Clostridium difficile toxins A and B inhibit human immune response in vitro.

Two Clostridium difficile toxins isolated from strain VPI 10463 were tested for their effect on different human T-cell proliferation systems. In mitogen- and antigen-driven T-cell proliferation systems, toxins inhibited the proliferative response in a dose-dependent fashion. In interleukin-2-driven culture systems, no effect of toxins could be found on preactivated T cells. We suspected that monocytes were the influenced cells, since in antigen- and mitogen-driven systems monocytes were necessary for the proliferative response, whereas the interleukin-2-driven system was independent of monocytes. To prove this concept, purified monocytes were treated with toxins. The treatment was found to markedly reduce the capacity of monocytes to stimulate T-cell proliferation. No inhibition of the proliferative response was measured when, instead of monocytes, resting or preactivated T cells were treated with toxins. These experiments clearly show that C. difficile toxins interact with monocytes and not with T cells. The effect of toxins on cells of the immune system might be one factor in the development of pseudomembranous colitis.     

Clostridium difficile toxins A and B directly stimulate human mast cells

Clostridium difficile toxins A and B (TcdA and TcdB) are the causative agents of antibiotic-associated pseudomembranous colitis. Mucosal mast cells play a crucial role in the inflammatory processes underlying this disease. We studied the direct effects of TcdA and TcdB on the human mast cell line HMC-1 with respect to degranulation, cytokine release, and the activation of proinflammatory signal pathways. TcdA and TcdB inactivate Rho GTPases, the master regulators of the actin cytoskeleton. The inactivation of Rho GTPases induced a reorganization of the actin cytoskeleton accompanied by morphological changes of cells. The TcdB-induced reorganization of the actin cytoskeleton in HMC-1 cells reduced the number of electron-dense mast cell-specific granules. Accordingly, TcdB induced the release of hexosaminidase, a marker for degranulation, in HMC-1 cells. The actin rearrangement was found to be responsible for degranulation since latrunculin B induced a comparable hexosaminidase release. In addition, TcdB as well as latrunculin B induced the activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase 1/2 and also resulted in a p38 MAPK-dependent increased formation of prostaglandins D(2) and E(2). The autocrine stimulation of HMC-1 cells by prostaglandins partially contributed to the degranulation. Interestingly, TcdB-treated HMC-1 cells, but not latrunculin B-treated HMC-1 cells, showed a strong p38 MAPK-dependent increase in interleukin-8 release. Differences in the mast cell responses to TcdB and latrunculin B are probably due to the presence of functionally inactive Rho GTPases in toxin-treated cells. Thus, the HMC-1 cell line is a promising model for studying the direct effects of C. difficile toxins on mast cells independently of the tissue context.     

Biological activities of toxins A and B of Clostridium difficile.

Examination of the biological activities of the two known toxins of Clostridium difficile revealed that one of the toxins (toxin A) elicited a hemorrhagic fluid response in rabbit intestinal loops and a positive fluid response in infant mice. The other toxin (toxin B) did not produce a significant fluid response in either model, although the toxin was more lethal in infant mice. Both toxins elicited erythematous and hemorrhagic skin reactions and increased vascular permeability in rabbit skin.     

The effects of Clostridium difficile toxins A and B on membrane integrity and protein synthesis in intestinal cells in vivo and in vitro and in McCoy cells in vitro

Clostridium difficile toxins A and B inhibited protein synthesis in McCoy tissue-culture cells but not in intestinal cells in vitro or in vivo. Toxins A and B had no effect on membrane permeability of either intestinal cells or McCoy cells.     

Purification and characterization of toxins A and B of Clostridium difficile.

