Clostridium difficile toxin A causes early damage to mitochondria in cultured cells.
OBJECTIVE
The mechanism by which Clostridium difficile toxin A causes actin depolymerization and cell rounding involves toxin internalization and subsequent monoglucosylation of the Rho family of proteins. This study explored toxin internalization and effects on mitochondrial function before cell rounding.


METHODS
Chinese hamster ovary (CHO) cells were exposed to toxin A, and mitochondrial localization was assayed by confocal microscopy. Mitochondrial function was measured by adenosine triphosphate (ATP) concentration, mitochondrial permeability, and leakage of cytochrome c.


RESULTS
Confocal microscopy showed toxin A colocalization with the mitochondrial protein GRP 75 at 5 minutes after toxin exposure. Between 5 and 15 minutes, toxin A caused an 80% diminution in cellular ATP levels; cell rounding and Rho glucosylation commenced between 15 and 30 minutes. Toxin A also resulted in reduction of mitochondrial membrane potential and a 2-3-fold increase in reactive oxygen radicals. Preincubation of CHO cells with the antioxidants butylated hydroxyanisole or butylated hydroxytoluene blocked the toxin A-induced increase in oxygen radicals and diminished cell rounding. Western blot analysis of toxin A-exposed isolated mitochondria showed a direct effect of toxin A on leakage of cytochrome c.


CONCLUSIONS
The results show that extensive mitochondrial damage occurs within 15 minutes in CHO cells exposed to toxin A. Diminished ATP concentrations and increased oxygen radicals are likely to contribute to cytotoxicity from this bacterial toxin.
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Division of Gastroenterology and Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.
Hagen
S J
SJ
Pothoulakis
C
C
Chen
M
M
Medina
N D
ND
Warny
M
M
LaMont
J T
JT
D000255:Adenosine Triphosphate / Q000037:antagonists & inhibitors; D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism / Q000494:pharmacology*; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003574:Cytochrome c Group / Q000378:metabolism; D004768:Enterotoxins / Q000378:metabolism / Q000494:pharmacology*; D008564:Membrane Potentials / Q000187:drug effects; D008854:Microscopy, Electron; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology / Q000648:ultrastructure; D017382:Reactive Oxygen Species / Q000378:metabolism; D014018:Tissue Distribution
D001427:Bacterial Toxins; D003574:Cytochrome c Group; D004768:Enterotoxins; D017382:Reactive Oxygen Species; C041348:tcdA protein, Clostridium difficile; D000255:Adenosine Triphosphate