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Isolation and molecular characterization of a surface-bound proteinase of Entamoeba histolytica
Abstract
Major pathogenic functions of Entamoeba histolytica involved in destruction of host tissues are the degradation of extracellular matrix proteins mediated by secreted cysteine proteinases and contact-dependent killing of host cells via membrane-active factors. A soluble protein with an affinity for membranes was purified from amoebic extracts to apparent homogeneity. N-terminal sequencing and subsequent molecular cloning of the factor revealed that it is a member of the cysteine proteinase family of E. histolytica, which we termed CP5. Further experiments with the purified protein showed that it has potent proteolytic activity that is abrogated in the presence of inhibitors specific for cysteine proteinases. The enzyme firmly associates with membranes retaining its proteolytic activity and it produces cytopathic effects on cultured monolayers. A model of the three-dimensional structure of CP5 revealed the presence of a hydrophobic patch that may account for the potential of the protein to associate with membranes. Immunocytochemical localization of the enzyme to the surface of the amoeba in combination with the recent finding that the gene encoding CP5 is missing in the closely related but non-pathogenic Entamoeba dispar suggests a potential role of the protein in host tissue destruction of E. histolytica.
... ehcp1 (acp3) is a second gene reported to be unique to E. histolytica (12), but sequences were detected in clinical strains of E. dispar (84). The third gene reported to be present only in E. histolytica is ehcp5, which encodes a surface-associated cysteine proteinase (43). Comparison of the deduced peptide sequences of the ehcp1 to ehcp6 genes indicated that the primary structures of the six ameba enzymes are similar and have sequence similarities of between 43 and 87% (11). ...
... The prodomain of eukaryotic cathepsins has two well-defined functions: (i) to maintain the enzyme in an inactive form (zy-mogen) until it reaches an appropriate site of protease function and (ii) to act as a structural template to ensure proper folding during translation (21). EhCP5 contains an Asn-X-(Ser/Thr) recognition sequences within the prosequence, which may be posttranslationally modified by glycosylation (43). The mature regions are homologous to cathepsin L-like cysteine proteinases and contain all conserved cysteine residues implicated in the maintenance of the three-dimensional structure. ...
... True lysosomes have not been identified in Entamoeba, although numerous acidic vesicles are present in the cytoplasm (78). Only one amebic cysteine proteinase, EhCP5, has a potential asparagine-linked glycosylation site, but it is not known if mannose-6-phosphate is present in the mature enzyme (43). Therefore, the mannose-6-phosphate-dependent pathway cannot be the major mechanism to target amebic cysteine proteinases. ...
Amebiasis is a major cause of morbidity and mortality throughout the tropical world. Entamoeba histolytica is now recognized as a separate species from the morphologically identical E. dispar, which cannot invade. Cysteine proteinases are a key virulence factor of E. histolytica and play a role in intestinal invasion by degrading the extracellular matrix and circumventing the host immune response through cleavage of secretory immunoglobulin A (sIgA), IgG, and activation of complement. Cysteine proteinases are encoded by at least seven genes, several of which are found in E. histolytica but not E. dispar. A number of new animal models, including the formation of liver abscesses in SCID mice and intestinal infection in human intestinal xenografts, have proven useful to confirm the critical role of cysteine proteinases in invasion. Detailed structural analysis of cysteine proteinases should provide further insights into their biochemical function and may facilitate the design of specific inhibitors which could be used as potential chemotherapeutic agents in the future.
... Although the corresponding proteins differ slightly in their molecular weight and enzymatic properties, it was postulated that all these are related [28]. Subsequent molecular cloning revealed a large number of cysteine peptidase genes in the E. histolytica genome [29][30][31][32][33][34][35]. Until 2003, in total 20 genes coding for cysteine peptidases of E. histolytica were described, all of which belonged to the C1 papain superfamily of cysteine peptidases [30]. ...
... They have a cathepsin L-like structure that is indicated by an ERFNIN motif in the pro-region of nearly all EhCP-A/-B enzymes [48]. Nevertheless, biochemical studies with purified EhCP-A1, -A2, and -A5 indicated a cathepsin B-like substrate specificity with a preference for arginine in the P2 position [11,27,31], likely the result of substitution of an alanine residue by acidic or charged amino acids in the postulated S2 pocket, corresponding to residue 209 of the papain sequence [48]. In contrast, EhCP-A4 has a preference for valine and isoleucine at P2, presumably the result of the hydrophobic nature of the S-pocket with valine at the base [49]. ...
... In addition, a punctate cytoplasmic localization has been observed and during phagocytosis a translocation of the cysteine peptidases to phagocytotic vesicles occurs [56,57]. Furthermore, cysteine peptidases have been found to be membrane associated [31,32,56,58] and -secreted [16,52,59,60]. Recently, an individual cysteine peptidase (EhCP-A4) was localized not only in cytoplasmic vesicles and was released extracellularly but localized also in the nuclear I. Bruchhaus and J. Matthiesen . ...
Entamoeba histolytica is characterized by its extraordinary capacity to invade and destroy human tissues. The main lytic activity has been attributed to cysteine peptidases, and a number of studies have shown that cysteine peptidases constitute major pathogenicity factors in E. histolytica. Interestingly, although most of the classes of peptidases are present in E. histolytica, only cysteine peptidases, and on a lesser scale, metallo-peptidases and serine peptidases, have been adequately studied. In this chapter, the peptidase families of E. histolytica are introduced, and their involvement in colonic invasion and in liver abscess formation are discussed.
... Von diesen ehcps gehören 47 in den Peptidase-Clan CA und drei zu dem Clan CE (siehe Tab.1). (Jacobs et al., 1998). Beiden Unterfamilien gemeinsam ist ein charakteristisches Motiv unbekannter Funktion in der Proregion, das die folgende konservierte Sequenz aufweist: ...
... Die Proteine EhCP-A1, EhCPA-2 und EhCP-A5 sind bereits gereinigt und näher charakterisiert worden (Jacobs et al., 1998;Luaces und Barrett, 1988;Scholze und Schulte, 1988). Alle drei Proteine sind innerhalb der Amöben in Vesikeln lokalisiert (Jacobs et al., 1998;Okada et al., 2005;Que et al., 2002), wobei EhCP-A5 zusätzlich eine Oberflächenlokalisation zugeschrieben wird, die auf einen hydrophoben N-terminalen Bereich des Protein zurückzuführen sein könnte (Jacobs et al., 1998). ...
... Die Proteine EhCP-A1, EhCPA-2 und EhCP-A5 sind bereits gereinigt und näher charakterisiert worden (Jacobs et al., 1998;Luaces und Barrett, 1988;Scholze und Schulte, 1988). Alle drei Proteine sind innerhalb der Amöben in Vesikeln lokalisiert (Jacobs et al., 1998;Okada et al., 2005;Que et al., 2002), wobei EhCP-A5 zusätzlich eine Oberflächenlokalisation zugeschrieben wird, die auf einen hydrophoben N-terminalen Bereich des Protein zurückzuführen sein könnte (Jacobs et al., 1998). Unter ...
... Sequence alignment and computer modeling confirmed that EhCP4 is a member of Clan CA, C1A subfamily with a cathepsin L-like structure [73,75,87], similar to previously characterized EhCPs. However, unlike previously characterized EhCPs, which have a preference for cathepsin B or L substrates with arginine in the P2 position, EhCP4 displays a unique preference for valine and isoleucine at P2 [14]. ...
... Immunocytochemical localization of the enzyme at the amoeba surface suggests a potential role in tissue destruction. The gene encoding EhCP5 is not expressed in the closely related but nonpathogenic species E. dispar [87,92]. Trophozoites of the virulent strain transfected with an antisense gene encoding EhCP5 had only 10% activity but retained their cytopathic effect on mammalian cell monolayers and were incapable of inducing the formation of liver lesions in hamsters. ...
... EhCP4 was recently shown to degrade IgA in vitro [14]; however, it is unclear if it is the same protease or if E. histolytica has more than one IgA-degrading protease. In fact, Garcia-Nieto identified this activity as a surface protease, and EhCP5, unlike EhCP4, is found in the amoeba plasma membrane [87], leading the authors to suggest that this enzyme could be the main protease involved in sIgA degradation [156]. ...
... The calculated Mr of the mature enzyme was 23 kDa, but the apparent Mr was 26 kDa, as assessed by SDS-PAGE [14]. Sequence alignment and computer modeling confirmed that EhCP4 is a member of Clan CA, C1A subfamily with a cathepsin L-like structure [73, 75, 87], similar to previously characterized EhCPs. However, unlike previously characterized EhCPs, which have a preference for cathepsin B or L substrates with arginine in the P2 position, EhCP4 displays a unique preference for valine and isoleucine at P2 [14]. ...
... Immunocytochemical localization of the enzyme at the amoeba surface suggests a potential role in tissue destruction. The gene encoding EhCP5 is not expressed in the closely related but nonpathogenic species E. dispar [87, 92]. Trophozoites of the virulent strain transfected with an antisense gene encoding EhCP5 had only 10% activity but retained their cytopathic effect on mammalian cell monolayers and were incapable of inducing the formation of liver lesions in hamsters. ...
... EhCP4 was recently shown to degrade IgA in vitro [14]; however, it is unclear if it is the same protease or if E. histolytica has more than one IgA-degrading protease. In fact, Garcia-Nieto identified this activity as a surface protease, and EhCP5, unlike EhCP4, is found in the amoeba plasma membrane [87], leading the authors to suggest that this enzyme could be the main protease involved in sIgA degradation [156]. Specific antiamoeba IgG responses are developed in >95% of patients with amoebiasis or even in individuals with E. histolytica asymptomatic colonization [164]. ...
