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Entamoeba histolytica: Correlation of the cytopathic effect of virulent trophozoites with secretion of a cysteine proteinase
Abstract
Work from several laboratories suggests a correlation between expression of cysteine proteinase activity and the cytopathic effect of virulent HM1 strain Entamoeba histolytica trophozoites on cultured cell monolayers. Consistent with this relationship, we find that L-6 trophozoites, mutants cloned from the HM1 parent strain, are deficient in both proteinase expression and cytopathic effect. Three other clones, with proteinase expression equal to or greater than that of the HM1 strain, express the cytopathic effect. Furthermore, a nontoxic specific proteinase inhibitor, Z-phenylalanyl-alanyl-CH2F, inhibits the cytopathic effect of live trophozoites in a dose-dependent manner. These results support the hypothesis that expression and release of the cysteine proteinase is an important factor in producing the cytopathic effect, presumably by its degradation of cell anchoring proteins.
... Entamoeba histolytica is characterized by its enormous capacity to destroy and invade human tissues. This ability is mainly attributed to their cysteine peptidases, which have been shown by various in vitro and in vivo studies [1][2][3][4][5][6][7][8][9]: ...
... 1. Fibroblast monolayers are disrupted by purified cysteine peptidases [3], likely because of their ability to degrade extracellular matrix components [5,8,10,11]; 2. A direct correlation between cysteine peptidase activity and pathogenicity was observed [2,3,6,12]; 3. The abscess formation can be inhibited by the use of specific cysteine peptidase inhibitors [4,9]; 4. It has been postulated that secreted cysteine peptidases interfere with the host immune system by cleaving immune molecules such as IgG and IgA [11,13,14], processing of complement C3 [15,16], inactivation of complement C3a and C5a [17], inactivation of pro-IL-18 [18], and generation of mature IL-1β from its pro-form [19]; ...
... 1. Fibroblast monolayers are disrupted by purified cysteine peptidases [3], likely because of their ability to degrade extracellular matrix components [5,8,10,11]; 2. A direct correlation between cysteine peptidase activity and pathogenicity was observed [2,3,6,12]; 3. The abscess formation can be inhibited by the use of specific cysteine peptidase inhibitors [4,9]; 4. It has been postulated that secreted cysteine peptidases interfere with the host immune system by cleaving immune molecules such as IgG and IgA [11,13,14], processing of complement C3 [15,16], inactivation of complement C3a and C5a [17], inactivation of pro-IL-18 [18], and generation of mature IL-1β from its pro-form [19]; ...
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.
... Experimental evidence supporting the role of this cysteine proteinase in the pathogenesis of amebiasis includes its ability to degrade fibronectin, collagen, and basement membrane matrix and to activate the third component of complement (Reed et al., 1989a,b). Purified proteinase reproduces the cytopathic effect of pathogenic amebae, and a specific irreversible inhibitor of the proteinase prevents destruction of cell monolayers by live axenic amebae (Keene et al., 1990; Hirata et al., 2007). Increased expression and excretion of the proteinase correlates with virulence of both axenic laboratory strains of E. histolytica, and fresh clinical isolates (Keene et al., 1986Keene et al., , 1990). ...
... Purified proteinase reproduces the cytopathic effect of pathogenic amebae, and a specific irreversible inhibitor of the proteinase prevents destruction of cell monolayers by live axenic amebae (Keene et al., 1990; Hirata et al., 2007). Increased expression and excretion of the proteinase correlates with virulence of both axenic laboratory strains of E. histolytica, and fresh clinical isolates (Keene et al., 1986Keene et al., , 1990). Antibodies to the proteinase were detected in 83% of patients with invasive disease but were not detected in patients with noninvasive infections (Reed et al., 1989a,b; Hellberg et al., 2000 ). ...
... The potential multifactorial roles of cysteine proteinases in invasion of pathogenic amebae are well documented (Keene et al., 1990; Riekenberg et al., 2005). They include degradation of host extracellular matrix and mucoproteins, dislodgment of epithelial cells and degradation of epithelial basement membrane, and possibly recruitment of inflammatory cells to sites of ameba invasion by activation of complement. ...
The amebiasis cysteine proteinase gene (ACP1) encoding an antigen from Entamoeba histolytica, as well as the recombinant ACP1, obtained by cloning and expression of the ACP1 gene in heterologous host Escherichia coli BL-21 (DE3), were used to evaluate their ability to induce immune protective responses in minipig against challenge infection in a minipig -E. histolytica model. There was a 64.52% reduction (P<0.001) in the group of recovery of challenged E. histolytica compared with that in the control group. Specific anti-ACP1 antibodies from immune protected minipig had significantly higher levels of immunoglobulin G (IgG) (P<0.001). Our data indicate recombinant ACP1 may be a potential target as a vaccine antigen.
... Trophozoites can damage host cells through direct contact or close proximity, as well as through phagocytic activity towards dead and dying host cells in a receptor-mediated fashion [14]. Of the molecules secreted by amoebas, cysteine proteases are particularly important [15][16][17][18][19][20]. They are responsible for a cytolytic effect on host cells [7], the modulation of the cellmediated immune response, and the proteolysis of the host extracellular matrix [16,[21][22][23][24][25]. ...
... Extensive tissue damage has been attributed to the cysteine proteinases of E. histolytica because they are (i) secreted in large quantities and can cleave extracellular matrix proteins, thus facilitating amebic invasion; (ii) secreted in higher quantities by virulent than nonvirulent trophozoites; and (iii) found to participate in the inflammation of the gut and ALA [15][16][17][18][19][20]27]. However, cysteine proteases are dispensable for phagocytosis and cytopathogenicity [28,29]. ...
The molecular mechanisms by which Entamoeba histolytica causes amebic liver abscess (ALA) are still not fully understood. Amebic mechanisms of adherence and cytotoxic activity are pivotal for amebic survival but apparently do not directly cause liver abscess. Abundant evidence indicates that chronic inflammation (resulting from an inadequate immune response) is probably the main cause of ALA. Reports referring to inflammatory mechanisms of liver damage mention a repertoire of toxic molecules by the immune response (especially nitric oxide and reactive oxygen intermediates) and cytotoxic substances released by neutrophils and macrophages after being lysed by amoebas (e.g., defensins, complement, and proteases). Nevertheless, recent evidence downplays these mechanisms in abscess formation and emphasizes the importance of peroxynitrite (ONOO(-)). It seems that the defense mechanism of amoebas against ONOO(-), namely, the amebic thioredoxin system (including peroxiredoxin), is superior to that of mammals. The aim of the present text is to define the importance of ONOO(-) as the main agent of liver abscess formation during amebic invasion, and to explain the superior capacity of amoebas to defend themselves against this toxic agent through the peroxiredoxin and thioredoxin system.
... As a consequence, numerous attempts to identify and characterize molecules involved in Entamoeba virulence have focussed on the role of cysteine proteinases. Following these investigations, amoeba cysteine proteinases appear to be indeed responsible for a number of effects considered important for E. histolytica pathogenicity, as outlined by the following results: (i) cultured fibroblast monolayers are disrupted by purified E. histolytica cysteine proteinases (Keene et al., 1990), probably by their activity to degrade extracellular matrix components such as fibronectin, laminin, and collagen types I, IV and V (Bracha and Mirelman, 1984;Luaces and Barrett, 1988;Schulte and Scholze, 1989); (ii) amoeba virulence is directly correlated with cysteine proteinase activity in respective trophozoite extracts (Gadasi and Kessler, 1983;Lushbaugh et al., 1985;Keene et al., 1990); and (iii) liver-abscess formation is inhibited by specific cysteine proteinase inhibitors Li et al., 1995). In addition, the importance of cysteine proteinases for amoeba pathogenicity was further supported by comparing E. histolytica with Entamoeba dispar. ...
