Distinct effects of Vibrio cholerae haemagglutinin/protease on the structure and localization of the tight junction-associated proteins occludin and ZO-1

Vibrio cholerae produces a little-studied cytotoxin, haemagglutinin/proteas e (HA/P), in addition to several better-characterized enterotoxins, i.e. ch olera toxin (CT), zonula occludens toxin (ZOT) and accessory cholera entero toxin (Ace). We have found recently that HA/P perturbs the barrier function of Mardin-Darby canine kidney epithelial cell line I (MDCK-I) by affecting the intercellular tight junctions (TJs) and the F-actin cytoskeleton. In t he present study we have assessed more specifically how TJs are affected by HA/P by investigating the cellular localization and biochemical integrity of two well-characterized TJ-associated proteins, occludin and ZO-1. Wester n blot analysis showed that occludin bands of 66-85 kDa were digested by HA /P to two predominant bands of around 50 kDa and 35 kDa, and that this degr adation was greatly attenuated when the specific bacterial metalloproteinas e inhibitor Zincov was co-administered. Trypsin, on the other hand, did not degrade occludin when it was applied in the same way, suggesting that the degradation of occludin by HA/P is an early and specific event. The other T J-associated protein ZO-1 was not degraded by HA/P in parallel experiments, suggesting the selectivity of HA/P-associated protein degradation. Moreove r, immunofluorescence labelling and confocal microscopy showed that ZO-1, b ut not occludin, around cell-cell boundaries was rearranged by HA/P treatme nt. Since ZO-1 is located on the inside of the plasma membrane and is direc tly associated with occludin, the results indicate that breakdown of occlud in may send signals to ZO-1 that affect its organization and the structure of the F-actin cytoskeleton. Our finding that the zinc-containing metallopr otease of V. cholerae specifically degraded occludin suggests that specific degradation of important host proteins by bacterial zinc-containing metall oproteases may be an important mechanism in microbial pathogenesis.