Clostridium difficile toxin B activates dual caspase-dependent and caspase-independent apoptosis in intoxicated cells.
Clostridium difficile toxin B (TcdB) inactivates the small GTPases Rho, Rac and Cdc42 during intoxication of mammalian cells. In the current work, we show that TcdB has the potential to stimulate caspase-dependent and caspase-independent apoptosis. The apoptotic pathways became evident when caspase-3-processed-vimentin was detected in TcdB-treated HeLa cells. Caspase-3 activation was subsequently confirmed in TcdB-intoxicated HeLa cells. Interestingly, caspase inhibitor delayed TcdB-induced cell death, but did not alter the time-course of cytopathic effects. A similar effect was also observed in MCF-7 cells, which are deficient in caspase-3 activity. The time-course to cell death was almost identical between cells treated with TcdB plus caspase inhibitor and cells intoxicated with the TcdB enzymatic domain (TcdB1-556). Unlike TcdB treated cells, intoxication with TcdB1-556 or expression of TcdB1-556 in a transfected cell line, did not stimulate caspase-3 activation yet cells exhibited cytopathic effects and cell death. Although TcdB1-556 treated cells did not demonstrate caspase-3 activation these cells were apoptotic as determined by differential annexin-V/propidium iodide staining and nucleosomal DNA fragmentation. These data indicate TcdB triggers caspase-independent apoptosis as a result of substrate inactivation and may evoke caspase-dependent apoptosis due to a second, yet undefined, activity of TcdB. This is the first example of a bacterial virulence factor with the potential to stimulate multiple apoptotic pathways in host cells.
Qa'Dan
Maen
M
Department of Botany and Microbiology, The University of Oklahoma, 770 Van Vleet Oval, GLCH 516, Norman, OK 73019, USA.
Ramsey
Matthew
M
Daniel
Jeremy
J
Spyres
Lea M
LM
Safiejko-Mroczka
Barbara
B
Ortiz-Leduc
William
W
Ballard
Jimmy D
JD
D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology*; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000235:genetics / Q000633:toxicity*; D016466:CHO Cells; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000172:deficiency / Q000378:metabolism*; D002460:Cell Line; D006224:Cricetinae; D004789:Enzyme Activation / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D014162:Transfection; D014746:Vimentin / Q000378:metabolism
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D061945:Caspase Inhibitors; D014746:Vimentin; C031815:toxB protein, Clostridium difficile; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases