Isolation of a fibroblast mutant resistant to Clostridium difficile toxins A and B.

A mutant of Chinese hamster lung fibroblasts (Don cells), resistant against Clostridium difficile toxins A and B, was isolated after mutagenization with ethylmethanesulphonate and a two-step selection with toxin B. The mutant, termed CdtR-Q, was 10(4) times more resistant to toxin B than wild-type cells and cross-resistant to toxin A (10(3) times more resistant). The resistance was overcome by increasing the dose of toxin. The resistance has been stable after cultivation for 40 generations in the absence of toxin. The morphology of the mutant was more epithelial-like than that of the fibroblast parental cells. The plating efficiency was about half that of the wild-type, whereas the growth rate was the same. The mutant was significantly less sensitive than the wild-type to the microfilament-interacting cytochalasins B and D. It was as sensitive as the wild-type to endocytosed toxins (diphtheria, pertussis, ricin), to microtubule-interacting agents (colchicine, gossypol, nocodazole, taxol, vinblastine), and to membrane-damaging toxins with different mechanisms of action, with one exception; the mutant was more highly sensitive to the action of phospholipase C (with broad substrate-specificity) than the wild-type. The results suggest that the mutant has a normal endocytosis, and that the mutation does not affect the microtubuli. The results are consistent with a mutation affecting the microfilaments in the cytoskeleton.