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

Abstract

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.

The specific fates of tight junction proteins in apoptotic epithelial cells

“…Similarly, treatment of MDCK cell layers with rat mucosal mast cell chymase (RMCP-II) altered the permeability and distribution of the tight junction proteins ZO-1 and occludin (Scudamore et al, 1998), which may also depend on an extracellular cleavage of occludin. Moreover, the bacterial cytotoxin hemagglutinin/ protease (HA/P) was shown to perturb barrier function by fragmentation of occludin (Wu et al, 2000). The electrophysiological data presented above are in line with these observations although other components of tight junctions and cell-cell contacts may contribute to the observed effects.…”

Haemagglutinin/protease expression and mucin gel penetration in El Tor biotype Vibrio cholerae

“…Hap can proteolytically activate cholera toxin A subunit (Booth et al, 1984) and the El Tor cytolysin/haemolysin (Nagamune et al, 1996), and hydrolyse several physiologically important proteins such as mucin, fibronectin and lactoferrin . Hap perturbs the paracellular barrier of cultured MDCK-1 and T84 intestinal epithelial cells (Wu et al, 1996;Mel et al, 2000) by acting on tight junction-associated proteins (Wu et al, 2000).…”

Transcriptional Regulation of Vibrio cholerae Hemagglutinin/Protease by the Cyclic AMP Receptor Protein and RpoS

“…HA/protease perturbs the paracellular barrier of cultured intestinal epithelial cells (31,50) by acting on tight-junction-associated proteins (51) and promotes the detachment of vibrios from monolayers and mucin (4,15). Importantly, HA/protease contributes to the reactogenicity of live attenuated cholera vaccine strains (3,40).…”