Glucosylation of Rho GTPases by Clostridium difficile toxin A triggers apoptosis in intestinal epithelial cells

Gerhard, Ralf; Nottrott, Stefanie; Schoentaube, Janett; Tatge, Helma; Olling, Alexandra; Just, Ingo

doi:10.1099/jmm.0.47769-0

Cited by 96 publications

(94 citation statements)

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“…3A) are consistent with reports that TcdA and TcdB can trigger apoptotic cell death. Apoptosis in response to the toxins is thought to be dependent upon glucosyltransferase activity and subsequent GTPase inactivation (19,24,29). To test if this is true in YAMC cells, we challenged cells with TcdA, TcdB, and their respective glucosyltransferase-deficient mutants and assayed for caspase-3/7 activation ( Fig.…”

Section: Figmentioning

confidence: 99%

“…The GTD has been shown to target and inactivate a number of Rho-family GTPases (19,20). This inactivation has been linked to a cell rounding or cytopathic effect (CPE) (21)(22)(23)(24) and to an apoptotic cytotoxic effect (25)(26)(27)(28)(29)(30)(31)(32)(33). In tissue culture models, the apoptotic effects of TcdA and TcdB occur at toxin concentrations of picomolar or lower and are evident at 24 to 48 h postintoxication (26,28,29,(34)(35)(36).…”

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confidence: 99%

“…This inactivation has been linked to a cell rounding or cytopathic effect (CPE) (21)(22)(23)(24) and to an apoptotic cytotoxic effect (25)(26)(27)(28)(29)(30)(31)(32)(33). In tissue culture models, the apoptotic effects of TcdA and TcdB occur at toxin concentrations of picomolar or lower and are evident at 24 to 48 h postintoxication (26,28,29,(34)(35)(36). TcdB also induces a glucosyltransferase-independent necrosis that is mediated by the assembly and activation of the NADPH oxidase (NOX) complex, subsequently producing high levels of reactive oxygen species (ROS) (34,(37)(38)(39)(40).…”

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Clostridium difficile Toxins TcdA and TcdB Cause Colonic Tissue Damage by Distinct Mechanisms

et al. 2016

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e As the major cause of antibiotic-associated diarrhea, Clostridium difficile is a serious problem in health care facilities worldwide. C. difficile produces two large toxins, TcdA and TcdB, which are the primary virulence factors in disease. The respective functions of these toxins have been difficult to discern, in part because the cytotoxicity profiles for these toxins differ with concentration and cell type. The goal of this study was to develop a cell culture model that would allow a side-by-side mechanistic comparison of the toxins. Conditionally immortalized, young adult mouse colonic (YAMC) epithelial cells demonstrate an exquisite sensitivity to both toxins with phenotypes that agree with observations in tissue explants. TcdA intoxication results in an apoptotic cell death that is dependent on the glucosyltransferase activity of the toxin. In contrast, TcdB has a bimodal mechanism; it induces apoptosis in a glucosyltransferase-dependent manner at lower concentrations and glucosyltransferase-independent necrotic death at higher concentrations. The direct comparison of the responses to TcdA and TcdB in cells and colonic explants provides the opportunity to unify a large body of observations made by many independent investigators. C lostridium difficile is the most common cause of antibioticassociated diarrhea in the United States, and C. difficile infection (CDI) has been steadily increasing in prevalence and severity over the last 15 years (1-3). Symptoms of CDI can range from mild diarrhea to pseudomembranous colitis, and hallmarks of the disease include neutrophil infiltration, fluid release, and necrotic lesions in the colonic epithelium (4, 5). The bacteria produce two main virulence factors, large toxins called TcdA and TcdB (6, 7).The respective function and relative importance of each toxin in pathogenesis have been active topics of investigation. Genetic knockout experiments in C. difficile have shown that both toxins are important for disease pathology, although TcdB alone is sufficient to cause death in both the hamster and mouse models (6-8). For many years, TcdA and TcdB have been thought to act synergistically, with TcdA acting as an enterotoxin and TcdB acting as a cytotoxin (9, 10). The general term enterotoxin refers to the capacity of TcdA to induce inflammation, cytokine release, and fluid secretion in animal intoxication models (11-13). While TcdB does not always induce these same phenotypes in models, such as the ileal loop model, it has been shown to disrupt the integrity of the epithelial structure in human explant and xenograft models (14, 15). TcdB is also notably more potent as a cytotoxin in cell culture models (9, 10, 16).The toxins have an N-terminal glucosyltransferase domain (GTD) that is delivered into the host cytosol by the C-terminal portion of the protein (17, 18). The GTD has been shown to target and inactivate a number of Rho-family GTPases (19,20). This inactivation has been linked to a cell rounding or cytopathic effect (CPE) (21-24) and to an apoptotic cytotoxic ef...

