Clostridium difficile toxin A causes early damage to mitochondria in cultured cells

Background & Aims: The mechanism by which Clostridium difficile toxin A cau ses 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 befor e 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) conce ntration, mitochondrial permeability, and leakage of cytochrome c, Results: Confocal microscopy showed toxin A colocalization with the mitochondrial p rotein 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 resulte d in reduction of mitochondrial membrane potential and a 2-3-fold increase in reactive oxygen radicals. Preincubation of CHO cells with the antioxidan ts 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 effe ct of toxin A on leakage of cytochrome c, Conclusions: The results show tha t extensive mitochondrial damage occurs within 15 minutes in CHO cells expo sed to toxin A. Diminished ATP concentrations and increased oxygen radicals are likely to contribute to cytotoxicity from this bacterial toxin.