The standard reference for pathogenic and nonpathogenic amoebae is the human parasite ; a direct correlation between virulence and protease expression has been demonstrated for this amoeba. Traditionally, proteases are considered virulence factors, including those that produce cytopathic effects in the host or that have been implicated in manipulating the immune response. Here, we expand the scope to other amoebae, including less-pathogenic species and highly pathogenic free-living amoebae. In this paper, proteases that affect mucin, extracellular matrix, immune system components, and diverse tissues and cells are included, based on studies in amoebic cultures and animal models. We also include proteases used by amoebae to degrade iron-containing proteins because iron scavenger capacity is currently considered a virulence factor for pathogens. In addition, proteases that have a role in adhesion and encystation, which are essential for establishing and transmitting infection, are discussed. The study of proteases and their specific inhibitors is relevant to the search for new therapeutic targets and to increase the power of drugs used to treat the diseases caused by these complex microorganisms.
... Cysteine proteases predominate in the trophozoites. Six cysteine proteases have been described: a 16-kDa cathepsin-B-like molecule [11], a 56-kDa neutral cysteine protease [12], a 22/27-kDa protease named amoebapain [13], a 26/29-kDa histolysain [14], a novel 30-kDa cysteine protease (CP 5 ) [15] and a cysteine protease forming part of the 112-kDa adhesin (EhCP112) [6]. ...
... It displays better affinity for the dipeptyl sequence RR (Arg-Arg) than RF (Arg-Phe), at present without more biochemical and structural data (complete gene sequence) it is not possible to conclude about the subfamily. This protease shares two common characteristics with other E. histolytica proteases described to date, namely the 16-kDa cathepsin-B-like protease, the 26/29 histolysain, the 22/27-kDa amoebapain, the 30-kDa CP5 and the 56-kDa neutral cysteine protease [11,12,14,15,36]. All are able to degrade peptides with arginine at the P 1 and P 2 position on fluorogenic substrate ZRR-AMC. ...
... The second difference concerns the translocation of EhCP60 from the internal vacuoles to the plasma membrane in the presence of target cells. Previous reports provide evidence for the existence of cysteine proteases on the surface of the trophozoites in the absence of a target cell [13,15,43,44]. Ostoa-Saloma et al. [43] showed that in amebic lysates fractionated according to Aley et al. [29] the intermediate (45,36 kDa) or low (30, 26, 23 kDa) molecular mass proteases were localized on the plasma membrane [29,43]. ...
Cysteine proteases are important virulence factors of Entamoeba histolytica, the causative agent of amoebiasis. A novel cysteine protease from parasite extracts was purified 15-fold by a procedure including concanavalin A–Sepharose, hydroxylapatite and DEAE–Sepharose chromatography. The purification resulted in the obtainment of an homogeneous protein with a molecular mass of 66 kDa on native PAGE. In 10% SDS/PAGE, three bands of 60, 54 and 50 kDa were evident. Each of the three specific mouse antisera raised against these proteins showed cross-reactivity with the three bands obtained from the purified eluate. The N-terminal sequencing of the first 10 amino acids from the three proteins showed 100% identity. These results support the hypothesis of a common precursor for the 60, 54 and 50-kDa proteins. Protease activity of the purified enzyme was demonstrated by electrophoresis in a gelatine-acrylamide copolymerized gel. Its activity was quantified by cleaving a synthetic fluorogenic peptide substrate such as N-carbobenzyloxy-arginyl-arginyl-7-amido-4-methylcoumarin. The optimum pH for the protease activity was 6.5; however, enzymatic activity was observed between pH 5 and pH 7.5. Typical of cysteine proteases, the enzyme was inhibited by 4-[(2S,3S)-carboxyoxiran-2-ylcarbonyl-l-leucylamido]butylguanidine and iodoacetamide, and activated by free sulfhydryl groups. The cellular location of the enzyme was examined on trophozoites before and after contact with red blood cells using indirect immunofluorescence and cellular fractionation. The 60-kDa cysteine protease translocated to the amoebic surface upon the interaction of trophozoites with red blood cells. This result provided evidence for participation of the 60-kDa protease in erythrophagocytosis.
... Other terms have been used to describe enzymes closely related or identical to this enzyme such as Cathepsin B [43], Neutral proteinase [44], Histolysin [45] (later changed to Histolysain [46]) and Amoebapain [47]. Cysteine endopeptidase enzymes secreted by E. histolytica [48] were found localized in lysosome-like vesicles or on the cell surface [49,50]. Molecular cloning revealed a large number of Cysteine endopeptidase genes in the genome of E. histolytica [49,51]. ...
... Cysteine endopeptidase enzymes secreted by E. histolytica [48] were found localized in lysosome-like vesicles or on the cell surface [49,50]. Molecular cloning revealed a large number of Cysteine endopeptidase genes in the genome of E. histolytica [49,51]. Interestingly, most of these genes are not expressed during culture of E. histolyticaIn vitro [51]. ...
The study in this review aims to know and study the parasite Entamoebahistolytica that causes Amoebiasis worldwide. During its life cycle, E. histolytica passes through several phases, trophozoite stage, precyst stage, the cyst stage, the Metacyst stage, and Metacystictrophozoite stage. In this study, the spread of the parasite globally and in the Arab world and methods of treating the disease were also discussed. The Genomic structureof E. histolytica, like other organisms, is characterized by diversity and heterogeneity in its genetic content, which is one of the most important reasons for its survival and its ability to infect. Interestingly, the genome of the E. histolytica contains a large amount of genes presumed to be of bacterial origin. The study of the genetic diversity of E. histolytica gives paths to the developmental change that resulted in the emergence of evolutionary features or traits. Understanding amoebic virulence is important. Several studies have shown that genetic factors influence the virulence of parasitic infections. Studying the virulence of E. histolytica by Serine-rich protein (REHP) gene is among the important issues used in molecular epidemiological studies.
... Cysteine proteases (CP) play a key role in the pathogenesis of E. histolytica [134]; among them, cysteine protease A5 (CP-A5, EHI_168240) is present only in E. histolytica [135]; it is an important amoebic factor for mucus depletion because it targets peptides in glycosylated poor regions of MUC2 [135,136]. This enzyme localizes to the surface of the trophozoite [137], contains an Arg-Gly-Asp (RGD) integrinbinding motif that has also been described in higher eukaryotic cathepsin X [138]. CP-A5 binds via its RGD motif to the α V β 3 integrin on enterocyte-like cells and stimulates inflammatory responses [139]. ...
The amoeba parasite Entamoeba histolytica is the causative agent of human amebiasis, an enteropathic disease affecting millions of people worldwide. This ancient protozoan is an elementary example of how parasites evolve with humans, e.g. taking advantage of multiple mechanisms to evade immune responses, interacting with microbiota for nutritional and protective needs, utilizing host resources for growth, division, and encystation. These skills of E. histolytica perpetuate the species and incidence of infection. However, in 10% of infected cases, the parasite turns into a pathogen; the host-parasite equilibrium is then disorganized, and the simple lifecycle based on two cell forms, trophozoites and cysts, becomes unbalanced. Trophozoites acquire a virulent phenotype which, when non-controlled, leads to intestinal invasion with the onset of amoebiasis symptoms. Virulent E. histolytica must cross mucus, epithelium, connective tissue and possibly blood. This highly mobile parasite faces various stresses and a powerful host immune response, with oxidative stress being a challenge for its survival. New emerging research avenues and omics technologies target gene regulation to determine human or parasitic factors activated upon infection, their role in virulence activation, and in pathogenesis; this research bears in mind that E. histolytica is a resident of the complex intestinal ecosystem. The goal is to eradicate amoebiasis from the planet, but the parasitic life of E. histolytica is ancient and complex and will likely continue to evolve with humans. Advances in these topics are summarized here.
... The no pathogenicity of E. dispar can be explained by multiple characteristics: poor adhesion to red blood cells leading to reduced haemolytic activity, low expression of phosphatidylserine (involved in the process of phagocytosis) [11], and the absence of a CP5-type protease [12,13]. The latter enzyme participates in the E. histolytica induced formation of amoebic liver abscesses [14]. ...
Entamoeba histolytica is the causative agent of amoebiasis, and Entamoeba dispar is its noninvasive morphological twin. Entamoeba invadens is a reptilian parasite. In the present study, Western blot, phosphatase activity, immunofluorescence, and bioinformatic analyses were used to identify PP2C phosphatases of E. histolytica, E. dispar, and E. invadens. PP2C was identified in trophozoites of all Entamoeba species and cysts of E. invadens. Immunoblotting using a Leishmania mexicana anti-PP2C antibody recognized a 45.2 kDa PP2C in all species. In E. histolytica and E. invadens, a high molecular weight element PP2C at 75 kDa was recognized, mainly in cysts of E. invadens. Immunofluorescence demonstrated the presence of PP2C in membrane and vesicular structures in the cytosol of all species analyzed. The ~75 kDa PP2C of Entamoeba spp. shows the conserved domain characteristic of phosphatase enzymes (according to in silico analysis). Possible PP2C participation in the encystation process was discussed.
... We have previously shown that surface EhCP-A5 degrades MUC2 mucin and binds macrophage integrins to activate inflammasome. 2,5,25 To determine the role of EhCP-A5, macrophages were stimulated with EhCP-A5 deficient parasites and similar ATG16L1 protein degradation was observed as wild type Eh, whereas E-64 treated Eh completely inhibited degradation (Fig. 2e). We have recently shown that following Ehmacrophage interaction, EhCP-A1 and EhCP-A4 are recruited to the intercellular junction that facilitated the degradation of cytoskeletal-associated proteins. ...
