In order for the protozoan parasite Entamoeba histolytica (E.h.) to cause invasive intestinal and extraintestinal infection, which leads to significant morbidity and mortality, it must disrupt the protective mucus layer by a previously unknown mechanism. We hypothesized that cysteine proteases secreted from the amoeba disrupt the mucin polymeric network, thereby overcoming the protective mucus barrier. The MUC2 mucin is the major structural component of the colonic mucus gel. Heavily O-glycosylated and protease-resistant mucin domains characterize gel-forming mucins. Their N-and C-terminal cysteine-rich domains are involved in mucin polymerization, and these domains are likely to be targeted by proteases because they are less glycosylated, thereby exposing their peptide chains. By treating recombinant cysteine-rich domains of MUC2 with proteases from E.h. trophozoites, we showed that the C-terminal domain was specifically targeted at two sites by cysteine proteases, whereas the N-terminal domain was resistant to proteolysis. The major cleavage site is predicted to depolymerize the MUC2 polymers, thereby disrupting the protective mucus gel. The ability of the cysteine proteases to dissolve mucus gels was confirmed by treating mucins from a MUC2-producing cell line with amoeba proteases. These findings suggest a major role for E.h. cysteine proteases in overcoming the protective mucus barrier in the pathogenesis of invasive amoebiasis. In this report, we identify a specific cleavage mechanism used by an enteric pathogen to disrupt the polymeric nature of the mucin gel.colon ͉ parasite
The adherent mucous gel layer lining the colonic epithelium is the first line of host defense against invasive pathogens, such as Entamoeba histolytica. The mucous layer prevents the attachment of amoeba to the colonic epithelium by trapping and aiding in the expulsion of the parasite. Disruption of the mucous layer is thought to occur in invasive amebiasis, and the mechanism by which the parasite overcomes this barrier is not known. The aim of this study was to characterize the specific interactions occurring between E. histolytica secreted cysteine proteinases and colonic mucin as a model to examine the initial events of invasive amebiasis. E. histolytica secreted products were examined for mucinase activity utilizing mucin metabolically labeled with [ 35 S]cysteine as a substrate. Cysteine proteinases degraded mucin in a time-and dose-dependent manner. A significant reduction (>50%) in high-molecular-weight mucin with altered buoyant density was observed when degraded mucin was analyzed by Sepharose 4B column chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, and CsCl density gradient centrifugation. Mucinase activity was eliminated by the specific cysteine protease inhibitor trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane and was independent of glycosidase activity. Moreover, the degraded mucin was 38% less effective than native mucin at inhibiting amebic adherence to target epithelial cells. These results are the first to show that E. histolytica cysteine proteinases alter the protective function of the mucous barrier by disrupting the structure of the MUC2 polymer. Mechanistically, the parasite achieves this via proteolytic degradation of the terminal cysteine-rich domains.The enteric protozoan parasite Entamoeba histolytica is the causative agent of human amebiasis. Infection with this parasite may result in amebic colitis and liver abscess formation, causing significant morbidity and mortality. More than 500 million people are infected with the parasite worldwide, resulting in an estimated 50 million cases of diarrhea and 100,000 deaths per year (36,38). Although less than 1% of E. histolytica infections result in invasive disease, amebiasis ranks second only to malaria as a cause of mortality due to a protozoan parasite.There are three separate and distinct phases in the pathogenesis of intestinal amebiasis: (i) colonization, (ii) mucous disruption and/or depletion, and (iii) binding to and cytolysis of host colonic epithelial cells. Histopathology studies in the gerbil model of invasive amebiasis suggest that amoeba first colonize the mucous layer by adherence via the parasite surface Gal-lectin to galactose (Gal) and N-acetyl-D-galactosamine (GalNAc) residues present on colonic mucin (9). Following colonization, the parasite causes a disruption and/or dissolution of the mucous layer to gain access to the underlying epithelium. This phenomenon may be a result of the concerted actions of a battery of cysteine proteinases (CPs) released by the parasite in...
Degradation of the mucus layer by Entamoeba histolytica is a prerequisite for invasion of the colonic mucosa. In this study, we demonstrate that amoeba-secreted products degrade 3 H-labeled and native colonic mucin oligosaccharides independently of proteolytic activity. We conclude that E. histolytica degrades mucin oligosaccharides, which may facilitate parasite invasion of the colon.Entamoeba histolytica is responsible for at least 50 million cases of diarrhea and an estimated 100,000 deaths per annum and ranks second only to malaria as a cause of mortality due to a protozoan parasite (12). Infection with the parasite leads to amebic colitis, colonic ulceration, and less frequently, dissemination to the liver, resulting in amebic liver abscess. The initial events leading to invasion of the colon by E. histolytica are poorly understood, and the mechanisms used by the parasite to overcome the innate host defenses of the gastrointestinal tract are currently under investigation. The parasite colonizes the colonic mucus layer by binding mucin oligosaccharides via a 170-kDa Gal/GalNAc lectin and must traverse this protective barrier in order to cause epithelial cell damage and colonic ulceration. Mucin oligosaccharides serve to protect the mucin core from proteases, preserving the integrity of the mucin polymer. Various O-linked glycan structures are attached to the apomucin via O-glycosidic linkage to serine and threonine residues, and these O-glycan branches contain N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), fucose, galactose, and sialic acid. The oligosaccharide component of gastrointestinal mucin has been reported to account for up to 90% of its dry weight, and the densely packed oligosaccharides are responsible for many intrinsic physical properties of the mucus gel, including hydration, gel-forming capacity, protease resistance, and rigidity (3). Previous studies have identified numerous glycosidase activities in E. histolytica lysates and secretory products. More specifically, the parasite has been found to produce a sialidase, an ␣-glucosidase, and -N-acetylhexosaminidase, enzymes which are released by the parasite and are hypothesized to be involved in amebic pathogenesis (7,9,14). These glycosidases may play a role in disrupting mucin by exposing the protein backbone to parasite proteases. Previously, we have shown that E. histolytica-secreted cysteine proteases degrade the poorly glycosylated regions of MUC2, and we hypothesize that the parasite may use the concerted actions of glycosidases and proteases to disassemble the mucin polymeric network (5).In the present study, we determined whether E. histolyticasecreted glycosidases could degrade colonic mucin oligosaccharides. Parasite secretory products were collected from trophozoites incubated in Hanks' balanced salt solution for 2 h, and Ͼ95% of trophozoites were viable as determined by trypan blue exclusion assay (13). Secreted products were assayed for activity against a panel of glycosidase substrates as previously described with s...
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