The Intestinal Protozoan ParasiteEntamoeba histolyticaContains 20 Cysteine Protease Genes, of Which Only a Small Subset Is Expressed during In Vitro Cultivation

Abstract

Cysteine proteases are known to be important pathogenicity factors of the protozoan parasite Entamoeba histolytica. So far, a total of eight genes coding for cysteine proteases have been identified in E. histolytica, two of which are absent in the closely related nonpathogenic species E. dispar. However, present knowledge is restricted to enzymes expressed during in vitro cultivation of the parasite, which might represent only a subset of the entire repertoire. Taking advantage of the current E. histolytica genome-sequencing efforts, we analyzed databases containing more than 99% of all ameba gene sequences for the presence of cysteine protease genes. A total of 20 full-length genes was identified (including all eight genes previously reported), which show 10 to 86% sequence identity. The various genes obviously originated from two separate ancestors since they form two distinct clades. Despite cathepsin B-like substrate specificities, all of the ameba polypeptides are structurally related to cathepsin L-like enzymes. None of the previously described enzymes but 7 of the 12 newly identified proteins are unique compared to cathepsins of higher eukaryotes in that they are predicted to have transmembrane or glycosylphosphatidylinositol anchor attachment domains. Southern blot analysis revealed that orthologous sequences for all of the newly identified proteases are present in E. dispar. Interestingly, the majority of the various cysteine protease genes are not expressed in E. histolytica or E. dispar trophozoites during in vitro cultivation. Therefore, it is likely that at least some of these enzymes are required for infection of the human host and/or for completion of the parasite life cycle.Cysteine proteases (EC 3.4.22) of the papain superfamily occur in a wide range of organisms including bacteria, plants, invertebrates, and vertebrates (7). In mammals, they are well documented as intracellular enzymes involved in protein turnover within lysosomes. In addition, extracellular cysteine proteases have been implicated in various physiological and pathophysiological processes, including tumor invasion and metastasis (33,43). Cysteine proteases are important virulence factors of various infectious agents and the main proteolytic enzymes in many protozoon parasites (22,29). The protozoan Entamoeba histolytica, the causative agent of human amoebiasis, is characterized by its great capacity to destroy host tissue, leading to potentially life-threatening diseases such as ulcerative colitis or liver abscess. Convincing evidence exists that cysteine proteases are essential for E. histolytica-induced pathology. Treatment of the ameba with sublethal doses of a specific cysteine protease inhibitor or the addition of laminin, which blocks the substrate-binding pocket of cysteine proteases, greatly reduces its ability to produce liver abscesses in laboratory animals (18, 37). Likewise, liver abscess formation was totally blocked and significantly less gut inflammation and damage to the intestinal permeability barrier were obser...

Recent Developments in Blastocystis Research

“…The genome is predicted to encode several hydrolases that might be involved in attacking host tissue (Denoeud et al, 2011), although tissue invasion by Blastocystis has never been observed in humans. The most likely possible effectors found in the Blastocystis genome are cysteine proteases, considering that these genes are also present in large numbers in the pathogenic protist Entamoeba histolytica (Bruchhaus et al, 2003). Nonetheless, these predictions all need further elucidation in the laboratory in order to determine whether they have any role in pathogenesis and disease and to prove that they are secreted.…”

Trophozoites ofEntamoeba histolyticaepigenetically silenced in several genes are virulence-attenuated

“…The CP-deficient trophozoites also failed to induce the intestinal epithelial cell production of the inflammatory cytokines IL-1β and IL-8 and caused significantly less intestinal inflammation and tissue damage (Zhang et al, 2000). More than twenty full-length CP genes have been identified to date in the E. histolytica genome but only about half of them have been found to be expressed in trophozoites cultured under axenic conditions (Bruchhaus et al, 2003;Tillack et al, 2006Tillack et al, , 2007. Four of the CPs (EhCP-1, EhCP-2, EhCP-3 and EhCP-5) have been purified and found to account for approximately 90 % of the total CP activity present in lysates of axenically cultured trophozoites (Scholze & Tannich, 1994;Bruchhaus et al, 1996Bruchhaus et al, , 2003Jacobs et al, 1998).…”

“…More than twenty full-length CP genes have been identified to date in the E. histolytica genome but only about half of them have been found to be expressed in trophozoites cultured under axenic conditions (Bruchhaus et al, 2003;Tillack et al, 2006Tillack et al, , 2007. Four of the CPs (EhCP-1, EhCP-2, EhCP-3 and EhCP-5) have been purified and found to account for approximately 90 % of the total CP activity present in lysates of axenically cultured trophozoites (Scholze & Tannich, 1994;Bruchhaus et al, 1996Bruchhaus et al, , 2003Jacobs et al, 1998). EhCP-5 which has significant homology to cathepsin L appears to be of special importance for pathogenicity because it is the only CP that was found to be present on the surface of the trophozoite (Jacobs et al, 1998).…”

The Entamoeba histolytica genome: something old, something new, something borrowed and sex too?

“…Redundancy also characterizes many of the genes encoding suspected or proven E. histolytica virulence factors. New cysteine proteinase genes were identified (there are now at least 20), some of which contain putative transmembrane domains -which are lacking in the cathepsins of higher eukaryotes [2,3]. Three new amebapore genes were identified (bringing the total to six) and 30 homologs of a gene encoding the intermediate subunit of an E. histolytica surface lectin were identified.…”

“…The issue of gene expression has been partially explored for the cysteine proteinase genes, in which it was observed that trophozoites in culture seem to express only eight of the 20 identified cysteine proteinase genes, with only six of the eight being expressed at significant levels [3]. It remains to be determined whether similar ratios exist for the other large gene families identified by the genome project.…”