The mechanism whereby Entamoeba histolytica (Eh) binding with macrophages at the intercellular junction triggers aggressive pro-inflammatory responses in disease pathogenesis is not well understood. The host intracellular protein degradation process autophagy and its regulatory proteins are involved in maintenance of cellular homeostasis and excessive inflammatory responses. In this study we unraveled how Eh hijacks the autophagy process in macrophages to dysregulate pro-inflammatory responses. Direct contact of live Eh with macrophages activated caspase-6 that induced rapid proteolytic degradation of the autophagy ATG16L1 protein complex independent of NLRP3 inflammasome and caspase-3/8 activation. Crohn’s disease susceptible ATG16L1 T300A variant was highly susceptible to Eh-mediated degradation that augmented pro-inflammatory cytokines in mice. Quantitative proteomics revealed downregulation of autophagy and vesicle-mediated transport and upregulation of cysteine-type endopeptidase pathways in response to Eh. We conclude during Eh-macrophage outside-in signaling, ATG16L1 protein complex plays an overlooked regulatory role in shaping the pro-inflammatory landscape in amebiasis.
... Il lui permet en effet de bien cibler l'extrémité C-terminale peu glycosylée des cellules caliciformes de l'hôte en vue de les lier puis les dégrader en présence d'un mucus abondant [52,53]. Dans certaines études in vivo, les souris infectées, après qu'elles aient été inhibées dans l'expression du gène codant pour ces enzymes, ont diminué leur capacité à produire des abcès hépatiques [54,55] [54][55][56][57][58][59]. Il a été démontré que la cystéine protéinase codé par ACP5 d'E. ...
Since the discovery of Entamoeba histolytica (E. histolytica), many concepts emerged on the relationship between protozoa and intestinal amoebosis. Existence of a significant proportion of asymptomatic subjects with E. histolytica were highlighted in many studies. Thus, three theories are evoked. The first one suggested E. histolytica is commensal of human bowel. The second considered this protozoan as binding parasite which symptoms only arise when certain host factors are present. Latter theory, suggested existence of two different species: one pathogenic, E. histolytica, and the other non-pathogenic, E. dispar. It took several decades to validate that theory. Moreover, a probable implication of E. dispar and E. moshkovskii in the onset of symptoms has been reported by some authors. In vivo and in vitro studies suggested microbiome involvement in the onset of invasive infection by E. histolytica in addition to other factors of parasite virulence. Other studies suggest complex interactions between immunity, nutrition and the intestinal host flora and parasite genotype for discriminative determination of symptoms in infected individuals. This makes it necessary to review what has been known hitherto about the relationship between the parasite and its host.
... Il lui permet en effet de bien cibler l'extrémité C-terminale peu glycosylée des cellules caliciformes de l'hôte en vue de les lier puis les dégrader en présence d'un mucus abondant [52,53]. Dans certaines études in vivo, les souris infectées, après qu'elles aient été inhibées dans l'expression du gène codant pour ces enzymes, ont diminué leur capacité à produire des abcès hépatiques [54,55] [54][55][56][57][58][59]. Il a été démontré que la cystéine protéinase codé par ACP5 d'E. ...
... Entamoeba histolytica harbors more than 50 cysteine proteases in sharp contrast to Trypanosoma cruzi in which cruzipain is its main cysteine protease [2,3,[40][41][42]. Seminal work by the Nozaki group demonstrated that EhICP1 and EhICP2 present similar inhibitory profiles towards EhCP1 and EhCP2, whereas EhICP1 exhibits a 12-fold less inhibitory constant towards EhCP5 than EhICP2 [5]. ...
The genome of Entamoeba histolytica encodes approximately 50 Cysteine Proteases (CPs) whose activity is regulated by two Inhibitors of Cysteine Proteases (ICPs), EhICP1 and EhICP2. The main difference between both EhICPs is the acquisition of a 17 N-terminal targeting signal in EhICP2 and three exposed cysteine residues in EhICP1. The three exposed cysteines in EhICP1 potentiate the formation of cross-linking species that drive heterogeneity. Here we solved the NMR structure of EhICP1 using a mutant protein without accessible cysteines. Our structural data shows that EhICP1 adopts an immunoglobulin fold composed of seven β-strands, and three solvent exposed loops that resemble the structures of EhICP2 and chagasin. EhICP1 and EhICP2 are able to inhibit the archetypical cysteine protease papain by intercalating their BC loops into the protease active site independently of the character of the residue (serine or threonine) responsible to interact with the active site of papain. EhICP1 and EhICP2 present signals of functional divergence as they clustered in different clades. Two of the three exposed cysteines in EhICP1 are located at the DE loop that intercalates into the CP substrate-binding cleft. We propose that the solvent exposed cysteines of EhICP1 play a role in regulating its inhibitory activity and that in oxidative conditions, the cysteines of EhICP1 react to form intra and intermolecular disulfide bonds that render an inactive inhibitor. EhICP2 is not subject to redox regulation, as this inhibitor does not contain a single cysteine residue. This proposed redox regulation may be related to the differential cellular localization between EhICP1 and EhICP2.
... EhCP5 is present on the surface of trophozoites and is also secreted [61]. In ehcp5-silenced trophozoites, mucin degradation is significantly diminished (>60%), compared to wild-type trophozoites. ...
The epithelium represents the first and most extensive line of defence against pathogens, toxins and pollutant agents in humans. In general, pathogens have developed strategies to overcome this barrier and use it as an entrance to the organism. Entamoeba histolytica, Naegleria fowleri and Acanthamoeba spp. are amoebae mainly responsible for intestinal dysentery, meningoencephalitis and keratitis, respectively. These amoebae cause significant morbidity and mortality rates. Thus, the identification, characterization and validation of molecules participating in host-parasite interactions can provide attractive targets to timely intervene disease progress. In this work, we present a compendium of the parasite adhesins, lectins, proteases, hydrolases, kinases, and others, that participate in key pathogenic events. Special focus is made for the analysis of assorted molecules and mechanisms involved in the interaction of the parasites with epithelial surface receptors, changes in epithelial junctional markers, implications on the barrier function, among others. This review allows the assessment of initial host-pathogen interaction, to correlate it to the potential of parasite invasion.
... Although, collectively, the cysteine proteases are regarded as virulence factors, evidence suggests that $90% of E. histolytica's cysteine protease-derived proteolytic activity is provided by just three proteins-EhCP-A1, EhCP-A2, and EhCP-A5 (Stanley et al. 1995;Bruchhaus et al. 1996;Ankri et al. 1999;Mel endez-L opez et al. 2007). EhCP-A5 is of particular interest as no functional ortholog exists in the nonpathogenic E. dispar (Jacobs et al. 1998) and expression of the protein is thought to be necessary for E. histolytica to invade the human intestinal mucosa (Thibeaux et al. 2014). In concert with amoebic glycosidases, an undefined number of cysteine proteases degrade the MUC2 polymers that constitute much of the mucosal layer (Moncada et al. 2003(Moncada et al. , 2005. ...
Amoebiasis is the third-most common cause of mortality worldwide from a parasitic disease. Whilst the primary aetiological agent of amoebiasis is the obligate human parasite Entamoeba histolytica, other members of the genus Entamoeba can infect humans and may be pathogenic. Here, we present the first annotated reference genome for Entamoeba moshkovskii, a species that has been associated with human infections, and compare the genomes of E. moshkovskii, E. histolytica, the human commensal Entamoeba dispar and the non-human pathogen Entamoeba invadens. Gene clustering and phylogenetic analyses show differences in expansion and contraction of families of proteins associated with host or bacterial interactions. They intimate the importance to parasitic Entamoeba species of surface-bound proteins involved in adhesion to extracellular membranes, such as the Gal/GalNAc lectin and members of the BspA and Ariel1 families. Furthermore, E. dispar is the only one of the four species to lack a functional copy of the key virulence factor cysteine protease CP-A5, whilst the gene’s presence in E. moshkovskii is consistent with the species’ potentially pathogenic nature. Entamoeba moshkovskii was found to be more diverse than E. histolytica across all sequence classes. The former is approximately 200 times more diverse than latter, with the 4 E. moshkovskii strains tested having a most recent common ancestor nearly 500 times more ancient than the tested E. histolytica strains. A four-haplotype test indicates that these E. moshkovskii strains are not the same species and should be regarded as a species complex.
... A total of six distinct genes (EhCP1 through EhCP6) encoding prepro forms of cysteine proteinases have been identified. The enzymes encoded by three of these genes have been purified and characterized, namely, EhCP1 (ACP3; previously known as amebapain) (138,156), EhCP2 (ACP2; earlier reported as histolysin) (80,157), and EhCP5, a membrane-bound protease (62). These three enzymes, all with a molecular mass of ϳ30 kDa, account for ϳ90% of cysteine protease transcripts and for virtually all cysteine protease activity found in E. histolytica lysates (24). ...
In spite of a wealth of knowledge on the biochemistry and cellular and molecular biology of Entamoeba histolytica, little has been done to apply these advances to our understanding of the lesions observed in patients with intestinal amebiasis. In this review, the pathological and histological findings in acute amebic colitis are related to the molecular mechanisms of E. histolytica pathogenicity described to date. Infection of the human colon by E. histolytica produces focal ulceration of the intestinal mucosa, resulting in dysentery (diarrhea with blood and mucus). Although a complete picture has not yet been achieved, the basic mechanisms involved in the production of focal lytic lesions include complex multifactorial processes in which lectins facilitate adhesion, proteases degrade extracellular matrix components, porins help nourish the parasite and may also kill incoming polymorphonuclear leukocytes and macrophages, and motility is used by the parasite to invade deeper layers of the colon. In addition, E. histolytica has developed mechanisms to modulate the immune response during acute infection. Nevertheless, much still needs to be unraveled to understand how this microscopic parasite has earned its well-deserved histolytic name.