... As a consequence, numerous attempts to identify and characterize molecules involved in Entamoeba virulence have focussed on the role of cysteine proteinases. Following these investigations, amoeba cysteine proteinases appear to be indeed responsible for a number of effects considered important for E. histolytica pathogenicity, as outlined by the following results: (i) cultured fibroblast monolayers are disrupted by purified E. histolytica cysteine proteinases (Keene et al., 1990), probably by their activity to degrade extracellular matrix components such as fibronectin, laminin, and collagen types I, IV and V (Bracha and Mirelman, 1984;Luaces and Barrett, 1988;Schulte and Scholze, 1989); (ii) amoeba virulence is directly correlated with cysteine proteinase activity in respective trophozoite extracts (Gadasi and Kessler, 1983;Lushbaugh et al., 1985;Keene et al., 1990); and (iii) liver-abscess formation is inhibited by specific cysteine proteinase inhibitors Li et al., 1995). In addition, the importance of cysteine proteinases for amoeba pathogenicity was further supported by comparing E. histolytica with Entamoeba dispar. ...
In order to identify molecules that might be responsible for the difference in pathogenicity between the two closely related protozoan parasites Entamoeba histolytica and Entamoeba dispar, we focussed on cysteine proteinases because this class of enzymes has been considered important for pathogenic tissue destruction. By screening a genomic library derived from an E. histolytica isolate, a total of six distinct genes (ehcp1–ehcp6) encoding typical prepro-forms of cysteine proteinases were identified which differed from each other by 40% to 85% of their nucleotide sequences. Three of these genes, ehcp1, ehcp2, and ehcp5, which exhibited high levels of expression, were found to be responsible for approximately 90% of cysteine proteinase transcripts, whereas the remaining three were either not or only marginally expressed. Expression of the different genes directly correlated with the level of activity of the respective enzymes in trophozoite lysates. Purification of the enzymes and N-terminal sequencing revealed that virtually all cysteine proteinase activity of E. histolytica can be attributed to three enzymes namely EhCP1, EhCP2 and EhCP5. Southern blot analysis indicated that just two of these abundantly expressed genes are missing in E. dispar. On the other hand, genes analogous to four of the six genes identified in E.histolytica were found to be present in E. dispar, but only two of these are expressed within the trophozoite stage.
... The main lytic activity has been attributed to cysteine endopeptidases. This class of enzymes, which is found in all organisms, plays a major role in the pathogenicity of E. histolytica as demonstrated in a large number of in vitro and in vivo studies (Ankri et al., 1999;Gadasi and Kessler, 1983;Keene et al., 1990;Li et al., 1995;Luaces and Barrett, 1988;Lushbaugh et al., 1985;Reed et al., 1989;Schulte and Scholze, 1989;Stanley et al., 1995). ...
... Thiol-dependent proteolytic activity in E. histolytica was first attributed to a neutral sulphydryl proteinase (McLaughlin and Faubert, 1977) and later to a cytotoxic proteinase (Lushbaugh et al., 1984). 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). ...
... As has been shown previously, the cysteine protease inhibitor E64 (100 M) almost completely blocked cell monolayer destruction. The fact that nearly all monolayer destruction can be blocked with E64 implies that EhMSP-1 does not participate directly in monolayer damage, which is due primarily to the cysteine proteases (32,47). Rather, EhMSP-1 likely plays an indirect role in monolayer destruction. ...
... Silencing EhMSP-1 expression dramatically increases E. histolytica adherence to fixed Chinese hamster ovary cell monolayers and live or apoptotic Jurkat lymphocytes, while also substantially decreasing cell monolayer destruction. The ability of the cysteine protease inhibitor E64 to block cell monolayer destruction almost completely in this study and in others suggests that EhMSP-1 silencing affects monolayer destruction indirectly and that EhMSP-1 itself is not likely to directly degrade cell monolayers (32,47). Silenced trophozoites were also less motile than control amebas when interacting with cell monolayers, less efficient at transwell migration, and more efficient at phagocytosis of Jurkat lymphocytes. ...