Toxin preparations were obtained by growing Clostridium difficile VPI strain 10463 in 2-liter brain heart infusion dialysis flasks at 37 degrees C for 3 days. The initial step of the purification scheme involved ultrafiltration through an XM-100 membrane filter. Two toxic activities, designated toxins A and B, were separated by ion-exchange chromatography on DEAE-NaCl gradients. Toxin A was purified to homogeneity by an acetic acid precipitation at pH 5.5. Other separation techniques, including CM Sepharose CL-6B, (NH4)2SO4 and acetic acid precipitations, and hydrophobic interaction chromatography, were examined in attempts to further purify toxin B. Although these methods failed to increase the specific activity of toxin B, they provided additional evidence that the two toxins are distinct molecules. The toxins are acid and heat labile and are inactivated by trypsin and chymotrypsin, but not by amylase. The molecular weight of toxin A, as estimated by gel filtration and gradient polyacrylamide electrophoresis, ranged from 440,000 to 500,000. The estimated molecular weight of toxin B was 360,000 to 470,000.     

Clostridium difficile toxin A. Interactions with mucus and early sequential histopathologic effects in rabbit small intestine

Clostridium difficile produces two toxins, A (enterotoxic) and B (cytopathic), that are implicated in the pathogenesis of pseudomembranous colitis. However, the relationship of the secretory effect and the early histopathologic events is still unclear. We examined the early histopathologic effect of purified C. difficile toxin A in rabbit ileal loops and correlated the mucosal damage with the secretory response. As early as 2 hours after inoculation, toxin A at 1.0 micrograms caused cytolysis of the basal portion of the apical epithelia cells. Electron microscopy showed the basal portions of the absorptive epithelial cells necrotic. No significant fluid accumulation occurred after 2 or 4 hours in ileal loops inoculated with either 0.1 or 1.0 micrograms toxin A. In contrast, with 3-10 micrograms toxin A there was significant hemorrhagic fluid accumulation after 2 hours that correlated with the severity of the histologic lesions. An early mucoid exudate was demonstrated not to be related to a mucus secretagogue effect of toxin A. Prior exposure of toxin A to rabbit intestinal mucus preparations did not alter its subsequent enterotoxic or cytopathic (Chinese hamster ovary cells) effects. These studies demonstrate that the early intestinal tissue damage by toxin A was dose and time dependent and involved disruption of the basal portions of the absorptive epithelial cells with marked separation of the cells along the mucosal surfaces that preceded the secretory response.     

Production and release of toxins A and B by Clostridium difficile

The production and release of toxins A and B by Clostridium difficile during in-vitro culture was investigated. Cell-associated toxin A was detected by immunoelectrophoresis of bacterial extracts released by ultrasonication and by fluorescent antibody labelling of whole cells. Extracellular toxin A was detected by immunoelectrophoresis and by enzyme-linked immunosorbent assay; extracellular toxin B was detected by cytotoxin assay. Both toxins A and B were produced and released during the decline phase of the bacterial growth cycle. The possible significance of these results in relation to the pathogenesis of pseudomembranous colitis is discussed.     

Characterization of toxins A and B of Clostridium difficile with monoclonal antibodies.

Two monoclonal antibodies (MAbs) were used to learn more about the structures of Clostridium difficile toxins A and B. One of the antibodies, the PCG-4 MAb, reacted specifically with toxin A. This MAb precipitated toxin A and neutralized the enterotoxic but not the cytotoxic activity of the toxin. The site to which the antibody bound was resistant to denaturation with sodium dodecyl sulfate; however, it was destroyed by N-bromosuccinimide. Immunoblot analysis with the PCG-4 MAb revealed the presence of a large number of bands in preparations of denatured toxin A, suggesting that toxin A exists as an aggregate of smaller components. The antibody was covalently coupled to Affi-Gel 10, and the gel was used to purify toxin A from the culture filtrate of a highly toxigenic strain of C. difficile by immunoaffinity chromatography. The second antibody, the G-2 MAb, cross-reacted with toxins A and B. The cross-reaction was confirmed by immunoblot analysis. These results show that toxins A and B share an epitope and suggest that they have a common subunit. The G-2 MAb did not neutralize or precipitate either toxin. The site to which the G-2 MAb bound was partially destroyed by sodium dodecyl sulfate and was resistant to oxidation with N-bromosuccinimide.     

Comparison of Clostridium sordellii toxins HT and LT with toxins A and B of C. difficile.