... 17 EHCP5 is also the most important pathogenicity factor in E. histolytica. 18 To the best of our knowledge, there was no study on the genetic polymorphism of CP5 gene and its sequences in different areas of Iran; therefore, the present study was developed to distinguish E. histolytica from E. dispar by single PCR using specific primer of CP5 gene. It was also studied the polymorphism of CP5 gene on E. histolytica isolated from 2 regions, Tabriz and Bandar Abbas. ...
Background: Amebiasis with up to 100 000 human deaths each year is the third cause of human deadly parasitic disease. With regard to the fact that cysteine protease 5 is known to be one of the most important pathogenicity factors of the Entamoeba histolytica and also, CP5 gene has been observed only in E. histolytica, hence we discriminated E. histolytica from E. dispar on CP5 gene by polymerase chain reaction (PCR) and characterized CP5 gene variation in E. histolytica isolated from patients in both cold regions and tropical regions of Iran at molecular level.
Materials and Methods: In the present study, a total of 2332 stool samples (1550 from Tabriz and 782 from Bandar Abbas) were studied microscopically. DNA extraction and PCR method were performed on the positive specimens, infected with E. histolytica/E. dispar. Finally we characterized CP5 gene in E. histolytica isolates from 10 positive samples in the cold regions (Tabriz) and 10 positive samples in the tropical regions (Bandar Abbas) by sequencing and studied the polymorphism of the gene.
Results: Of 1550 subjects studied from Tabriz and 782 from Bandar Abaas, 83/1550 (8.3%) and 65/782 (5.35%) persons were infected with E. histolytica/E. dispar, respectively. The molecular results on 20 E. histolytica PCR positive isolates from both regions revealed that nucleotides substitution and polymorphism on CP5 gene was more in samples from Bandar Abbas than those from Tabriz.
Conclusion: Prevalence of amebiasis was high in the tropical region (Bandar Abbas) compared with the cold region (Tabriz). In this study, CP5 gene variation in the pathogenicity and virulence of this parasite in the tropical region was higher than that in the cold region.
... Entamoeba histolytica is invasive species and cause symptomatic disease characterized with abdominal pain, cramps, blood diarrhea. Entamoeba dispar is non-invasive species which is useful as a study model for E. histolytica because both species have a lot of identical gens regions and similar immunogenic effect (Willhoeft et al. 1999;Bruchhaus et al. 1996;Jacobs et al. 1998). IBD is a chronic inflammatory condition of the intestines caused by inadequate mucosal immune response to antigenic components. ...
AimTo explore the prevalence of amebiasis in inflammatory bowel disease (IBD), Crohn’s disease and ulcerative colitis, in patients in Clinical hospital Mostar (Bosnia and Herzegovina, region of Herzegovina). Methods
In this study, Entamoeba histolytica/dispar prevalence was investigated in fresh faeces by native microscopy and immunochromatographic rapid assay “RIDA®QUICK Entamoeba test”, in 119 cases of new found IBD patients, 84 of ulcerative colitis and 35 of Crohn’s disease and in control group who had also 119 patients who didn’t have any gastrointestinal complaints. IBD diagnosis was established by standard diagnostic procedures (anamnesis, clinical manifestations, laboratory, endoscopy and biopsy). ResultsEntamoeba histolytica/dispar were found in 19 (16.0 %) of a total of 119 cases, 12 (14.3 %) of the 84 patients with ulcerative colitis and 7 (20.0 %) of the 35 patients with Crohn’s disease. As for the 119 patients in the control group who had not any gastrointestinal complaints, 2 (1.7 %) patients were found to have E. histolytica/dispar in their faeces. Amoeba prevalence in the patient group was determined to be significantly higher in group with Crohn’s disease, ulcerative colitis and IBD total than in the control group (p < 0.001). Conclusion
Ameba infections in patients with Crohn’s disease and ulcerative colitis, have a greater prevalence compared to the normal population.
... Among different CPs, EhCP5 has emerged as one of the major virulence-related CP. EhCP5 is present on the surface of the amoeba and is thought to participate in the disruption of the mucin barrier of the colon [92]. It also interacts with the colonic epithelial cell integrins, thus activating NFkappaβmediated inflammatory response in host cells [93]. ...
Amoebiasis is one of the major public health problems in developing countries. In spite of the availability of an effective drug and absence of overt drug resistance, the disease is still prevalent among large population and spread over a number of countries. It is caused by the protist parasite Entamoeba histolytica that essentially infects humans, though other species that infect a few animals have been reported. A number of molecular techniques have recently been developed. These have helped in understanding biological processes in E. histolytica and in the identification of key molecules that are involved in amoebic virulence and invasion. Moreover, developments in the area of disease and invasion models have allowed understanding of these processes at molecular level and circumvented lack of a good animal model of amoebiasis. All these knowledge will help us to design better therapeutics and allow us to control this important disease.
... These results suggest G. duodenalis trophozoites possess multiple types of cathepsin cysteine proteases, and each may have a different role in disease pathogenesis. Indeed, single-celled parasites can possess both surface-associated and secreted cathepsin cysteine proteases which may have unique roles in pathogenesis [89][90][91][92]. ...
Giardia duodenalis infections are among the most common causes of waterborne diarrhoeal disease worldwide. At the height of infection, G. duodenalis trophozoites induce multiple pathophysiological processes within intestinal epithelial cells that contribute to the development of diarrhoeal disease. To date, our understanding of pathophysiological processes in giardiasis remains incompletely understood. The present study reveals a previously unappreciated role for G. duodenalis cathepsin cysteine proteases in intestinal epithelial pathophysiological processes that occur during giardiasis. Experiments first established that Giardia trophozoites indeed produce cathepsin B and L in strain-dependent fashion. Co-incubation of G. duodenalis with human enterocytes enhanced cathepsin production by Assemblage A (NF and S2 isolates) trophozoites, but not when epithelial cells were exposed to Assemblage B (GSM isolate) trophozoites. Direct contact between G. duodenalis parasites and human intestinal epithelial monolayers resulted in the degradation and redistribution of the intestinal epithelial cytoskeletal protein villin; these effects were abolished when parasite cathepsin cysteine proteases were inhibited. Interestingly, inhibition of parasite proteases did not prevent degradation of the intestinal tight junction-associated protein zonula occludens 1 (ZO-1), suggesting that G. duodenalis induces multiple pathophysiological processes within intestinal epithelial cells. Finally, this study demonstrates that G. duodenalis-mediated disruption of villin is, at least, in part dependent on activation of myosin light chain kinase (MLCK). Taken together, this study indicates a novel role for parasite cathepsin cysteine proteases in the pathophysiology of G. duodenalis infections.
... Protease activation and transactivation experiments. CP-A5 proteolytic activity with the synthetic peptides ZRR-AMC (Bachem) was determined as described 34 . Specific enzymatic activity was expressed in micromoles of released For MMP activation, purified pro-MMPs-1 and -3 were diluted to 50 mg ml À 1 in assay buffer (50 mM Tris, 10 mM CaCl 2 , 150 mM NaCl and 0.05% Brij-35, pH 7.5). ...
Intestinal invasion by the protozoan parasite Entamoeba histolytica is characterized by remodelling of the extracellular matrix (ECM). The parasite cysteine proteinase A5 (CP-A5) is thought to cooperate with human matrix metalloproteinases (MMPs) involved in ECM degradation. Here, we investigate the role CP-A5 plays in the regulation of MMPs upon mucosal invasion. We use human colon explants to determine whether CP-A5 activates human MMPs. Inhibition of the MMPs' proteolytic activities abolishes remodelling of the fibrillar collagen structure and prevents trophozoite invasion of the mucosa. In the presence of trophozoites, MMPs-1 and -3 are overexpressed and are associated with fibrillar collagen remodelling. In vitro, CP-A5 performs the catalytic cleavage needed to activate pro-MMP-3, which in turn activates pro-MMP-1. Ex vivo, incubation with recombinant CP-A5 was enough to rescue CP-A5-defective trophozoites. Our results suggest that MMP-3 and/or CP-A5 inhibitors may be of value in further studies aiming to treat intestinal amoebiasis.
... To date, only about 20 proteins or protein families have been identified as exposed on the plasma membrane of the parasite. These proteins include EhADH112 and the cysteine peptidase EhCP112 (EhCP-B9), which form a 112 kDa adhesion protein (3,4); the serine-rich E. histolytica protein (SREHP) (5); a calreticulin (6); an as-yet unidentified mannose binding lectin (7); transmembrane kinases, including phagosome-associated TMK96 (PATMK), TMK39 and TMK54 (8,9); a family of Bsp-A-like molecules (10); a rhomboid protease (11); an EhRab7 molecule (12); an actinin-like protein (AAF20148) (13); the lysine (K) and glutamic acid (E) enriched proteins KERP-1 and KERP-2 (13); the cysteine peptidases EhCP-A2 and EhCP-A5 (14,15); a peroxiredoxin (29 kDa thiol-dependent peroxidase) (16); the transcription factor URE3-BP, which localizes to the cytoplasm and inner surface of the plasma membrane (17); the ARIEL antigen (18,19); a LIM protein (EhLimA) associated with lipid rafts in the plasma membrane (20); the M8 family surface metalloprotease (EhMSP-1) (21); alcohol dehydrogenase 3 (22); and syntaxin 1 and SNAP-25 (23). ...