Invasive amebiasis due to Entamoeba histolytica infection is an important cause of morbidity in developing countries. The E. histolytica genome contains two homologues to the metalloprotease leishmanolysin gene, Entamoeba histolytica MSP-1 (EhMSP-1) and EhMSP-2, while the commensal ameba Entamoeba dispar has lost EhMSP-1. In this study, we sought to characterize E. histolytica metallosurface protease 1 (EhMSP-1). Using immunoprecipitation and a model substrate, we found that EhMSP-1 was a functional
metalloprotease. Confocal microscopy and flow cytometry revealed that EhMSP-1 localized to the cell surface and revealed the
existence of distinct, nonclonal trophozoite populations with high and low EhMSP-1 surface abundance that became synchronized
following serum starvation. Phenotypic assays were performed after silencing EhMSP-1. Adherence of EhMSP-1-deficient trophozoites to tissue culture cell monolayers was more than five times greater than that
of control amebas, but surface staining of several antigens, including the galactose adherence lectin, was unchanged. EhMSP-1 silencing similarly increased adherence to both viable and apoptotic Jurkat lymphocytes. Tissue culture cell monolayer destruction
was reduced by EhMSP-1 silencing, although it was blocked almost completely by inhibiting cysteine proteases. Consistent with a primary defect in
regulation of amebic adherence, EhMSP-1 silencing also resulted in reduced mobility on tissue culture cell monolayers and in increased phagocytosis. In conclusion,
EhMSP-1 was shown to be a surface metalloprotease involved in regulation of amebic adherence, with additional effects on cell
motility, cell monolayer destruction, and phagocytosis.
... The glycolytic activity of the thiol-dependent protein (-SH) in E. histolytica is attributed first to the neutralizing sulphydryl proteinase [41] and then to the cytotoxic proteinase [42]. 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]. ...
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.
... A heavy chain of Gal/GalNAc lectin (Hgl) is attached to the intracellular action, and a signal is transmitted to epithelial cells, and β 2 , β 7 integrinsare activated. These events contribute to the increase of cell membrane permeability of Ca 2+ and the synthesis of miR643 that indirectly triggers apoptosis [9][10][11][12]. One of the apoptosis effects on PMS2, MSH1, MSH2, MSH3, and MSH6 genes is a mutation in the function of these genes, which destabilize the repair of nucleic acid, nucleotides elimination, and insufficiency of DNA mismatch repairing. ...
The mortality rate of Entamoeba histolytica is still high and approximately 100,000 per year. Environmental factors and different pathogens can cause microsatellite instability (MSI) positive, which may be one reason for colorectal cancer. MSI status can play an essential role in treatment. Moreover, E. histolytica might be one of the pathogens which raise the incidence of colorectal cancer. Therefore, the probable relationship of E. histolytica with MSI production was evaluated. Four hundred samples of colorectal biopsies based on pathological reports were divided into four groups: colitis, polyps, hyperplasia or dysplasia, and adenocarcinoma. The prevalence of E. histolytica was examined with PCR and immunohistochemical staining (IHC) for the light chain lectin HK-9. The adenocarcinoma formalin-fixed paraffin-embedded colorectal tumours sections were tested for MSI genes. We detected E. histolytica in 6% and 4% of colitis samples by PCR and IHC technique, respectively. However, it did not identify in polyp and hyperplasia samples. The MSI test was examined in the colorectal cancer group, which became positive in 19%. Entamoeba histolytica was detected in 26.3% (5/19) of MSI-positive and 2.5% (2/81) of MSI-negative cases by IHC technique however was not identified by PCR assay in this group. It is concluded PCR and IHC assay is recommended as complementary tests in colitis biopsies. Simultaneous PCR and IHC negative results could confirm the non-existence of the parasite with more confidence. Consequently, E. histolytica might be one of the biotic factors which raise the incidence of colorectal cancer because of the coincidence of the IHC positive results in MSI-positive adenocarcinoma.
... The other essential virulence factor in the pathogenesis of Entamoeba is its secretion of cysteine proteases (EhCP),which digests proteins of the extracellular matrix, enabling trophozoites to penetrate deeper into the tissue of intestinal submucosa and disperse this layer [31,32]. Through in vitro assays, digestion of purified proteins (collagen, elastin, fibrinogen and laminin) has been measured and the results compared between E. histolytica and E. dispar, as well as between strains of different virulence [33]. ...
[This corrects the article DOI: 10.1371/journal.pone.0181962.].
... The cytopathic effect correlates with the amount of cysteine proteinase activity released into the medium by clinical isolates of E. histolytica (89) and can be inhibited by specific peptide inhibitors (84). Mutants of E. histolytica strain HM-1 which are deficient in both proteinase expression and cytopathic effect have been identified (45). The in vitro cytopathic effect correlates with the early pathology of invasion in animal models in which the intestinal epithelial cells separate before making direct contact with trophozoites, presumably from disruption of the extracellular matrix (118). ...