Clostridium sordellii produces two toxins, designated HT (haemorrhagic toxin) and LT (lethal toxin), that are similar to toxins A and B of C. difficile. The physicochemical properties of toxins HT and A were remarkably similar. The specific biological activities of toxin HT were almost the same as those of toxin A, and their NH2-terminal sequences shared close homology. The properties of toxins LT and B were similar, as were their NH2-terminal sequences, but toxin B was much more cytotoxic than toxin LT. Immunodiffusion analysis with specific antibodies showed that although toxins B and LT shared major antigenic determinants, each had unique epitopes. The results suggest that toxins B and LT have diverged more than toxins A and HT. Immunoblotting with antibodies to the toxins of C. difficile showed that toxins HT and LT had common antigenic determinants.     

Association between production of toxins A and B and types of Clostridium difficile.

One hundred and seventy two strains of Clostridium difficile isolated from 62 patients with antibiotic associated diarrhoea or pseudomembranous colitis were analysed for the production of toxins A and B and typed using 35S-methionine labelling followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). There was a correlation between production of toxins A and B and the type of C difficile. One hundred and forty four of 172 strains were either high or low producers of both toxins. Toxins were not detected in 28 of 172 strains. Types A and Y were consistently non-toxin producers; types B and E were high toxin producers. Type X, the epidemic strain, showed variable toxin production. Symptoms of 49 patients with haematological malignant pathology who were part of a documented outbreak of antibiotic associated diarrhoea, were analysed in relation to toxin production and type of the clinical strains isolated. In general, there was a correlation between symptoms of antibiotic associated diarrhoea, the type of C difficile, and its potential for producing toxins.     

Effects of Clostridium difficile toxins given intragastrically to animals.

We examined the activities of Clostridium difficile toxin preparations given intragastrically to hamsters, mice, and rats. The culture filtrate from a highly toxigenic strain of C. difficile caused hemorrhage and accumulation of fluid in the small intestine and cecum, diarrhea, and death in hamsters and mice. In rats, the culture filtrate caused only a small amount of fluid accumulation and slight hemorrhage along the small intestine. When toxin A was removed from the culture filtrate, the filtrate lost its activity. Preparations of homogeneous toxin A caused a response similar to that observed after the administration of culture filtrate. Hamsters were more sensitive to toxin A than mice or rats were. When hamsters were given multiple low doses of toxin A 1 week apart at a concentration which singly caused no response, they became ill and died, indicating that the toxin may have long-term effects. High amounts of toxin B did not cause any significant response when given intragastrically, unless initially mixed with low amounts of toxin A or given to hamsters with bruised ceca. These results suggest that toxins A and B act synergistically and that the action of toxin B may occur via the tissue damage caused by toxin A.     

Differential cytotoxic effects of toxins A and B isolated from Clostridium difficile

Toxin A and toxin B preparations of Clostridium difficile have been shown to affect metabolic functions of intact HeLa cells with different kinetics. The cytotoxins were purified from dialyzed filtrates of C. difficile strain VPI 10463 by hydrophobic interaction chromatography and ion-exchange chromatography and were concentrated by dialysis or by ultrafiltration. The toxins, which are immunologically unrelated, were analyzed by polyacrylamide gel electrophoresis and by immunochemistry with the Western blot technique. Toxin A was resolved into one major cytotoxic protein and a minor, rapidly migrating species that did not comigrate with toxin B. Toxin B was resolved into one major and three minor cytotoxic proteins. One protein comigrating with toxin A had no cytotoxic activity. The highly purified toxin A at 1.0 mg/ml caused loss of intracellular K+ and inhibition of protein synthesis in HeLa cells within 1 h. These effects correlated with morphological changes indicating cytotoxicity. At lower protein concentrations of toxin A (10- to 100-fold less), however, cytotoxic effects were seen at 120 min, whereas no changes in K+ levels or protein synthesis were yet evident. The toxin B preparation, 1,000-fold more toxic than toxin A, was diluted to equivalent cytotoxicity as measured in the overnight assay. Toxin B caused loss of K+ and inhibition of protein synthesis well after cytotoxic morphological changes were complete. In contrast, at higher protein concentrations (2- to 2,000-fold more), intracellular K+ was lost completely by 120 min. The effects on cell rounding and protein synthesis were incomplete at 120 min, but increased with the toxin B concentration.     

