SummaryVibrio cholerae produces a little-studied cytotoxin, haemagglutinin/protease (HA /P), in addition to several better-characterized enterotoxins, i.e. cholera toxin (CT), zonula occludens toxin (ZOT) and accessory cholera enterotoxin (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 the present study we have assessed more speci®cally how TJs are affected by HA /P by investigating the cellular localization and biochemical integrity of two wellcharacterized TJ-associated proteins, occludin and ZO-1. Western 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 degradation was greatly attenuated when the speci®c bacterial metalloproteinase 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 speci®c event. The other TJassociated protein ZO-1 was not degraded by HA /P in parallel experiments, suggesting the selectivity of HA /P-associated protein degradation. Moreover, immuno¯uorescence labelling and confocal microscopy showed that ZO-1, but not occludin, around cell±cell boundaries was rearranged by HA /P treatment. Since ZO-1 is located on the inside of the plasma membrane and is directly associated with occludin, the results indicate that breakdown of occludin may send signals to ZO-1 that affect its organization and the structure of the F-actin cytoskeleton. Our ®nding that the zinc-containing metalloprotease of V. cholerae speci®-cally degraded occludin suggests that speci®c degradation of important host proteins by bacterial zinccontaining metalloproteases may be an important mechanism in microbial pathogenesis.

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