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.
... The other essential virulence factor in the pathogenesis of Entamoeba is its secretion of cysteine proteases (EhCP),which digests proteins of the extracellular matrix, enabling trophozoites to penetrate deeper into the tissue of intestinal submucosa and disperse this layer [31,32]. Through in vitro assays, digestion of purified proteins (collagen, elastin, fibrinogen and laminin) has been measured and the results compared between E. histolytica and E. dispar, as well as between strains of different virulence [33]. ...
We sought to establish an ex vivo model for examining the interaction of E. histolytica with human tissue, using precision-cut liver slices (PCLS) from donated organs. E. histolytica- or E. dispar-infected PCLS were analyzed at different post-infection times (0, 1, 3, 24 and 48 h) to evaluate the relation between tissue damage and the expression of genes associated with three factors: a) parasite survival (peroxiredoxin, superoxide dismutase and 70 kDa heat shock protein), b) parasite virulence (EhGal/GalNAc lectin, amoebapore, cysteine proteases and calreticulin), and c) the host inflammatory response (various cytokines). Unlike E. dispar (non-pathogenic), E. histolytica produced some damage to the structure of hepatic parenchyma. Overall, greater expression of virulence genes existed in E. histolytica-infected versus E. dispar-infected tissue. Accordingly, there was an increased expression of EhGal/GalNAc lectin, Ehap-a and Ehcp-5, Ehcp-2, ehcp-1 genes with E. histolytica, and a decreased or lack of expression of Ehcp-2, and Ehap-a genes with E. dispar. E. histolytica-infected tissue also exhibited an elevated expression of genes linked to survival, principally peroxiredoxin, superoxide dismutase and Ehhsp-70. Moreover, E. histolytica-infected tissue showed an overexpression of some genes encoding for pro-inflammatory interleukins (ILs), such as il-8, ifn-γ and tnf-α. Contrarily, E. dispar-infected tissue displayed higher levels of il-10, the gene for the corresponding anti-inflammatory cytokine. Additionally, other genes were investigated that are important in the host-parasite relationship, including those encoding for the 20 kDa heat shock protein (HSP-20), the AIG-1 protein, and immune dominant variable surface antigen, as well as for proteins apparently involved in mechanisms for the protection of the trophozoites in different environments (e.g., thioredoxin-reductase, oxido-reductase, and 9 hypothetical proteins). Some of the hypothetical proteins evidenced interesting overexpression rates, however we should wait to their characterization. This finding suggest that the present model could be advantageous for exploring the complex interaction between trophozoites and hepatocytes during the development of ALA, particularly in the initial stages of infection.
... CPs have been described as major pathogenicity factors of E. histolytica. In several studies, a direct correlation between CP activity and ALA formation was observed [19,[28][29][30][31]. In addition, ALA formation can be inhibited by specific cysteine peptidase inhibitors, and overexpression and silencing of individual E. histolytica cp genes can alter the ALAs-inducing ability of amoebae [3,6,[32][33][34][35][36]. Furthermore, several studies indicate that especially EhCP-A5 is involved in the invasion process into the intestinal mucosa [37][38][39]. ...
We here compared pathogenic (p) and non-pathogenic (np) isolates of Entamoeba histolytica to identify molecules involved in the ability of this parasite to induce amoebic liver abscess (ALA)-like lesions in two rodent models for the disease. We performed a comprehensive analysis of 12 clones (A1-A12) derived from a non-pathogenic isolate HM-1:IMSS-A and 12 clones (B1-B12) derived from a pathogenic isolate HM-1:IMSS-B. "Non-pathogenicity" included the induction of small and quickly resolved lesions while "pathogenicity" comprised larger abscess development that overstayed day 7 post infection. All A-clones were designated as non-pathogenic, whereas 4 out of 12 B-clones lost their ability to induce ALAs in gerbils. No correlation between ALA formation and cysteine peptidase (CP) activity, haemolytic activity, erythrophagocytosis, motility or cytopathic activity was found. To identify the molecular framework underlying different pathogenic phenotypes, three clones were selected for in-depth transcriptome analyses. Comparison of a non-pathogenic clone A1np with pathogenic clone B2p revealed 76 differentially expressed genes, whereas comparison of a non-pathogenic clone B8np with B2p revealed only 19 differentially expressed genes. Only six genes were found to be similarly regulated in the two non-pathogenic clones A1np and B8np in comparison with the pathogenic clone B2p. Based on these analyses, we chose 20 candidate genes and evaluated their roles in ALA formation using the respective gene-overexpressing transfectants. We conclude that different mechanisms lead to loss of pathogenicity. In total, we identified eight proteins, comprising a metallopeptidase, C2 domain proteins, alcohol dehydrogenases and hypothetical proteins, that affect the pathogenicity of E. histolytica.
