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Interaction of cytopathogenic toxin from Clostridium difficile with cells in tissue culture
Abstract:
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.
Insights
Clostridium difficile toxin causes cell damage by disrupting actin filaments, similar to cytochalasin B. This toxin affects cell shape and attachment, with potential for transmembrane or intracellular action.
Area of Science:
- Cell biology
- Microbiology
Background:
- Clostridium difficile is a significant pathogen.
- Cytopathogenic toxins contribute to C. difficile pathogenesis.
- Understanding toxin mechanisms is crucial for therapeutic development.
Purpose of the Study:
- To investigate the cytopathogenic effects of Clostridium difficile toxin.
- To elucidate the mechanism of toxin-induced cellular damage.
- To compare the toxin's effects with known cellular disruptors.
Main Methods:
- Tissue culture of five cell lines exposed to partially purified C. difficile toxin.
- Assessment of morphological changes and cell viability.
- Investigation of toxin binding and effect of temperature and metabolic inhibitors.
- Immunofluorescence microscopy to analyze actin filament integrity.
- Short-term and long-term exposure experiments to determine irreversible intoxication.
Main Results:
- C. difficile toxin induced morphological changes in human lung/intestinal fibroblasts, Chinese hamster ovary cells, mouse adrenal cells, and mouse neuroblastoma cells.
- Fibroblast sensitivity was highest, with effects inhibited at 0°C and by 2,4-dinitrophenol.
- Toxin disrupted actin filament bundles, similar to cytochalasin B, affecting cell shape and attachment.
- Irreversible intoxication occurred within 20 minutes, preceding apparent cytopathogenic effects at 3-5 hours.
Conclusions:
- Clostridium difficile toxin disrupts fibroblast actin cytoskeleton, leading to cytopathogenic effects.
- The toxin's action involves disruption of actin microfilaments, potentially through transmembrane signaling or intracellular penetration.
- Further research is needed to confirm the precise mechanism of action and identify toxin receptors.