Expression of Vibrio cholerae zonula occludens toxin and analysis of its subcellular localization

Vibrio cholerae elaborates zonula occludens toxin (Zot), a protein that increases the permeability of small intestinal mucosa by opening intercellular tight junctions. The zot gene is located, together with the genes encoding CT and Ace enterotoxins, within the genome of V. cholerae filamentous phage CTXsmall ef, Cyrillic. Interestingly, Zot appears to be structurally and functionally related to the gene I product of other filamentous phages and it has been shown to be required for CTXsmall ef, Cyrillic morphogenesis. In this study we described the cloning of zot in several expression plasmid systems and we examined the subcellular localization of Zot by using affinity purified anti-Zot antibodies. We found that Zot localizes in the V. cholerae cell envelope with M(r);45 kDa which is consistent with the predicted primary translation product from the first methionine of zot (44.8 kDa). A second molecule, corresponding to the 33 kDa N-terminal region of Zot, was also detected. Both molecules are exposed at the bacterial cell surface. The production of the 33 kDa Zot, that might represent a processing product, was abolished in mutant ZotG59. N-terminal tagged 6xHis-Zot fusion protein retained the capability to reach the outer membrane and the 6xHis tag was not cleaved off during the translocation to the periplasm, whereas the presence of the tag partially blocked the formation of the 33 kDa molecule. Zot secretion and anchorage to the bacterial outer membrane was also observed in E. coli strains expressing Zot, suggesting that the toxin may be directed to the outer membrane via the same pathway in E. coli and V. cholerae. Zot cleavage might be due to a V. cholerae specific protease activity, since the 33 kDa protein was not efficiently produced in E. coli. On the basis of these data and Zot amino acid sequence analysis, we suggest that while the N-terminal part of the molecule is involved in the morphogenesis of CTXsmall ef, Cyrillic, the C-terminal region might carry the domain(s) responsible for Zot enterotoxic activity.