Surface molecules are of major importance for host-parasite interactions. During Entamoeba histolytica infections, these interactions are predicted to be of prime importance for tissue invasion, induction of colitis and liver abscess formation. To date, however, little is known about the molecules involved in these processes, with only about 20 proteins or protein families found exposed on the E. histolytica surface. We have therefore analyzed the complete surface proteome of E. histolytica. Using cell surface biotinylation and mass spectrometry, 694 putative surface-associated proteins were identified. In silico analysis predicted that approximately 26% of these proteins are membrane-associated, as they contain transmembrane domains and/or signal sequences, as well as sites of palmitoylation, myristoylation or prenylation. An additional 25% of the identified proteins likely represent non-classical secreted proteins. Surprisingly, no membrane-association sites could be predicted for the remaining 49% of the identified proteins. To verify surface localization, 23 proteins were randomly selected and analyzed by immunofluorescence microscopy. Of these 23 proteins, 20 (87%) showed definite surface localization. These findings indicate that a far greater number of E. histolytica proteins than previously supposed are surface-associated, a phenomenon that may be based on the high membrane turnover of E. histolytica.
... Tannich and colleagues have now identified up to six different CP genes that encode typical papain family proteinases with a high degree of conservation in all active site residues (Bruchhaus et al., 1996). One of them, ehcp5, is missing in the non-pathogenic E. dispar, suggesting a potential role of the protein in host tissue destruction of E. histolytica (Jacobs et al., 1998). Because of the present lack of genetic tools to manipulate the genome of E. histolytica, mutants in which the CP genes were knocked out are not yet available. ...
Inhibition of most of the expression of the cysteine proteinases of Entamoeba histolytica strain HM-1: IMSS was successfully performed by transcription of ehcp5 antisense RNA using the promoter of ehg34, which encodes a L21 ribosomal protein of E. histolytica. We have generated a stable transfectant in which the overall level of cysteine proteinase activity is strongly reduced (≈ 90%). This transfectant has a normal growth rate in Diamond's TYI-S-33 medium, a cytopathic and haemolytic activity similar to the control HM-1:IMSS pEhAct-Neo transfectant but with a significantly lower phagocytic activity.
... Until recently, it has been suggested that no qualitative differences are likely to exist between the two species because, for every putative pathogenicity factor of E. histolytica so far characterized at the molecular level, a counterpart has been recognized in E. dispar [4±6]. However, in recent studies on cysteine proteinases we found isoforms exclusively present in E. histolytica, indicating that such an assumption is premature until an extensive survey has been undertaken [7,8]. In vivo, the enormous cytolytic potency of the pathogenic amoeba is considered essential for tissue destruction and for the ability to escape from the cellular immune system of the host after invasion. ...
Amoebapore, a 77-residue peptide with pore-forming activity from the human pathogen Entamoeba histolytica, is implicated in the killing of phagocytosed bacteria and in the cytolytic reaction of the amoeba against host cells. Previously, we structurally and functionally characterized three amoebapore isoforms in E. histolytica but recognized only one homolog in the closely related but non-pathogenic species Entamoeba dispar. Here, we identified two novel amoebapore homologs from E. dispar by molecular cloning. Despite strong resemblance of the primary structures of the homologs, molecular modeling predicts a species-specific variance between the peptide structures. Parallel isolation from trophozoite extracts of the two species revealed a lower amount of pore-forming peptides in E. dispar and substantially higher activity of the major isoform from E. histolytica towards natural membranes than that from E. dispar. Differences in abundance and activity of the lytic polypeptides may have an impact on the pathogenicity of amoebae.
... The E. dispar chromosomal locus homologus to that in which EhCP5 is found has been sequenced, and a degenerate version of the gene was found that contained numerous mutations and had no possibility of encoding a protein. The corresponding protein in E. histolytica is found on the surface of trophozoites (Jacobs et al. 1998) and is therefore suspected of playing a role in tissue invasion. It is anticipated that additional differences will be uncovered as genome sequencing progresses. ...
Over the past 25 years a fundamental change has taken place in our understanding of amebiasis, through the recognition of
Entamoeba dispar as a species that is distinct from Entamoeba histolytica but is morphologically indistinguishable. This change in taxonomy has significant implications for the diagnosis and treatment
of infections, as well as for our ability to interpret the earlier literature. The defining characteristic of the two species
remains the ability of E. histolytica to cause invasive disease while E. dispar cannot, but the underlying genetic differences between the two that are responsible for this remain to be defined. The ongoing
comparative genome sequencing will hopefully shed light on the dichotomy.
... Other terms that have been used to describe closely related or identical enzymes are cathepsin B (Lushbaugh et al., 1985), neutral proteinase (Keene et al., 1990), histolysin (Luaces and Barrett, 1988) (later changed to histolysain; Luaces et al., 1992) and amoebapain (Scholze et al., 1992). E. histolytica cysteine endopeptidases were found to be secreted (Leippe et al., 1995) and localised in lysosome-like vesicles or at the surface of the cell (Garcia-Rivera et al., 1999;Jacobs et al., 1998). Molecular cloning has revealed a large number of cysteine endopeptidase genes in the E. histolytica genome (Bruchhaus et al., 2003;Garcia-Rivera et al., 1999;Reed et al., 1993;Tannich et al., 1991cTannich et al., ., 1992. ...
... Localization of EhCP-A5 demonstrated in this study does not support the inference by the previous study (Jacobs et al., 1998), in which EhCP-A5 was identified as a membrane-binding protein and localized to the plasma membrane by transmission electron microscopy using an antibody raised against 12-a.a.-long peptide specific to EhCP-A5. In the present study, we used both anti-HA and anti-nEhCP-A5 recombinant antibodies for immunofluorescence assay. ...
The transport of lysosomal proteins is, in general, mediated by mannose 6-phosphate receptors via carbohydrate modifications. Here, we describe a novel class of receptors that regulate the transport of lysosomal hydrolases in the enteric protozoan Entamoeba histolytica, which is a good model organism to investigate membrane traffic. A novel 110 kDa cysteine protease (CP) receptor (CP-binding protein family 1, CPBF1) was initially discovered by affinity co-precipitation of the major CP (EhCP-A5), which plays a pivotal role in the pathogenesis of E. histolytica. We demonstrated that CPBF1 regulates EhCP-A5 transport from the endoplasmic reticulum to lysosomes and its binding to EhCP-A5 is independent of carbohydrate modifications. Repression of CPBF1 by gene silencing led to the accumulation of the unprocessed form of EhCP-A5 in the non-acidic compartment and the mis-secretion of EhCP-A5, suggesting that CPBF1 is involved in the trafficking and processing of EhCP-A5. The CPBF represents a new class of transporters that bind to lysosomal hydrolases in a carbohydrate-independent fashion and regulate their trafficking, processing and activation and, thus, regulate the physiology and pathogenesis of E. histolytica.
... Several studies have shown that peptidases (particularly cysteine peptidases ) are major pathogenicity factors in E. histolytica [54]. CP-A5 is the prime candidate, (although we only found one peptide in experiment I which however covers 5% of the protein), since itlocalizes at the amoebic surface [55] and is involved in human colon invasion [18] and ALA formation [56]. This protease contains an Arg-Gly-Asp (RGD) integrin binding motif which has also been found in the proregion of cathepsin X from higher eukaryotes [57]. ...
Exposure of Entamoeba histolytica to specific ligands induces cell polarization via the activation of signalling pathways and cytoskeletal elements. The process leads to formation of a protruding pseudopod at the front of the cell and a retracting uropod at the rear. In the present study, we show that the uropod forms during the exposure of trophozoites to serum isolated from humans suffering of amoebiasis. To investigate uropod assembly, we used LC-MS/MS technology to identify protein components in isolated uropod fractions. The galactose/N-acetylgalactosamine lectin, the immunodominant antigen M17 (which is specifically recognized by serum from amoeba-infected persons) and a few other cells adhesion-related molecules were primarily involved. Actin-rich cytoskeleton components, GTPases from the Rac and Rab families, filamin, α-actinin and a newly identified ezrin-moesin-radixin protein were the main factors found to potentially interact with capped receptors. A set of specific cysteine proteases and a serine protease were enriched in isolated uropod fractions. However, biological assays indicated that cysteine proteases are not involved in uropod formation in E. histolytica, a fact in contrast to the situation in human motile immune cells. The surface proteins identified here are testable biomarkers which may be either recognized by the immune system and/or released into the circulation during amoebiasis.
... These proteinases are not expressed during in vitro culture (Bruchhaus et al. 2003). Although, the disruption of raft-like domains by methyl-b-cyclodextrin and filipin cholesterol binding agents do not inhibit constitutive secretion of cysteine proteases (Laughlin et al. 2004 ), other membrane cysteine proteinases, such as EhCP2 (ACP2), EhCP5, and CP112 (Jacobs et al. 1998; García-Rivera et al. 1999; Que et al. 2002 ), could participate in amoebic virulence because of their role in lipid rafts. Recently, Biller et al. (2009) found hemolytic and high cystine peptidase activities in an E. histolytica virulent isolate compared with an avirulent parasite isolate. ...