Effects of Clostridium perfringens beta-toxin on the rabbit small intestine and colon

Clostridium perfringens type B and type C isolates, which produce beta-toxin (CPB), cause fatal diseases originating in the intestines of humans or livestock. Our previous studies demonstrated that CPB is necessary for type C isolate CN3685 to cause bloody necrotic enteritis in a rabbit ileal loop model and also showed that purified CPB, in the presence of trypsin inhibitor (TI), can reproduce type C pathology in rabbit ileal loops. We report here a more complete characterization of the effects of purified CPB in the rabbit small and large intestines. One microgram of purified CPB, in the presence of TI, was found to be sufficient to cause significant accumulation of hemorrhagic luminal fluid in duodenal, jejunal, or ileal loops treated for 6 h with purified CPB, while no damage was observed in corresponding loops receiving CPB (no TI) or TI alone. In contrast to the CPB sensitivity of the small intestine, the colon was not affected by 6 h of treatment with even 90 μg of purified CPB whether or not TI was present. Time course studies showed that purified CPB begins to induce small intestinal damage within 1 h, at which time the duodenum is less damaged than the jejunum or ileum. These observations help to explain why type B and C infections primarily involve the small intestine, establish CPB as a very potent and fast-acting toxin in the small intestines, and confirm a key role for intestinal trypsin as an innate intestinal defense mechanism against CPB-producing C. perfringens isolates.     

Positive regulation of Clostridium difficile toxins.

The toxigenic element of Clostridium difficile VPI 10463 contains a small open reading frame (ORF) immediately upstream of the toxin B gene (G. A. Hammond and J. L. Johnson, Microb. Pathog. 19:203-213, 1995). The deduced amino acid sequence of the ORF, which we have designated txeR, encodes a 22-kDa protein which contains a helix-turn-helix motif with sequence identity to DNA binding regulatory proteins. We used a DNA fragment containing the C. difficile toxin A repeating units (ARU) as a reporter gene to determine if txeR regulates expression from the toxin A and toxin B promoters in Escherichia coli. To test the affect of txeR on expression, we fused the ARU gene fragment in frame with the toxin promoters. The fusions expressed a 104-kDa protein that contained the epitopes for monoclonal antibody PCG-4, which we used to measure levels of recombinant ARU by enzyme-linked immunosorbent assay. When txeR was expressed in trans with the toxin B promoter-ARU fusion contained on separate low-copy-number plasmid, expression of ARU increased over 800-fold. Furthermore, when we tested the toxin A promoter fused to ARU, expression increased over 500-fold with txeR supplied in trans. Our results suggest that TxeR is a positive regulator that activates expression of the C. difficile toxins.     

Enzyme immunoassay for detection of antibody to toxins A and B of Clostridium difficile.

An enzyme immunoassay (enzyme-linked immunosorbent assay [ELISA]) to detect hamster antibody to toxins A and B of Clostridium difficile was developed in which toxin preparations are used to coat the solid phase. The specificity of the assay was supported by blocking tests with the toxin preparations and proteins from a nontoxigenic strain. Sera from immunized and control hamsters were tested by this technique, and results were compared with those from a cytotoxicity neutralization assay. Antibody to toxins A and B assayed by ELISA showed a close quantitative correlation with antibody titers obtained by cytotoxicity neutralization. The ELISA assays described appear to provide a sensitive, specific, and practical method to define the prevalence of antibody to C. difficile toxins. These assays could be readily applied to human sera to examine and study the immune response of patients with C. difficile-induced disease.     

Biochemical studies on the effect of Clostridium difficile toxin B on actin in vivo and in vitro.

