Clostridium difficile toxin A-induced apoptosis is p53-independent but depends on glucosylation of Rho GTPases.
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.
Nottrott
Stefanie
S
Institute of Toxicology, Hannover Medical School, 30625 Hannover, Germany.
Schoentaube
Janett
J
Genth
Harald
H
Just
Ingo
I
Gerhard
Ralf
R
D000199:Actins / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D001427:Bacterial Toxins / Q000378:metabolism* / Q000494:pharmacology*; D020169:Caspases / Q000378:metabolism; D002403:Cathepsins / Q000037:antagonists & inhibitors; D002453:Cell Cycle / Q000187:drug effects; D004768:Enterotoxins / Q000378:metabolism* / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D005960:Glucosides / Q000378:metabolism*; D016695:Glycosyltransferases / Q000378:metabolism / Q000502:physiology; D019073:HT29 Cells; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D008954:Models, Biological; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D016159:Tumor Suppressor Protein p53 / Q000502:physiology*; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
D000199:Actins; D001427:Bacterial Toxins; D004768:Enterotoxins; D005960:Glucosides; D007976:Leupeptins; D016159:Tumor Suppressor Protein p53; C041348:tcdA protein, Clostridium difficile; C063261:acetylleucyl-leucyl-norleucinal; D016695:Glycosyltransferases; D002403:Cathepsins; D020169:Caspases; D020741:rho GTP-Binding Proteins