... However, occasionally E. histolytica penetrates the intestinal mucosa, which leads to ulcerative colitis or it disseminates to other organs, most commonly to the liver, where it induces abscess formation. Cysteine peptidases are considered to play a major role for the pathogenicity of E. histolytica as suggested by a large number of in vitro and in vivo studies [1][2][3][4][5][6][7][8][9]. Most convincing are results from infections of laboratory animals indicating that E. histolytica trophozoites that have reduced cysteine peptidase activity are greatly impaired in their ability to induce amoebic liver abscesses [8,9]. ...
... essential for the establishment and survival of the parasites; they play an important role in the development and pathogenesis of several parasitic infections and have been proposed as targets in the structure-based strategy of drug design (Sakanari et al., 1997;Schaeffer et al., 2011). Cysteine peptidases from trophozoites of Entamoeba histolytica were found to have a cytopathic effect on mammalian cells (Keene et al., 1990); cysteine proteases of Plasmodium falciparum are involved in the degradation of host haemoglobin (Rosenthal, 1995); cathepsin B has been purified from intracellular and extracellular extracts from all stages of Trypanosoma cruzi (Nóbrega et al., 1998); cysteine peptidase involvement in host cell invasion by sporozoites of Eimeria tenella was deduced because cell invasion was inhibited by cysteine peptidase inhibitors (Schaeffer et al., 2011). In the case of the genus Perkinsus, other type of proteases, serine peptidases, have been found in P. marinus ECPs and been proposed as possible virulence factors responsible for tissue degradation in infected oysters (La Peyre et al., 1995, 1996; the major extracellular protease (a N-glycosylated serine peptidase) produced by P. marinus in vitro was characterised and designed as perkinsin . ...
The variability of the protein expression profiling in the extracellular products (ECPs) of in vitro cultured Perkinsus olseni deriving from 4 regions of the Spanish coast was evaluated. The regions involved were the rías of Arousa and Pontevedra (Galicia, NW Spain), Carreras River (Andalusia, SW Spain) and Delta de l'Ebre (Catalonia, NE Spain). P. olseni in vitro clonal cultures were produced from parasite isolates from four clams from each region. Proteins released by the in vitro cultured parasites were isolated and separated by two dimensional electrophoresis (2DE). Qualitative comparison of protein expression profiles in the P. olseni ECPs among clones from all the regions was performed with PD Quest software. Around 130 spots were counted in the gels from ECPs of P. olseni clones from each region, of which 23 spots were shared by clones from all the regions and various spots were representative from clones of one region (appear in every clonal culture from that region but did not in every one of the other regions). A total of 34 spots were excised from the gels and analysed for sequencing. The protein cathepsin B, involved in proteolysis, the signal recognition particle receptor subunit β, involved in protein transport through membranes, and a protein belonging to N-acetyl transferase superfamily, involved in biosynthesis, were identified in spots shared by P. olseni ECPs from all regions. Pepsin A precursor, involved in proteolysis; heat shock protein (HSP) 60; and phosphoserin aminotransferase, involved in biosynthesis, were representative of P. olseni ECPs from Ría de Arousa, while peroxiredoxin V, involved in oxidation-reduction, was representative of P. olseni ECPs from Ría de Pontevedra. Differences in released proteins suggest different virulence or resistance to host attack between parasites from different locations.
