Glucosylation of Rho GTPases by Clostridium difficile toxin A triggers apoptosis in intestinal epithelial cells.
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).
Gerhard
Ralf
R
Institute of Toxicology, Hannover Medical School, 30625 Hannover, Germany.
Nottrott
Stefanie
S
Institute of Toxicology, Hannover Medical School, 30625 Hannover, Germany.
Schoentaube
Janett
J
Institute of Toxicology, Hannover Medical School, 30625 Hannover, Germany.
Tatge
Helma
H
Institute of Toxicology, Hannover Medical School, 30625 Hannover, Germany.
Olling
Alexandra
A
Institute of Toxicology, Hannover Medical School, 30625 Hannover, Germany.
Just
Ingo
I
Institute of Toxicology, Hannover Medical School, 30625 Hannover, Germany.
D017209:Apoptosis / Q000187:drug effects*; D001427:Bacterial Toxins / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism; D004768:Enterotoxins / Q000378:metabolism*; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000187:drug effects*; D006031:Glycosylation; D019073:HT29 Cells; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology* / Q000187:drug effects; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
D001427:Bacterial Toxins; D004768:Enterotoxins; C041348:tcdA protein, Clostridium difficile; D020169:Caspases; D020741:rho GTP-Binding Proteins