Clostridium difficile toxin A-induced apoptosis is p53-independent but depends on glucosylation of Rho GTPases

Nottrott, Stefanie; Schoentaube, Janett; Genth, Harald; Just, Ingo; Gerhard, Ralf

doi:10.1007/s10495-007-0074-8

Cited by 62 publications

(103 citation statements)

References 44 publications

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“…41 Contributing to the heterogeneity of LatB effect in cells of different origin, our data also show that HBMEC apoptosis induced by LatB, which was mediated by c-Abl, was not a universal effect because tumor cell lines we and others examined did not exhibit LatB-induced apoptosis. 30 Further specificity of the role of c-Abl in HBMEC apoptosis is suggested because, whereas LatB-induced apoptosis in HBMECs was inhibited by STI-571 and siRNA to c-Abl, etoposide-induced PARP cleavage was not inhibited by STI-571, and etoposide-and vincristine-induced caspase-8 cleavage was also not inhibited by siRNA to c-Abl (supplemental Figure 2).…”

Section: Discussionmentioning

confidence: 99%

c-Abl mediates endothelial apoptosis induced by inhibition of integrins αvβ3 and αvβ5 and by disruption of actin

Xu¹,

Millard²,

Ren³

et al. 2010

Blood

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Inhibition of integrins IntroductionAngiogenesis and endothelial cell responses are essential processes in diseases, such as cancer, and ischemic conditions. Integrins, heterodimeric cell-surface receptors composed of ␣ and ␤ subunits, are central regulators of angiogenesis and endothelial cell functions. 1 Integrins enable cells to adhere to extracellular matrix (ECM), migrate over ECM substrates, and respond to ECM contact by proliferation, differentiation, and protection from apoptosis mediated by regulation of a number of intracellular signaling pathways. 1-3 Inhibition of integrins ␣v␤3 and ␣v␤5, which are preferentially expressed and activated on angiogenic endothelial cells, induces endothelial apoptosis and impairs tumor angiogenesis. 4,5 ␣v␤3/␣v␤5-integrin signaling is mediated through interactions with an arginine-glycine-aspartic acid (RGD) peptide sequence in matrix proteins, such as vitronectin (VN), and can be abrogated by soluble function-blocking RGD peptides, such as cyclic RGDfV. 4 Indeed, inhibitors of integrin ␣v␤3 are undergoing clinical trials in cancer patients, and cilengitide (EMD 121974; Merck KGaA), an integrin ␣v␤3/␣v␤5 function-blocking RGDfV peptide, has encouraging activity in phase 1 and 2 trials against brain tumors in children and adult cancer patients. 6,7 Integrin ␣v␤3 mechanism of action is complex. ␣v␤3 participates in pathologic angiogenesis, 4,8 supporting its development as a target for therapy. To mediate its function, integrin ␣v␤3 requires activation and phosphorylation of the ␤3 integrin tail on Tyr747 and Tyr459, which signal downstream to pathways involving, among others, Src, FAK, Shc, p53, and p21 WAF . 8,9 Complicating matters, integrin ␣v␤3 cosignals with growth factor receptors, such as vascular endothelial growth factor receptor-2 and others. 8 The intracellular signaling events mediating outside-in and inside-out signaling are complex and depend on the context of activation of the integrin and the cell type studied. Therefore, it is not surprising that the precise molecular mechanisms induced by engagement, crosstalk, or inhibition of ␣v␤3 integrin remain only partially understood.Engagement of integrins with the ECM allows cells to adhere and spread, inducing changes in the actin cytoskeleton. Actin is an abundant cytoskeletal protein important in cell spreading and motility. 10,11 Engagement of integrins with the ECM generates complex bidirectional signaling cascades between integrins and the actin cytoskeleton, which serve to transmit both force and biochemical signals. The interaction of integrins with actin is mostly through a number of intermediary proteins that can be cell-specific and/or stimulus-specific. 11 A critical molecule that interacts with F-actin in fibroblasts is c-Abl. c-Abl integrates multiple signals to coordinate F-actin dynamics, whereas F-actin itself has an inhibitory effect on c-Abl kinase activity. 12-14 Therefore, it is anticipated that actindependent signaling, including that by c-Abl, may mediate at least some of the phenotypes ...

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Section: Discussionmentioning

confidence: 99%

c-Abl mediates endothelial apoptosis induced by inhibition of integrins αvβ3 and αvβ5 and by disruption of actin

Xu¹,

Millard²,

Ren³

et al. 2010

Blood

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“…The large T antigen binds several cellular factors, including p53, a tumor suppressor known to regulate cell cycle, mitotic division, and apoptotic cell death pathways. There are conflicting reports regarding the function of p53 in TcdA-induced apoptosis (35,45). One study using a nontransformed colonic cell line concluded that TcdA-induced apoptosis is dependent upon p53 function (45).…”

Section: Discussionmentioning

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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“…The proteins are homologous glucosyltransferases that inactivate small GTPases of the Rho/Rac family. The resulting disruption in signaling causes a loss of cell-cell junctions, dysregulation of the actin cytoskeleton, and apoptosis (4,5).…”

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confidence: 99%

Structural organization of the functional domains of Clostridium difficile toxins A and B

Pruitt¹,

Chambers

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

133

149

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Clostridium difficile toxins A and B are members of an important class of virulence factors known as large clostridial toxins (LCTs). Toxin action involves four major steps: receptor-mediated endocytosis, translocation of a catalytic glucosyltransferase domain across the membrane, release of the enzymatic moiety by autoproteolytic processing, and a glucosyltransferase-dependent inactivation of Rho family proteins. We have imaged toxin A (TcdA) and toxin B (TcdB) holotoxins by negative stain electron microscopy to show that these molecules are similar in structure. We then determined a 3D structure for TcdA and mapped the organization of its functional domains. The molecule has a "pincher-like" head corresponding to the delivery domain and two tails, long and short, corresponding to the receptor-binding and glucosyltransferase domains, respectively. A second structure, obtained at the acidic pH of an endosome, reveals a significant structural change in the delivery and glucosyltransferase domains, and thus provides a framework for understanding the molecular mechanism of LCT cellular intoxication.bacterial toxin | electron microscopy | pore formation

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Clostridium difficile toxin A-induced apoptosis is p53-independent but depends on glucosylation of Rho GTPases

Cited by 62 publications

References 44 publications

c-Abl mediates endothelial apoptosis induced by inhibition of integrins αvβ3 and αvβ5 and by disruption of actin

c-Abl mediates endothelial apoptosis induced by inhibition of integrins αvβ3 and αvβ5 and by disruption of actin

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

Structural organization of the functional domains of Clostridium difficile toxins A and B

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