Difference in the biological effects of Clostridium difficile toxin B in proliferating and non-proliferating cells

Toxin A (TcdA) and toxin B (TcdB) from Clostridium difficile are the causative agents of the C. difficile-associated diarrhea (CDAD) and its severe form, the pseudomembranous colitis. TcdA and TcdB both glucosylate and thereby inactivate low molecular weight GTP-binding proteins of the Rho, Rac, and Cdc42 subfamilies. In cultured cell lines, TcdB induces actin re-organization and bi-nucleation ("cytopathic effects") and cell death ("cytotoxic effects"). In this study, the role of cell cycle progression in the cytopathic and the cytotoxic effects of TcdB is evaluated by a differential analysis of these effects in proliferating and non-proliferating cells. Density-synchronized murine fibroblasts and confluent HT29 colonocytes are exploited as cell culture models for non-proliferating cells. Cell death is analyzed in terms of a loss of cell viability, phosphatidylserine exposure, and DNA fragmentation. In proliferating cells, TcdB blocks cell proliferation and induces apoptotic cell death. In contrast, TcdB induces non-apoptotic cell death in non-proliferating cells. TcdB-induced cell rounding turns out to be independent of cell cycle progression. Cell cycle progression is an important determinant in the biological effects of TcdB. With respect to the pathology of CDAD, this study leads to the new hypothesis that necrotic cell death of terminally differentiated colonocytes and inhibition of epithelial renewal of the colon contribute to the pathogenesis of CDAD.