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CYSTEINE PROTEINASES SECRETED BY ENTAMOEBA HISTOLYTICA ALTER INTESTINAL EPITHELIAL BARRIER FUNCTION

A Stillar, D Moncada, K Chadee
Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta

Entamoeba histolytica secretes cysteine proteases that may play a major role in disrupting the epithelial barrier of the gut. The epithelial layer of the colon forms a selective barrier against immune cells, molecules and pathogens, which includes both transcellular and paracellular regulation. Tight junction proteins (TJP) are the first line of paracellular regulation of the epithelial barrier. In this study we investigated whether amoeba cysteine proteases can alter TJP and disrupt the epithelial barrier. In response to wild-type (WT) amoeba, transepithelial electrical resistance (TER) of T84 colonic epithelial cell monolayers decreased by 70% after 120 min exposure as compared with controls. In contrast, in cysteine protease-deficient pSA8 amoebae-expressing antisense to cysteine protease 5 (90% reduction of cysteine protease secretion and enzyme activity), TER was decreased by 30%. To address alterations in TJ proteins, the permeability of the epithelial barrier were assessed by ¹⁴C-mannitol fluxes. In response to WT amoebae there was a 15-fold increase in the permeability of the T84 monolayer after 3 h and only a 5-fold change in change in permeability in response to the pSA8 amoebae at the same time point. Western blot analysis revealed there was a significant reduction of the TJP claudin-1 after 30-min exposure to WT amoeba compared with T84 cells not exposed to amoeba. Exposure to WT amoeba secreted products also caused an increase in occludin after 120 min while TJP's ZO-1, claudin-2, claudin-4 and JAM were not affected. RNA of T84 colonic epithelial cells was not altered in response to WT and cysteine protease-deficient pSA8 amoebae regardless to time of exposure. These results indicate that cysteine proteases secreted by amoebae can alter TJP independent of TJP gene regulation to affect intestinal barrier function.
Supported by CIHR

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