of a 20-kDa N-terminal peptide, the activated "B" components form homoheptameric rings that subsequently dock with an "A" component(s) on the cell surface. By following an acidified endosomal route and translocation into the cytosol, "A" molecules disable a cell (and host organism) via disruption of the actin cytoskeleton, increasing intracellular levels of cyclic AMP, or inactivation of signaling pathways linked to mitogen-activated protein kinase kinases. Recently, B. anthracis has gleaned much notoriety as a biowarfare/bioterrorism agent, and of primary interest has been the edema and lethal toxins, their role in anthrax, as well as the development of efficacious vaccines and therapeutics targeting these virulence factors and ultimately B. anthracis. This review comprehensively surveys the literature and discusses the similarities, as well as distinct differences, between each Clostridium and Bacillus binary toxin in terms of their biochemistry, biology, genetics, structure, and applications in science and medicine. The information may foster future studies that aid novel vaccine and drug development, as well as a better understanding of a conserved intoxication process utilized by various gram-positive, spore-forming bacteria.     

Phosphorylation of cellular proteins in response to treatment with Clostridium difficile toxin B and Clostridium sordellii toxin L.

Toxin B from Clostridium difficile induces typical morphological changes of cultured cells consisting of rounding up and arborization, which are associated with a dramatic disruption of microfilaments. In this study, we show that toxin L, a cytotoxin produced by bacterial strain Clostridium sordellii, has similar effects on cultured cells including the redistribution of F-actin and of the adhesion plaque protein vinculin. It has been assumed that the mechanisms involved in cytopathic effects of toxin B are related to the function of an unidentified component that regulates the organization of the actin cytoskeleton. We demonstrate that the treatment of cultured astrocytes with toxin B or toxin L alters the incorporation of inorganic phosphate into several proteins. Immunoblot analysis revealed that one of these proteins is tropomyosin. Since tropomyosin stabilizes microfilaments and inhibits the severing activity of gelsolin, the toxin-induced phosphorylation may counteract this inhibition resulting in severing of microfilaments and capping of short filaments. A decrease in the radioactivity associated with intermediate filament protein vimentin was also detected using a monoclonal antibody which specifically recognizes a phosphorylated epitope of vimentin. Since vimentin is an in vivo substrate for various protein kinases, these data are in favor of broad effects of these toxins. Direct measurement of protein kinase C in cells exposed to toxin B or to toxin L did not reveal a significant change in protein kinase C activity. Furthermore, treatments with toxins do not increase cAMP levels, suggesting that toxins do not activate protein kinase A. Although further studies are required to determine the primary target site for the clostridial cytotoxin B and L, our results show that they provoke the alteration in the phosphorylation of cellular proteins.