Abstract
Invasive microorganisms efface enteric microvilli to establish intimate contact with the apical surface of enterocytes. To understand the molecular basis of this effacement in amebic colitis, we seeded Entamoeba histolytica trophozoites on top of differentiated human Caco-2 cell layers. Western blots of detergent lysates from such cocultures showed proteolysis of the actin-bundling protein villin within 1 min of direct contact of living trophozoites with enterocytes. Mixtures of separately prepared lysates excluded detergent colysis as the cause of villin proteolysis. Caspases were not responsible as evidenced by the lack of degradation of specific substrates and the failure of a specific caspase inhibitor to prevent villin proteolysis. A crucial role for amebic cysteine proteinases was shown by prevention of villin proteolysis and associated microvillar alterations through the treatment of trophozoites before coculture with synthetic inhibitors that completely blocked amebic cysteine proteinase activity on zymograms. Moreover, trophozoites of amebic strains pSA8 and SAW760 with strongly reduced cysteine proteinase activity showed a reduced proteolysis of villin in coculture with enteric cells. Salmonella typhimurium and enteropathogenic Escherichia coli disturb microvilli without villin proteolysis, indicating that the latter is not a consequence of the disturbance of microvilli. In conclusion, villin proteolysis is an early event in the molecular cross-talk between enterocytes and amebic trophozoites, causing a disturbance of microvilli.
Highlights
An analysis of the early contact between pathogenic microorganisms and their hosts has advanced our understanding of the pathogenesis of infectious disease and of cellular microbiology [1,2,3]
Specific Proteolysis of Villin in Enterocytes in Direct Contact with Trophozoites—To evaluate the effect of E. histolytica HM1:IMSS on enteric villin, the lysates of 3-week-old Caco-2 cell layers cocultured or not with 2 ϫ 106 trophozoites for 1–5 min were analyzed on Western blots immunostained with a monoclonal antibody against villin
Western blots of lysates from cocultures with antibodies against ezrin, gelsolin, or ␣-catenin showed little or no amebic proteolysis (Fig. 1B), suggesting that among actin-binding proteins villin is a specific early target for trophozoites adhering to the apical side of enterocytes
Summary
An analysis of the early contact between pathogenic microorganisms and their hosts has advanced our understanding of the pathogenesis of infectious disease and of cellular microbiology [1,2,3]. Molecular changes associated with such adhesion implicate the transfer of the Gal/N-acetylgalactosamine-specific amebic lectin onto the lateral side of enterocytes [5], insertion of amebic poreforming proteins into the host cell membrane [6], dephosphorylation and degradation of host cell proteins [7, 8], and activation of caspase-3 like caspases [9]. The purpose of this study was to investigate the molecular mechanism by which trophozoites of E. histolytica efface the brush border microvilli of enteric cells. Villin is a major actin-bundling protein and a member of the family of Ca2ϩ-regulated actin-binding proteins. Our experimental model consists of 3-week-old well differentiated Caco-2 cell layers on top of which we seed trophozoites of various strains, namely pathogenic E. histolytica HM-1:IMSS, transfected E. histolytica pSA8 [18], or non-pathogenic Entamoeba dispar SAW760, and we examined villin by immunocytochemistry and Western blotting
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