Clostridium difficile toxin A (TcdA) is one of two homologous glucosyltransferases that mono-glucosylate Rho GTPases. HT29 cells were challenged with wild-type and mutant TcdA to investigate the mechanism by which apoptosis is induced. The TcdA-induced re-organization of the actin cytoskeleton led to an increased number of cells within the G2/M phase. Depolymerization of the actin filaments with subsequent G2/M arrest, however, was not causative for apoptosis, as shown in a comparative study using latrunculin B. The activation of caspase-3, -8, and -9 strictly depended on the glucosylation of Rho GTPases. Apoptosis measured by flow cytometry was completely abolished by a pan-caspase inhibitor (z-VAD-fmk). Interestingly, cleavage of procaspase-3 and Bid was not inhibited by z-VAD-fmk, but was inhibited by the calpain/cathepsin inhibitor ALLM. Cleavage of procaspase-8 was susceptible to inhibition by z-VAD-fmk and to the caspase-3 inhibitor Ac-DMQD-CHO, indicating a contribution to the activation of caspase-3 in an amplifying manner. Although TcdA induced mitochondrial damage and cytochrome c release, p53 was not activated or up-regulated. A p53-independent apoptotic effect was also checked by treatment of HCT 116 p53(-/-) cells. In summary, TcdA-induced apoptosis in HT29 cells depends on glucosylation of Rho GTPases leading to activation of cathepsins and caspase-3.

The intestinal epithelial cell line HT-29 was used to study the apoptotic effect of Clostridium difficile toxin A (TcdA). TcdA is a 300 kDa single-chain protein, which glucosylates and thereby inactivates small GTPases of the Rho family (Rho, Rac and Cdc42). The effect of TcdA-catalysed glucosylation of the Rho GTPases is well known: reorganization of the actin cytoskeleton with accompanying morphological changes in cells, leading to complete rounding of cells and destruction of the intestinal barrier function. Less is known about the mechanism by which apoptosis is induced in TcdA-treated cells. In this study, TcdA induced the activation of caspase-3, -8 and -9. Apoptosis, as estimated by the DNA content of cells, started as early as 24 h after the addition of TcdA. The impact of Rho glucosylation was obvious when mutant TcdA with reduced or deficient glucosyltransferase activity was applied. TcdA mutant W101A, with 50-fold reduced glucosyltransferase activity, induced apoptosis only at an equipotent concentration compared with wild-type TcdA at a 50 % effective concentration of 0.2 nM. The enzyme-deficient mutant TcdA D285/287N was not able to induce apoptosis. Apoptosis induced by TcdA strictly depended on the activation of caspases, and was completely blocked by the pan-caspase inhibitor z-VAD-fmk. Destruction of the actin cytoskeleton by latrunculin B was not sufficient to induce apoptosis, indicating that apoptosis induced by TcdA must be due to another mechanism. In summary, TcdA-induced apoptosis (cytotoxic effect) depends on the glucosylation of Rho GTPases, but is not triggered by destruction of the actin cytoskeleton (cytopathic effect).

SummaryClostridium difficile toxin A and B (TcdA/TcdB) are glucosyltransferases that glucosylate GTPases of the Rho family. The epidermal growth factor (EGF) positively modulates C. difficile toxin-induced disturbance of the intestinal barrier function by an unknown mechanism. We found that EGF-treated CaCo-2 monolayers were less susceptible to TcdA-catalysed glucosylation of Rac1 but not of RhoA, which correlated with phosphorylation of Rac1 at Ser-71. Phospho-Rac1/phospho-Cdc42 (Ser-71) still bound to the PAK-CRIB domain indicating an active state. A more detailed characterization of phospho-Rac1 was performed using the phosphomimetic mutant Rac1 S71E. Ectopic expression of Rac1 S71E induced a specific phenotype of cells showing an increase in filopodial structures that were also induced by EGF. Rac1 S71E (and Cdc42 S71E) but not Rac1 S71A was at least fivefold weaker substrate for TcdAcatalysed glucosylation compared with wild type Rac1. The protective effect was checked in transfection experiments where Rac1 S71E and, to a lesser extent, Cdc42 S71E reduced the TcdA-induced cytopathic effect. Thus, Ser-71 phosphorylation of Rac1 might be interesting for modulation of microbial pathogenesis where Rho GTPases, especially Rac1 and Cdc42, are involved. In addition, this is the first description of a specific functional outcome of Rac1 phosphorylation at Ser-71.

Mutation of tryptophan-101 in Clostridium difficile toxin A, a 308-kDa glucosyltransferase, resulted in a 50-fold-reduced cytopathic activity in cell culture experiments. The mutant toxin A was characterized and applied to distinguish between glucosyltransferase-dependent and -independent effects with respect to RhoB up-regulation as a cellular stress response.Clostridium difficile toxins A and B (TcdA and TcdB, respectively) are the major pathogenicity factors that are causative for antibiotic-associated pseudomembranous colitis (19). Several reports of the in vivo effects of TcdA in animal models reflect efforts to understand the cellular mechanism leading to clinical symptoms as well as to the release of mediators that are involved in the inflammatory process (2,13,15,18,20). The inherent glucosyltransferase