Trophozoites of Entamoeba histolytica HM-1:IMSS become less virulent after long-term maintenance in axenic cultures. The factors responsible for the loss of virulence during in vitro cultivation remain unclear. However, it is known that in vitro cultivation of amoeba in culture medium supplemented with cholesterol restores their virulence. In this study, we analyzed the effect of adding phosphatidylcholine-cholesterol (PC-Chol) liposomes to the culture medium and evaluated the effect of this lipid on various biochemical and biological functions of E. histolytica HM-1:IMSS in terms of its virulence. The addition of PC-Chol liposomes to the culture medium maintained the virulence of these parasites against hamster liver at the same level as the original virulent E. histolytica strain, even though these amoebae were maintained without passage through hamster liver for 18 months. The trophozoites also showed increased endocytosis, erythrophagocytosis, and carbohydrate residue expression on the amoebic surface. Protease activities were also modified by the presence of cholesterol in the culture medium. These findings indicate the capacity of cholesterol to preserve amoeba virulence and provide an alternative method for the maintenance of virulent E. histolytica trophozoites without the need for in vivo procedures.
Protozoan parasites have led to worldwide devastation because of their ability to cause infectious diseases. They have evolved as successful pathogens in part because of their remarkable and sophisticated ways to evade innate host defenses. This holds true for both intracellular and extracellular parasites that deploy multiple strategies to circumvent innate host defenses for their survival. The different strategies protozoan parasites use include hijacking the host cellular signaling pathways and transcription factors. In particular, the nuclear factor-κB (NF-κB) pathway seems to be an attractive target for different pathogens owing to their central role in regulating prompt innate immune responses in host defense. NF-κB is a ubiquitous transcription factor that plays an indispensable role not only in regulating immediate immune responses against invading pathogens but is also a critical regulator of cell proliferation and survival. The major immunomodulatory components include parasite surface and secreted proteins/enzymes and stimulation of host cells intracellular pathways and inflammatory caspases that directly or indirectly interfere with the NF-κB pathway to thwart immune responses that are directed for containment and/or elimination of the pathogen. To showcase how protozoan parasites exploits the NF-κB signaling pathway, this review highlights recent advances from Entamoeba histolytica and other protozoan parasites in contact with host cells that induce outside-in and inside-out signaling to modulate NF-κB in disease pathogenesis and survival in the host.
While Entamoeba histolytica ( Eh )-induced pro-inflammatory responses are critical in disease pathogenesis, the downstream signaling pathways that subsequently dampens inflammation and the immune response remains unclear. Eh in contact with macrophages suppresses NF-κB signaling while favoring NLRP3-dependent pro-inflammatory cytokine production by an unknown mechanism. Cullin-1 and cullin-5 (cullin-1/5) assembled into a multi-subunit RING E3 ubiquitin ligase complex are substrates for neddylation that regulates the ubiquitination pathway important in NF-κB activity and pro-inflammatory cytokine production. In this study, we showed that upon live Eh contact with human macrophages, cullin-1/4A/4B/5 but not cullin-2/3, were degraded within 10 minutes. Similar degradation of cullin-1/5 were observed from colonic epithelial cells and proximal colonic loops tissues of mice inoculated with live Eh . Degradation of cullin-1/5 was dependent on Eh -induced activation of caspase-1 via the NLRP3 inflammasome. Unlike cullin-4B, the degradation of cullin-4A was partially dependent on caspase-1 and was inhibited with a pan caspase inhibitor. Cullin-1/5 degradation was dependent on Eh cysteine proteinases Eh CP-A1 and Eh CP-A4, but not Eh CP-A5, based on pharmacological inhibition of the cysteine proteinases and Eh CP-A5 deficient parasites. siRNA silencing of cullin-1 /5 decreased the phosphorylation of pIκ-Bα in response to Eh and LPS stimulation and downregulated NF-κB-dependent TNF-α mRNA expression and TNF-α and MCP-1 pro-inflammatory cytokine production. These results unravel a unique outside-in strategy employed by Eh to attenuate NF-κB-dependent pro-inflammatory responses via NLRP3 activation of caspase-1 that degraded cullin-1/5 from macrophages.
BACKGROUND: Tritrichomonas foetus is a protozoa flagellated that reside as a parasite or commensal in organ cavities such as the gastrointestinal and reproductive tracts of their hosts. While this parasite is an important venereal pathogen in cattle and the causative agent of chronic diarrhea in the domestic cat, the mechanisms that define the host specificity of this parasite are still unknown.
METHODS: Here, we integrate the genomic and transcriptomic information of the parasites obtained from different hosts (bovine, feline and porcine), to determine the gene expression profiles of T. foetus associated with host adaptation.
RESULTS: We demonstrated the existence of patterns of co-expressed genes specific to each strain and related to known transcription factors (Myb DNA-binding proteins), proteases and genes related to protein phosphorylation process. Also, the genes related to Myb DNA-binding proteins and protein kinases were differentially expressed between strains being those genes different for each strain.
CONCLUSIONS: On the basis of the expression profile variability of genes involved in transcription, intracellular signaling and proteases between the strains (pathogenic and non‐pathogenic), we propose that these genes have roles in T. foetus adaptation to different hosts. This integrated approach will serve as a useful resource for future studies about the host-parasite interaction and for the future identification of new targets for diagnosis, vaccines and therapeutic intervention to control the bovine and feline tritrichomonosis.
The human protozoan parasite Entamoeba histolytica can live in the human intestine for months or years without generating any symptoms in the host. For unknown reasons, amoebae can suddenly destroy the intestinal mucosa and become invasive. This can lead to amoebic colitis or extraintestinal amoebiasis whereby the amoebae spread to other organs via the blood vessels, most commonly the liver where abscesses develop. Entamoeba nuttalli is the closest genetic relative of E. histolytica and is found in wild macaques. Another close relative is E. dispar, which asyptomatically infects the human intestine. Although all three species are closely related, only E. histolytica and E. nuttalli are able to penetrate their host’s intestinal epithelium. Lineage-specific genes and gene families may hold the key to understanding differences in virulence among species. Here we discuss those genes found in E. histolytica that have relatives in only one or neither of its sister species, with particular focus on the peptidase, AIG, Ariel, and BspA families.
Several members of the genus Entamoeba infect humans including E. histolytica, E. moshkovskii, and E. dispar. Among these, only E. histolytica is considered pathogenic and is the causal agent of amebiasis. Amebiasis is the second most common cause of death from a parasite worldwide after malaria with considerable morbidity and mortality [1, 2]. Based on the site of infection, the clinical features of amebiasis range from asymptomatic colonization to amebic colitis (dysentery or diarrhea) and invasive extraintestinal amebiasis, which appears most commonly in the form of liver abscesses [2]. According to the World Health Organization, amebiasis occurs worldwide, with approximately 50 million people infected annually, causing close to 100,000 deaths per year. Therefore, it is important to understand the epidemiology, infection, and control of the disease. The aim of this chapter is to provide a current understanding of human amebiasis, which illustrates the parasite biology, mechanism of pathogenesis, diagnosis, and prevention.
Entamoeba histolytica (Eh) is the causative agent of amebiasis, one of the major causes of dysentery-related morbidity worldwide. Recent studies have underlined the importance of the intercellular junction between Eh and host cells as a determinant in the pathogenesis of amebiasis. Despite the fact that direct contact and ligation between Eh surface Gal-lectin and EhCP-A5 with macrophage α5β1 integrin are absolute requirements for NLRP3 inflammasome activation and IL-1β release, many other undefined molecular events and downstream signaling occur at the interface of Eh and macrophage. In this study, we investigated the molecular events at the intercellular junction that lead to recognition of Eh through modulation of the macrophage cytoskeleton. Upon Eh contact with macrophages key cytoskeletal-associated proteins were rapidly post-translationally modified only with live Eh but not with soluble Eh proteins or fragments. Eh ligation with macrophages rapidly activated caspase-6 dependent cleavage of the cytoskeletal proteins talin, Pyk2 and paxillin and caused robust release of the pro-inflammatory cytokine, IL-1β. Macrophage cytoskeletal cleavages were dependent on Eh cysteine proteinases EhCP-A1 and EhCP-A4 but not EhCP-A5 based on pharmacological blockade of Eh enzyme inhibitors and EhCP-A5 deficient parasites. These results unravel a model where the intercellular junction between macrophages and Eh form an area of highly interacting proteins that implicate the macrophage cytoskeleton as a sensor for Eh contact that leads downstream to subsequent inflammatory immune responses.
Galactose and N-acetyl-D-galactosamine-inhibitable lectin of Entamoeba histolytica has roles in pathogenicity and induction of protective immunity in rodent models of amoebiasis. Recently, the intermediate subunit of the lectin, Igl1, of E. histolytica has been shown to have hemolytic activity. However, the corresponding lectin is also expressed in a non-virulent species, Entamoeba dispar, and another subunit, Igl2, is expressed in the protozoa. Therefore, in this study, we compared the activities of Igl1 and Igl2 subunits from E. histolytica and E. dispar using various regions of recombinant Igl proteins expressed in Escherichia coli. The recombinant E. dispar Igl proteins had comparable hemolytic activities with those of E. histolytica Igl proteins. Furthermore, Igl1 gene-silenced E. histolytica trophozoites showed less hemolytic activity compared with vector-transfected trophozoites, indicating that the expression level of Igl1 protein influences the activity. These results suggest that the lower hemolytic activity in E. dispar compared with E. histolytica reflects the lower expression level of Igl1 in the E. dispar parasite. © 2017 Kato et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Entamoeba histolytica (Eh) is the protozoan parasite responsible for intestinal amebiasis and interacts dynamically with the host intestinal epithelium during disease pathogenesis. A multifaceted pathogenesis profile accounts for why 90% of individuals infected with Eh are largely asymptomatic. For 100 millions individuals that are infected each year, key interactions within the intestinal mucosa dictate disease susceptibility. The ability for Eh to induce amebic colitis and disseminate into extraintestinal organs depends on the parasite competing with indigenous bacteria and overcoming the mucus barrier, binding to host cells inducing their cell death, invasion through the mucosa and outsmarting the immune system. In this review we summarize how Eh interacts with the intestinal epithelium and subverts host defense mechanisms in disease pathogenesis.
