Morphologic and functional effects of Clostridium difficile enterotoxin in tissue culture

The effects of the Clostridium difficile toxin were examined in HeLa and mouse adrenal tumor (MAT) cells. Cytotoxicity was evaluated by vital dye exclusion and 51Cr release. In both HeLa and MAT cells, C. difficile toxin caused rounding of virtually 100% of cells. This rounding was distinguishable from rounding produced by the Escherichia coli heat-labile enterotoxin (LT): (1) LT was inactive in HeLa cells; (2) in MAT cells, C. difficile toxin produced uniformly rounded cells, while LT-rounded cells usually had cytoplasmic extensions and a regular background of flattened cells. In C. difficile toxin affected HeLa cells, there were less than mitotic figures per 1000 cells compared with 15-18 in controls. Clostridium difficile toxin treated HeLa cells showed less than 10% cytotoxicity in 24 h and no more than 30% by 74 h, similar to control cells. However, paralleling the suppression of mitotic figures, cell multiplication was inhibited in C. difficile toxin treated cells when subcultured in a short-term (72 h) assay compared with up to a 500% increase in control cells. This inhibition was also seen in a 6-week cloning assay in which C. difficile toxin treated cells had a cloning efficiency of less than 1% compared with approximately 10% in controls. We conclude that the major effect of the C. difficile toxin is inhibition of growth rather than immediate cell death. The relationship of this growth inhibition to colitis remains to be elucidated.