We describe a simplified procedure for purification of Clostridium difficile toxin B. In this procedure, cytotoxicity is associated with a single protein band with a molecular mass of 230 kilodaltons. We used direct fluorescent staining of actin filaments to study the effect of this toxin on cultured cells. Morphologic changes were preceded by a decrease in the number and length of stress fibers followed by their disappearance with condensation of cellular actin around the nucleus. We then showed that cells treated with either cytochalasin B or toxin B had a significant increase in the monomeric actin pool as quantitated by DNase I inhibition. In contrast to the cytochalasins, toxin B had no direct effect on the rate or extent of actin polymerization or network formation in vitro. Cytoplasmic extracts of toxin B-treated cells had a significantly lower level of modulating activity on actin assembly and interactions in vitro compared with extracts of untreated cells. These results suggest that the action of toxin B on cells is due to direct or indirect effects on cellular proteins involved in controlling the state of actin assembly in the cells.     

Evaluation of four immunoenzymatic tests for detecting Clostridium difficile toxins A and B

Four immunoenzymatic tests for detecting Clostridium difficile toxins A and B were studied: two rapid tests (Tox A/B QUIK CHEK-Techlab and NoviView Toxine-A-Hiss diagnostics) and two Elisa tests (C. difficile TOX A/B II -Techlab and Toxin A+B Elisa Test, Novitec-Hiss diagnostics). The results were compared to those obtained with ImmunoCard Tox A+B -ICTAB (Meridian), C. difficile Toxine A (Oxoid) for rapid test and Elisa Premier A+B Meridian for Elisa. A total of 41 stools and 16 isolates were studied with rapid tests. On stools, the sensitivity and specificity of QUIK CHEK test was 94.1% and 100% respectively compared to the test ICTAB. On the isolates, sensitivity and specificity was 100%. With the Noviview test, the sensitivity on stools and isolates was respectively 88.2 and 85.7% and the specificity was 100% compared to Oxoid. A total of 38 stools were studied with Elisa tests. With Techlab test compared to the test Premier, sensitivity and specificity was 100%. The Novitec test gave five false negative reactions with consequently a sensitivity of 70.6%.     

Clostridium difficile: its disease and toxins.

Clostridium difficile is the etiologic agent of pseudomembranous colitis, a severe, sometimes fatal disease that occurs in adults undergoing antimicrobial therapy. The disease, ironically, has been most effectively treated with antibiotics, although some of the newer methods of treatment such as the replacement of the bowel flora may prove more beneficial for patients who continue to relapse with pseudomembranous colitis. The organism produces two potent exotoxins designated toxin A and toxin B. Toxin A is an enterotoxin believed to be responsible for the diarrhea and mucosal tissue damage which occur during the disease. Toxin B is an extremely potent cytotoxin, but its role in the disease has not been as well studied. There appears to be a cascade of events which result in the expression of the activity of these toxins, and these events, ranging from the recognition of a trisaccharide receptor by toxin A to the synergistic action of the toxins and their possible dissemination in the body, are discussed in this review. The advantages and disadvantages of the various assays, including tissue culture assay, enzyme immunoassay, and latex agglutination, currently used in the clinical diagnosis of the disease also are discussed.     

Isolation of a fibroblast mutant resistant to Clostridium difficile toxins A and B

A mutant of Chinese hamster lung fibroblasts (Don cells), resistant against Clostridium difficile toxins A and B, was isolated after mutagenization with ethylmethanesulphonate and a two-step selection with toxin B. The mutant, termed CdtR-Q, was 10(4) times more resistant to toxin B than wild-type cells and cross-resistant to toxin A (10(3) times more resistant). The resistance was overcome by increasing the dose of toxin. The resistance has been stable after cultivation for 40 generations in the absence of toxin. The morphology of the mutant was more epithelial-like than that of the fibroblast parental cells. The plating efficiency was about half that of the wild-type, whereas the growth rate was the same. The mutant was significantly less sensitive than the wild-type to the microfilament-interacting cytochalasins B and D. It was as sensitive as the wild-type to endocytosed toxins (diphtheria, pertussis, ricin), to microtubule-interacting agents (colchicine, gossypol, nocodazole, taxol, vinblastine), and to membrane-damaging toxins with different mechanisms of action, with one exception; the mutant was more highly sensitive to the action of phospholipase C (with broad substrate-specificity) than the wild-type. The results suggest that the mutant has a normal endocytosis, and that the mutation does not affect the microtubuli. The results are consistent with a mutation affecting the microfilaments in the cytoskeleton.