Entamoeba histolytica is an obligate human intestinal parasite causing amoebic dysentery and liver abscess. Entamoeba dispar is its close largely nonpathogenic relative. Both species are well adapted to the human host. The motile amoebae called trophozoites dwell in the lumen of the large intestine; in its distal part, they differentiate to cysts, which are excreted and can survive in the environment and infect new hosts. Although metronidazole and other nitroimidazoles represent an efficient treatment, there are more than 55,000 deaths worldwide every year.
The E. histolytica genome project led to a better understanding of the molecular equipment; two surprises were the significant number of genes acquired from bacteria by lateral gene transfer and a very high number of protein kinases and phosphatases underscoring the complex signal transduction pathways in this parasite.
The parasite attacks human cells by first attaching to them via a galactose and N-acetylgalactose (Gal/GalNAc)-specific lectin. The host cell membrane is pierced by a pore-forming protein called amoebapore, and cysteine proteinases destroy cellular components, antibodies and complement.
The human host mounts a massive humoral and cellular immune response against E. histolytica with macrophages and NKT cells being the most efficient defenders. The antibody response is limited in its impact through the ability of the amoebae to cap and shed antibodies bound to their surface. In addition, E. histolytica has developed a number of mechanisms to cope with immune cells.
E. histolytica belongs to the microaerophilic microorganisms devoid of classical mitochondria and devoid of oxidative phosphorylation. So the main pathway to extract energy is by glycolysis, and when not needed, the energy is stored in the form of glycogen. The biosynthetic abilities are quite crippled; most useful molecules, sugars, amino acids, lipids and nucleotides are acquired from the host or from phagocytosed bacteria.
Entamoeba histolytica is a protozoan parasite and the causative agent of amebiasis. It is estimated approximately 1% of humans are infected with E. histolytica, resulting in an estimate of 100,000 deaths annually. Clinical manifestations of amebic infection range widely from asymptomatic to severe symptoms, including dysentery and extra-intestinal abscesses. Like other infectious diseases, it is assumed that only ~20% of infected individuals develop symptoms, and genetic factors of both the parasite and humans as well as the environmental factors, e.g., microbiota, determine outcome of infection. There are multiple essential steps in amebic infection: degradation of and invasion into the mucosal layer, adherence to the intestinal epithelium, invasion into the tissues, and dissemination to other organs. While the mechanisms of invasion and destruction of the host tissues by the amebae during infection have been elucidated at the molecular levels, it remains largely uncharacterized how the parasite survive in the host by evading and attacking host immune system. Recently, the strategies for immune evasion by the parasite have been unraveled, including immunomodulation to suppress IFN-γ production, elimination of immune cells and soluble immune mediators, and metabolic alterations against reactive oxygen and nitrogen species to fend off the attack from immune system. In this review, we summarized the latest knowledge on immune reaction and immune evasion during amebiasis.
Cysteine proteases of the protozoan parasite Entamoeba histolytica are key virulence factors involved in overcoming host defences. These proteases are cathepsin-like enzymes with a cathepsin-L like structure, but cathepsin-B substrate specificity. In the host intestine, amoeba cysteine proteases cleave colonic mucins and degrade secretory immunoglobulin (Ig) A and IgG rendering them ineffective. They also act on epithelial tight junctions and degrade the extracellular matrix to promote cell death. They are involved in the destruction of red blood cells and the evasion of neutrophils and macrophages and they activate pro-inflammatory cytokines IL-1 beta and IL-18. In short, amoeba cysteine proteases manipulate and destroy host defences to facilitate nutrient acquisition, parasite colonization and/or invasion. Strategies to inhibit the activity of amoeba cysteine proteases could contribute significantly to host protection against E. histolytica.
The intestinal parasite Entamoeba histolytica (Eh) is a significant health risk in the developing world where infection occurs via ingestion of contaminated food or water. The resulting disease, amebiasis, claims 100,000 lives each year. In the colon, the parasite colonizes the mucous layer and interacts with the underlying mucosa to induce a variety of symptoms. The intestinal barrier has three main components: Bacteria and host proteins found within the lumen, a protective layer of mucus, and a single layer of intestinal epithelial cells (IEC) connected by tight junctions (TJ). Eh possesses a variety of virulence components that target the intestinal barrier to cause increased gut permeability, resulting in diarrhea. In the colon Eh can phagocytose pathogenic bacteria, which may increase the virulence of the parasite. Moreover, Eh binds colonic mucins via the parasite surface Gal/GalNAc lectin to facilitate colonization of the mucous layer. Subsequently, the virulence factor, Eh CP-A5, degrades the MUC2 polymer, allowing the parasite access to the epithelia where it binds to and destroys IEC by apoptosis or alters the expression of TJ proteins, causing secretory diarrhea. The sequence of events in intestinal amebiasis is not well understood and is based largely on in vitro and in vivo models of the disease.
Tight junctions and microvilli constitute an anti-invasive barrier at the luminal side of enteric cell layers. Both subcellular structures are disrupted following adhesion of Entamoeba histolytica trophozoites to enteric cell layers in vitro. It was our aim to analyse the molecular mechanism underlying this disruption. Therefore, we cocultured enteric T84 cell layers established on filter inserts with E. histolytica trophozoites and tested various modulators of enteric molecules, involved in the functional regulation of tight junctions, as well as inhibitors of trophozoite virulence factors on their capacity to maintain the transepithelial electrical resistance. Pretreatment of trophozoites with the proteinase inhibitor N-Tosyl-Phenylalanine chloromethyl ketone or N-Tosyl-l-Lysine chloromethyl ketone prevented the decrease in transepithelial electrical resistance whereas none of the modulators used to pretreat enterocytes were successful. Moreover, zymography and Western blot analysis revealed that both N-Tosyl-Phenylalanine chloromethyl ketone and N-Tosyl-l-Lysine chloromethyl ketone inhibited E. histolytica cysteine proteinases and prevented proteolysis of tight junction molecules ZO-1 and ZO-2 and of villin, the major actin bundling molecule in microvilli. Immunocytochemistry with an antibody against ezrin, an actin-binding molecule in microvilli, and phase contrast microscopy demonstrated that pretreatment of trophozoites with N-Tosyl-Phenylalanine chloromethyl ketone or N-Tosyl-l-Lysine chloromethyl ketone also prevented disturbance of microvilli and destruction of Caco-2 enteric cell layers in cocultures. Taken together, our results indicate that trophozoites use their proteinases to overcome microvilli and tight junction barriers during the invasion of enteric cell layers, that these phenomena could be prevented by pretreatment of trophozoites with N-Tosyl-Phenylalanine chloromethyl ketone or N-Tosyl-l-Lysine chloromethyl ketone, and that such pretreatment disabled trophozoites to destroy enteric cell layers in vitro.
Entamoeba histolytica is an enteric tissue-invading protozoan parasite that causes amoebic colitis and occasionally liver abscess in humans. During tissue invasion, amoebic adhesion to host components is an important event for host cell death leading to successful invasion and infection. Among amoebic virulence factors, Gal/GalNAc lectin is known to be major adhesion factor to host cells. In this study, we investigated the role of amoebic secreted CP (Cysteine Proteases) in amoebic adhesion to extracellular matrix (ECM) protein using CP inhibitor and E. histolytica strains in which the endogenous inhibitor of cysteine protease (ICP) 1 gene was overexpressed (ICP1(+)) or repressed by antisense small RNA-mediated gene silencing (ICP1(-)). We found that pretreatment of wild-type amoebae with CP inhibitor E64, or thiol-group modifiers such as diamide and N-Ethylmaleimide resulted in a significant decrease in adhesion to laminin and collagen ECM proteins. Furthermore, ICP1(+) strain, with a reduction of secreted CP activity, exhibited reduced ability by 40 percent to adhere to laminin. In contrast, ICP1(-) strain, with an 1.9-fold increase of secreted CP activity, showed a two-fold increase in amoebic adherence to laminin compared to the control strain. In addition, total amount of secreted CP5 was decreased in ICP1(+) amoeba. Conversely, total amount of secreted CP1 and mature-form CP5 were increased in ICP1(-) amoeba. We also found that ICP1 was secreted into extracellular milieu. These results suggest that secreted CP activity by E. histolytica may be an important factor affecting adhesion to host proteins, and regulation of CP secretion by ICP plays a major role in pathogenesis. This study provides insight into the CP-mediated tissue pathogenesis in amoeba-invaded lesions during human amoebiasis.
Copyright © 2014. Published by Elsevier Inc.
Cysteine peptidases of Entamoeba histolytica (EhCPs) are considered to be important pathogenicity factors. It has been described that under standard axenic culture conditions, only three (ehcp-a1, ehcp-a2 and ehcp-a5) out of approximately 50 cys-teine peptidase genes present in the E. histolytica genome are substantially expressed, thus representing the set of major EhCPs. In this study, transcrip-tional silencing of the major peptidase genes was used to characterize their physiological role in more detail. Analysing the transfectants a fourth major cysteine peptidase activity belonging to EhCP-A7 could be characterized. Neither cytopathic activity nor phagocytosis of erythrocytes was altered in CP-inactivated amoebae. However, a significant difference in haemolytic activity was observed. EhCP-A1 and EhCP-A7 apparently had no influence on haemolytic activity, whereas transfectants silenced for ehcp-a5 as well as those silenced for all major peptidases showed a significant reduction in their haemolytic activity. Furthermore, cells silenced for ehcp-a1 and ehcp-a7 and more effectively cells silenced in all major ehcps were impaired in digesting of phagocytosed erythrocytes. Moreover, amoebae silenced for all major peptidase genes lost the ability to form aggregates of erythrocytes prior to phagocytosis.
