title: Distinct effects of Vibrio cholerae haemagglutinin/protease on the structure and localization of the tight junction-associated proteins occludin and ZO-1.
abstract: Vibrio 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 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. 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 specific 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 specific event. The other TJ-associated protein ZO-1 was not degraded by HA/P in parallel experiments, suggesting the selectivity of HA/P-associated protein degradation. Moreover, immunofluorescence 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 finding that the zinc-containing metalloprotease of V. cholerae specifically degraded occludin suggests that specific degradation of important host proteins by bacterial zinc-containing metalloproteases may be an important mechanism in microbial pathogenesis.
authors.affiliation: Department of Health and Environmental, Linköping University, Sweden.
authors.forename: Z
authors.initials: Z
authors.lastname: Wu
authors.forename: P
authors.initials: P
authors.lastname: Nybom
authors.forename: K E
authors.initials: KE
authors.lastname: Magnusson
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004285:Dogs; D004847:Epithelial Cells / Q000378:metabolism / Q000648:ultrastructure; D005780:Gelatin / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000494:pharmacology*; D018613:Microscopy, Confocal; D062793:Occludin; D010750:Phosphoproteins / Q000378:metabolism*; D019108:Tight Junctions / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism; D062826:Zonula Occludens-1 Protein
chemical_list: D008565:Membrane Proteins; D062793:Occludin; D010750:Phosphoproteins; D062826:Zonula Occludens-1 Protein; D005780:Gelatin; D014357:Trypsin; D008666:Metalloendopeptidases; C039814:hemagglutinin-protease