Studies of Vibrio cholerae toxins (zonula occludens toxin and hemagglutinin/protease) and their effects on epithelial cells

Sammanfattning: Intestinal epithelium is important for absorption of nutrients and for defense against harmful substances in the gut. The permeability and barrier functions of epithelia are mainly controlled by intercellular tight junctions. Vibrio cholerae, a noninvasive enteropathogen, produces several toxins which affect the intestinal epithelium Besides the hypertoxic cholera toxin (CT), it has also been suggested to produce two other putative enterotoxins, zonula occludens toxin (ZOT) and accessory cholera enterotoxin (Ace). Since the suggested effects ofZOT on the tight junctions have been shown to be reversible, ZOT could be useful for studying the mechanisms of regulation of the tight junctions. The gene encoding ZOT has been cloned and sequenced, but the ZOT protein was not purified or characterized. Therefore, the first task in this Ph. D. study was isolation and characterization of WT. PCR-derived fragments containing zot were cloned into expression vectors. These vectors were then introduced into E. coli or V. cholerae. However, ZOT could only be expressed as insoluble inclusion bodies in E. coli. When extra signal sequences were used to export it, no stable and reproducible ZOT signals could be found. Neither was there any detectable amount of ZOT -protein in culture supematants of V. cholerae. Moreover, no ZOT-specific effects on cultured epithelial cells, i.e. MDCK-I, Caco-2, and HT-29, could be found in the culture supematants of V. cholerae stralu 395, in which the ZOT-activities were initially found.On the other hand, a V. cho/erae stralu that did not contalu any of the known enterotoxins showed severe cytotoxic effects on the epithelial cells. Further investigations revealed that the V. cholerae hemagglutinin/protease (HA/P) was the responsible agent. HA/P did not affect the apical microvilli of the cells but caused distinct reorganization of a tight junction-associated protein Z0-1, as well as reanangement of the F-actin cytoskeleton, as studied with confocal microscopy. These cytotoxic effects of HA/P were reduced by endogenous nitric oxide (NO). HA/P could only cause negligible alterations in the lateral diffusion of ganglioside GMI receptors of er on the cell surface, as assessed with fluorescence recovery after photo bleaching (FRAP). However, extended challenge of MDCK-I cells with HA/P inhibited the uptake of CT by the cells.