Entamoeba histolytica and E. dispar are genetically distinct but closely related protozoan species. Both colonize the human gut but only E. histolytica is able to invade tissues and cause disease. Comparison of the 2 species may help to elucidate the specific mechanisms involved
in the pathogenicity of E. histolytica. During the last few years, various amoeba molecules considered to be important for pathogenic tissue invasion have been
identified and characterized, such as a galactose-inhibitable surface lectin, pore-forming peptides and cysteine proteinases.
This review summarizes present knowledge about the structure and function of these molecules, with emphasis on the differences
between E. histolytica and E. dispar.
Entamoeba histolytica has diverse distribution and is a substantial risk in almost all the countries where barrier between human feces, food and water source are ordinary. There are at least 8 different amoebas that live in the human intestinal lumen however those are generally accepted as commensals except for E. histolytica. The parasite imposes a major threat to public health in most parts of world and has re-emerged in some previously dormant areas as it is categorized as second leading cause of death from parasitic disease worldwide. In most of E. histolytica infection, symptoms remain absent or very mild whereas most frequent clinical manifestation are colitis and liver abscess due to amoebic infection. Laboratory diagnosis of amoebiasis is usually made on the basis of microscopical and serological methods Nitromidazole derivatives like metronidazole, tinidazole and ornidazole are considered as foundation stone of the treatment for amoebiasis. Lack of effective vaccination is one of the major hurdles for the control of amoebiasis that may prevent transmission of the parasite and or at least progression of the infected individuals into active invasive disease. The aim of this article is to comprehensively review the epidemiology, disease pathology and treatment of this parasitic zoonotic disease.
Entamoeba histolytica has diverse distribution and is a substantial risk in almost all the countries where barrier between human feces, food and water source are ordinary. There are at least 8 different amoebas that live in the human intestinal lumen however those are generally accepted as commensals except for E. histolytica. The parasite imposes a major threat to public health in most parts of world and has re-emerged in some previously dormant areas as it is categorized as second leading cause of death from parasitic disease worldwide. In most of E. histolytica infection, symptoms remain absent or very mild whereas most frequent clinical manifestation are colitis and liver abscess due to amoebic infection. Laboratory diagnosis of amoebiasis is usually made on the basis of microscopical and serological methods Nitromidazole derivatives like metronidazole, tinidazole and ornidazole are considered as foundation stone of the treatment for amoebiasis. Lack of effective vaccination is one of the major hurdles for the control of amoebiasis that may prevent transmission of the parasite and or at least progression of the infected individuals into active invasive disease. The aim of this article is to comprehensively review the epidemiology, disease pathology and treatment of this parasitic zoonotic disease.
Abstract Enterocyte turnover along with proper epithelial barrier function are crucial aspects of mucosal defense. Apoptosis is a highly regulated type of programmed cell death that allows for the homeostatic turnover of the epithelial layer. Recent studies have suggested that microbial modulation of enterocyte apoptosis can result in increased epithelial permeability, leading to gastrointestinal pathophysiology. In this review, we highlight key mechanisms and pathways via which various viral, bacterial and parasitic pathogens are able to modulate enterocyte apoptosis. We also discuss how these alterations to enterocyte apoptosis can result in the activation of chronic gastrointestinal disorders, such as allergies, irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD). The role of proteinase-activated receptors in the pathogenesis of modulated apoptosis-induced pathogenesis is also discussed. Newly discovered processes, through which host epithelial cells may have evolved, rescue mechanisms from microbe-induced apoptosis are discussed. Together, these mechanisms are key to our ever-increasing understanding of host-microbe interactions in the gut.
Human infections with Entamoeba histolytica sporadically become pathogenic, unknown triggers converting the parasite to its invasive phenotype. Parasite virulence results from complex host-parasite interactions implicating multiple amoebic and host factors, eliciting host defence responses and parasite resistance to stress caused by the host reactions and changing environments during tissue invasion.
An alignment/phylogeny of the papain superfamily of cysteine proteases was created using an initial structure-based alignment followed by successive iterations of sequence alignment and phylogenetic inference. The iterative approach resulted in significant improvements in the alignment/phylogeny. There were three groups of cysteine proteases that were distantly related and which could be aligned against each other only in the active site regions: the papain group, which included such stereotypical cysteine proteases as cathepsins B, C, H, L and S; and the bleomycin hydrolase and calpain groups. There was one bacterial sequence in each of the bleomycin hydrolase and calpain groups. The former probably arose by lateral gene transfer, the latter possibly by direct evolution from an ancestral protease predating the eukaryote/prokaryote divergence. The phylogeny of the papain group indicated that many families diverged almost simultaneously early during eukaryotic evolution. In mammals there are at least 12 distinct families of cysteine proteases, possibly many more, including at least two as yet uncharacterized enzymes.
One of the three cysteine proteinase genes, ACP1 (or CP 3), has been reported to be missing in non-pathogenic strains of Entamoeba histolytica (or Entamoeba dispar as recently labeled). Unexpectedly, a gene fragment very similar in its sequence (95% homology) to ACP1 of pathogenic strains was obtained by use of the polymerase chain reaction from genomic DNA and cDNA of various cloned non-pathogenic strains as well as in 23 clinical isolates from asymptomatic carriers. The finding of the ACP1 homologue in non-pathogenic or E. dispar strains rules out the proposed use of its absence for diagnostic purposes.
Tachystoscopic stereo can be used to greatly enhance the performance and usability of low-cost molecular graphics systems. Here, a simple way to connect and control three-dimensional liquid-crystal glasses from a PC/AT with EGA graphics capabilities is described. The method makes elegant use of the screen's vertical retrace for synchronization purposes, allowing left and right views to be alternated every refresh cycle.
Crude lysates of Entamoeba histolytica (strain HM 1:IMSS) analyzed by substrate gel electrophoresis in 12.5% acrylamide separating gels with reducing agents showed six hydrolysis zones with apparent molecular weights of 73,000 (high), 45,000, 36,000 (intermediate), 30,000, 26,000 and 23,000 (low molecular weight proteinases). Amebic lysates fractionated using the procedure of Aley et al. or the procedure of Rosenberg and Gitler and analyzed by the same method show all enzymes in the fractions with the soluble components and only the intermediate and low molecular weight proteinases in the fraction containing internal vesicles or membranes and plasma membrane. Some of these proteinases seem to be integral membrane proteins since they resist treatment with high salt, high urea buffer. All fractions are capable of digesting azocasein. Fractionation of amebic lysates by hydrophobic chromatography using phenyl-Sepharose or phase separation of amebic extracts with Triton X-114 show that proteinases with high, intermediate and low molecular weight behave as hydrophilic proteins while only proteinases of intermediate and low molecular weight behave as hydrophobic proteins. These results suggest that some proteinases are segregated in different compartments of the cell.
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We report the construction of two plasmid vectors, pJC20 and pJC40, for the expression of recombinant genes in Escherichia coli under the control of T7 RNA polymerase. Their small sizes of ca. 2.4 kb ease the subcloning of large inserts and the high copy numbers obtained result in satisfactory yields in all plasmid preparations. A multiple-cloning site offers sites for directional cloning and nested deletions. In addition, pJC40 encodes a cleavable amino-terminal histidine tail of 10 residues which is added to the gene product, thus allowing purification by metal chelate chromatography. Observed expression yields are in the range of 10% of total bacterial protein for all genes tested in our laboratory.
The purpose of this work was to define cell surface proteases on Entamoeba histolytica trophozoites. Molecular sizes of 2-mercaptoethanol-activated proteases were determined in several ameba cell fractions. Inhibited proteases were resolved in SDS-polyacrylamide gels and then defined by their ability to digest bovine albumin during electrophoretic migration of ameba bands through a stacking gel containing albumin. This second gel revealed nine gaps of digestion along the horizontal albumin line corresponding to proteases with molecular weights of 195, 175, 150, 124, 102, 70, 45, 36, and 28 kDa. The 70-kDa protease proved to be the most active in plasma membrane, in whole membrane fractions, and in total extracts of ameba. This protease appears to be an integral membrane component as it was reconstituted in an artificial membrane system in its active form, as well as because it was present on the surface of glutaraldehyde-fixed amebas. These results demonstrate that amebic trophozoites contain on their surface a very active protease, which may play a role in the digestion of host components.
We have cloned the complete gene encoding cysteine proteinase 3 in E. histolytica as well as a cDNA encoding a highly homologous protein in E. dispar. In addition, we have demonstrated the presence and expression of the CP3 gene in various E. histolytica and E. dispar isolates. Since the expression of the gene is rather similar within both Entamoeba species, we assume that it does not constitute the proposed virulence factor of E. histolytica.
During the past few years, the introduction of DNA-mediated gene transfer into parasite research has permitted subtle studies on fundamental aspects of parasite biology. In this paper, Egbert Tannich describes the recent breakthrough of successful Entamoeba histolytica transfection, and the subsequent developments in this field.
The enormous cytolytic potential of Entamoeba histolytica appeals to parasitologists and immunologists because it kills target cells in a contact-dependent reaction resembling that of cytotoxic lymphocytes. In this review, Matthias Leippe summarizes what is currently known about a family of pore-forming peptides termed 'amoebapores', to which the cytolytic effect has been attributed, and describes the structural and functional properties of these potent factors, as well as their structure-activity relationships. Finally, a comparison is made with effector molecules of the mammalian defensive system.