Proteolysis of Enteric Cell Villin by Entamoeba histolytica Cysteine Proteinases

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.

A Novel Entamoeba histolytica Cysteine Proteinase, EhCP4, Is Key for Invasive Amebiasis and a Therapeutic Target

“…Thus, EhCP4 was apparently secreted into the microenvironment in vivo. During the cross-talk between enterocytes and trophozoites, villin proteolysis is one of the early events causing disruption of microvilli, due to EhCP activity (44). Although we found that either EhCP1 or EhCP4 could digest human villin-1 and laminin (supplemental Fig.…”

Interaction of Phospholipase C-γ1with Villin Regulates Epithelial Cell Migration

“…For instance gelsolin could be a substrate for caspase-3, whereas villin could be a substrate for other proteases. Such proteolytic cleavage of villin has been reported in enteric cells infected with Entamoeba histolytica (17). It may also be noted that in villin-gelsolin, where the S4 -S6 domains of villin were substituted with the second half of the gelsolin core, chimeras failed to function like full-length villin protein, suggesting that although these two proteins share significant structural and Mathew, and S. Khurana, submitted for publication).…”

Regulation of intestinal epithelial cell cytoskeletal remodeling by cellular immunity following gut infection

“…24 These authors also showed that cysteine proteinases of enteric origin were likely not responsible for the proteolysis. The results of the current study, however, showed the lack of villin proteolysis in absence of host adaptive immunity in SCID mice in spite of massive infection burdens in these hosts (data not shown).…”