HUMAN INTESTINAL EPITHELIAL-CELLS SWELL AND DEMONSTRATE ACTIN REARRANGEMENT IN RESPONSE TO THE METALLOPROTEASE TOXIN OF BACTEROIDES-FRAGILIS

Enterotoxigenic Bacteroides fragilis (ETBF) cells produce a 20-kDa hea t-labile metalloprotease toxin which is potentially important in the p athogenesis of diarrhea associated with this infection. Previous studi es indicate that subconfluent HT29/C1 cells treated with the B. fragil is toxin (BFT) develop morphologic changes with dissolution of tight c lusters and apparent swelling. Such alterations suggest toxin-stimulat ed reorganization of the cellular cytoskeleton. The purpose of the cur rent study was to evaluate the effect of BFT on actin microfilaments ( F-actin) and cell volume. As assessed by fluorescent phallicidin stain ing which detects F-actin, BFT treatment of HT29/C1 cells resulted in redistribution of F-actin with loss of stress fibers, a floccular stai ning pattern, and cellular membrane blebbing without quantitative chan ges in F-actin fluorescence intensity. The F-actin redistribution was time and concentration dependent, In contrast to the cell shrinkage ob served in response to the F-actin-depolymerizing agents cytochalasin D and Clostridium difficile toxin A, BFT stimulated an increase in HT29 /C1 cell volume of 10 to 25% (compared with control cells) over a 24-h time course. only 10 to 30 ng of BFT per ml was necessary to stimulat e a maximal increase in HT29/C1. cell volume, The effect of BFT on cel l volume was persistent and dependent on the proteolytic activity of B FT. In agreement with cell viability assays indicating that BFT did no t injure HT29/C1 cells, intoxicated cells exhibited regulatory volume decrease, suggesting that toxin-treated cells remain physiologically d ynamic, We conclude that BFT acts on the intestinal epithelial cell cy toskeleton to alter F-actin structure and to stimulate an increase in HT29/C1 cell volume, Although these two activities of BFT appear to be linked, the precise sequence of cellular events following intoxicatio n of HT29/C1 cells with BFT remains unclear, We hypothesize that these F-actin and cell volume changes may lead to an alteration in tight ju nction function in the polarized intestinal epithelium, contributing t o the pathogenesis of diarrhea in ETBF infections.