Entamoeba histolytica cysteine proteinases (EhCPs) play a key role in disrupting the colonic epithelial barrier and the innate host immune response during invasion of E. histolytica, the protozoan cause of human amebiasis. EhCPs are encoded by 50 genes, of which ehcp4 (ehcp-a4) is the most up-regulated during invasion and colonization in a mouse cecal model of amebiasis. Up-regulation of ehcp4 in vivo correlated with our finding that co-culture of E. histolytica trophozoites with mucin-producing T84 cells increased ehcp4 expression up to 6-fold. We have expressed recombinant EhCP4, which was autocatalytically activated at acidic pH but had highest proteolytic activity at neutral pH. In contrast to the other amebic cysteine proteinases characterized so far, which have a preference for arginine in the P2 position, EhCP4 displayed a unique preference for valine and isoleucine at P2. This preference was confirmed by homology modeling, which revealed a shallow, hydrophobic S2 pocket. Endogenous EhCP4 localized to cytoplasmic vesicles, the nuclear region, and perinuclear endoplasmic reticulum (ER). Following co-culture with colonic cells, EhCP4 appeared in acidic vesicles and was released extracellularly. A specific vinyl sulfone inhibitor, WRR605, synthesized based on the substrate specificity of EhCP4, inhibited the recombinant enzyme in vitro and significantly reduced parasite burden and inflammation in the mouse cecal model. The unique expression pattern, localization, and biochemical properties of EhCP4 could be exploited as a potential target for drug design.
The enteric protozoan parasite Entamoeba histolytica is the cause of potentially fatal amebic colitis and liver abscesses. E. histolytica trophozoites colonize the colon, where they induce inflammation, penetrate the mucosa, and disrupt the host immune system. The early establishment of E. histolytica in the colon occurs in the presence of antimicrobial human (LL-37) and murine (CRAMP [cathelin-related antimicrobial peptide]) cathelicidins, essential components of the mammalian innate defense system in the intestine. Studying this early step in the pathogenesis of amebic colitis, we demonstrate that E. histolytica trophozoites or their released proteinases, including cysteine proteinase 1 (EhCP1), induce intestinal cathelicidins in human intestinal epithelial cell lines and in a mouse model of amebic colitis. Despite induction, E. histolytica trophozoites were found to be resistant to killing by these antimicrobial peptides, and LL-37 and CRAMP were rapidly cleaved by released amebic cysteine proteases. The cathelicidin fragments however, did maintain their antimicrobial activity against bacteria. Degradation of intestinal cathelicidins is a novel function of E. histolytica cysteine proteinases in the evasion of the innate immune system in the bowel. Thus, early intestinal epithelial colonization of invasive trophozoites involves a complex interplay in which the ultimate outcome of infection depends in part on the balance between degradation of cathelicidins by amebic released cysteine proteinases and upregulation of proinflammatory mediators which trigger the inflammatory response.T he organism Entamoeba histolytica is a protozoan parasite that causes amebic colitis and liver abscesses through water-and food-borne infection. Approximately 10% of the world's population is infected with Entamoeba, and it is a major cause of death from parasitic infections (29). Colonization of the intestinal tract by E. histolytica follows binding of the amebic surface Gal/GalNAc adherence lectin to epithelial mucin oligosaccharides, with subsequent degradation of the mucin polymer network, extracellular matrix proteins, and components of the innate host defense by released cysteine proteinases (17,20,21,27,28). This early establishment of E. histolytica triggers an inflammatory response, which plays a role in the ultimate outcome of infection (4, 13).Cathelicidins are small cationic antimicrobial peptides of the mammalian innate immune system with broad activity against bacteria (6,10,11,15) and protozoa (7,9,19). LL-37 is the only cathelicidin described in humans (8) and CRAMP (cathelinrelated antimicrobial peptide) is the cathelicidin found in mice (6). Both LL-37 and CRAMP have related structure, function, and distribution in epithelial cells, including the intestine of humans and mice, and are part of the defense against microbial epithelial infections (32). For example, expression of LL-37 mRNA and protein was increased by Helicobacter pylori in gastric epithelial cells (11), and CRAMP protected mice from colonic colonizati...
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