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Section: Figmentioning

confidence: 99%

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Clostridium difficile Toxins TcdA and TcdB Cause Colonic Tissue Damage by Distinct Mechanisms

et al. 2016

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“…9E. The actin cytoskeleton is a known target of toxins A and B (14,22). To visualize the distribution of intracellular actin fibers, we stained 3T3-L1 fibroblasts with the ActinGreen reagent.…”

Section: Live But Not Heated S Boulardii Significantly Inhibited Cmentioning

confidence: 99%

ProbioticSaccharomyces boulardiiCNCM I-745 prevents outbreak-associatedClostridium difficile-associated cecal inflammation in hamsters

Koon

et al. 2016

American Journal of Physiology-Gastrointestinal and Liver Physi

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. difficile infection (CDI) is a common debilitating nosocomial infection associated with high mortality. Several CDI outbreaks have been attributed to ribotypes 027, 017, and 078. Clinical and experimental evidence indicates that the nonpathogenic yeast Saccharomyces boulardii CNCM I-745 (S.b) is effective for the prevention of CDI. However, there is no current evidence suggesting this probiotic can protect from CDI caused by outbreak-associated strains. We used established hamster models infected with outbreak-associated C. difficile strains to determine whether oral administration of live or heat-inactivated S.b can prevent cecal tissue damage and inflammation. Hamsters infected with C. difficile strain VPI10463 (ribotype 087) and outbreakassociated strains ribotype 017, 027, and 078 developed severe cecal inflammation with mucosal damage, neutrophil infiltration, edema, increased NF-B phosphorylation, and increased proinflammatory cytokine TNF␣ protein expression. Oral gavage of live, but not heated, S.b starting 5 days before C. difficile infection significantly reduced cecal tissue damage, NF-B phosphorylation, and TNF␣ protein expression caused by infection with all strains. Moreover, S.b-conditioned medium reduced cell rounding caused by filtered supernatants from all C. difficile strains. S.b-conditioned medium also inhibited toxin A-and B-mediated actin cytoskeleton disruption. S.b is effective in preventing C. difficile infection by outbreak-associated via inhibition of the cytotoxic effects of C. difficile toxins. (46). C. difficile is an anaerobic bacterium that produces two toxins -toxin A and toxin B -that mediate diarrhea, inflammation, and apoptosis of the mucosal epithelium in animals and humans (32). The effects of the toxin in target intestinal cells involve inactivation of the Rho family of GTPase, leading to cytoskeletal disorganization, epithelial cell apoptosis, and ultimately cell death (42, 54). C. difficile toxins also stimulate transcription of several proinflammatory genes, including tumor necrosis factor-␣ (TNF␣) (19) and activate transcription factors and mitogen-activated protein kinases involved in their proinflammatory effects (18,25). The mainstream CDI regimen includes the use of metronidazole, vancomycin, and fidaxomicin (55). However, many C. difficileinfected patients may also suffer from recurrent infections (13), while the recent epidemics that also involve new epidemic outbreak-associated strains (29) pose a major medical problem and epidemiological concern. These challenges have been met with a broad range of preventive approaches against CDI, including the use of probiotics, most notably Saccharomyces boulardii (S.b) alone or in combination with established antibiotic treatment (23,24).S.b is a nonpathogenic yeast that represents one of the well-studied probiotics against CDI in both experimental and clinical settings (24,40). Randomized double-blind placebocontrolled clinical trials have shown that S.b CNCM I-745 is an effective probiotic in the prophylaxis of antibi...

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“…32 Prolonged exposure to the toxins leads to host-cell apoptosis. 33 TcdC and TcdR are two other PaLoc-encoded proteins. TcdC is a negative regulator and modulates toxin gene expression.…”

Section: Pathogenesismentioning

confidence: 99%

Clostridium difficileinfection

2010

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Glucosylation of Rho GTPases by Clostridium difficile toxin A triggers apoptosis in intestinal epithelial cells

Cited by 96 publications

References 37 publications

Clostridium difficile Toxins TcdA and TcdB Cause Colonic Tissue Damage by Distinct Mechanisms

Clostridium difficile Toxins TcdA and TcdB Cause Colonic Tissue Damage by Distinct Mechanisms

ProbioticSaccharomyces boulardiiCNCM I-745 prevents outbreak-associatedClostridium difficile-associated cecal inflammation in hamsters

Clostridium difficileinfection

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