Interaction of cytopathogenic toxin from Clostridium difficile with cells in tissue culture.

Partially purified cytopathogenic toxin from Clostridium difficile induced morphological changes in five cell lines in tissue culture. The relative sensitivity scale of the cell lines was human lung and intestinal fibroblasts greater than Chinese hamster ovary cells much greater than mouse adrenal cells greater than mouse neuroblastoma cells. The cytopathogenic effect did not occur in toxin-treated lung fibroblasts incubated at 0 degree C. Pre-incubation of lung fibroblasts with 2,4-dinitrophenol prevented the cytopathogenic effect. The toxin bound to as yet unidentified receptors at the surface of human lung and intestinal fibroblasts. The toxin-induced morphological (actinomorphic) changes in lung and intestinal fibroblasts closely resembled the effects induced by the fungal metabolite cytochalasin B (CB), which is known to disrupt microfilaments reversibly. Indirect immunofluorescence with anti-actin antiserum demonstrated that the C. difficile toxin disrupted the straight actin filament bundles seen in normal fibroblasts. The cytopathogenic effect became apparent 3--5 h after exposure to toxin. However, irreversible intoxication occurred already within 20 min of exposure, as toxin-treated fibroblasts which were trypsinized and reseeded were not able to attach to the solid substratum and regenerate their typical shape, a process requiring reorganization of actin into microfilament bundles. Two possible different modes of action of the toxin, leading to microfilament disruption, are suggested: 1) Transmembrane signal by surface-bound toxin via microfilament-linked integral membrane protein(s) and 2) Penetration of surface-bound whole toxin or an active fragment, followed by its intracellular action. The experimental evidence so far is consistent with either of these mechanisms.