... However, it is known that EhMSP-1 knockdown does not affect steady-state surface abundance of previously identified adhesins such as the GalNAc-specific lectin and the serine-rich E. histolytica protein (SREHP) [102][103][104]. The ability of the CP inhibitor E-64 to almost completely block cell monolayer destruction suggests that EhMSP-1 silencing affects monolayer destruction indirectly and that EhMSP-1 itself likely does not directly degrade cell monolayers [91,102,105]. Apparently, EhMSP-1 plays no role in in vitro resistance to complement, in contrast to leishmanolysin. ...
... However, it is known that EhMSP-1 knockdown does not affect steady-state surface abundance of previously identified adhesins such as the GalNAc-specific lectin and the serine-rich E. histolytica protein (SREHP)102103104. The ability of the CP inhibitor E-64 to almost completely block cell monolayer destruction suggests that EhMSP-1 silencing affects monolayer destruction indirectly and that EhMSP-1 itself likely does not directly degrade cell monolayers [91, 102, 105]. Apparently, EhMSP-1 plays no role in in vitro resistance to complement, in contrast to leishmanolysin. ...
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.
... Interesting findings have been reported concerning the CPs of protozoa such as Plasmodium [39], Trichomonads [40], Entamoeba [41], Cryptosporidium [42], Eimeria [43], Toxoplasma [44], and Naegleria [45]. The majority of the proteinases detected in T. cruzi, T. brucei and various Leishmania species are also cysteine proteinases. ...
Whole genome sequences of microbial pathogens present new opportunities for clinical application. Presently, genome sequencing of the human protozoan parasite Leishmania major is in progress. The driving forces behind the genome project are to identify genes with key cellular functions and new drug targets, to increase knowledge on mechanisms of drug resistance and to favor technology transfer to scientists from endemic countries. Sequencing of the genome is also aimed at the identification of genes that are expressed in the infectious stages of the parasite and in particular in the intracellular form of the parasite. Several protective antigens of Leishmania have been identified. In addition to these antigens, lysosomal cysteine proteinases (CPs) have been characterized in different strains of Leishmania and Trypanosoma, as new target molecules. Recently, we have isolated and characterized Type I (CPB) and Type II (CPA) cysteine proteinase encoding genes from L. major. The exact function of cysteine proteinases of Leishmania is not completely understood, although there are a few reports describing their role as virulence factors. One specific feature of CPB in Leishmania and other trypanosomatids, is the presence of a Cterminal extension (CTE) which is possibly indicative of conserved structure and function. Recently, we demonstrated that DNA immunization of genetically susceptible BALB / c mice, using a cocktail of CPB and CPA genes, induced long lasting protection against L. major infection. This review intends to give an overview of the current knowledge on genetic vaccination used against leishmaniasis and the importance of CP genes for such an approach.
... They have been isolated from axenic cultures of E. histolytica and have the property to degrade collagen, fibrinogen, elastin and laminin, extracellular matrix elements that trophozoites have to break through in order to cause invasive disease (Keene et al., 1986;Luaces and Barrett, 1988). These proteins are involved in the disruption of cellular monolayers (Keene et al., 1990;Lauwaet et al., 2004). Its inhibition with antisense codons decreases amebic phagocytosis, inflammation of the intestine, and the formation of ALA. ...
Precision-cut liver slices (PCLS) are mainly used to evaluate hepatotoxicity and metabolism of chemicals, as well as to study mechanisms of liver damage and repair. However, recently they have been used as a system to study amoebic infections. The aim of this study was to validate this model as an alternative for experimental amoebic liver absess (ALA) in animals. To do this, the PCLS was analyzed for the expression of amoebapore and cysteine proteinases 1 and 5, three of the most studied virulence factors of Entamoeba histolytica, as well as the induction of apoptosis and cytokines production in response to the ex vivo infection. PCHLS were prepared with the Brendel-Vitron tissue slicer and then, infected with 200,000 trophozoites of E. histolytica. Samples were taken at 0, 6, 12, 18, and 24h and compared to control non-infected slices. Morphological studies were performed in order to verify the infection; while apoptosis was studied by TUNEL and PAS techniques. The expression of cysteine proteinases (1 and 5), and amoebapore, was analyzed by real-time PCR. By using ELISA assays, the production of cytokines was also studied. PCHLS were found to be a reproducible infection system, and E. histolytica caused the expression of cysteine proteinases and amoebapore in infected slices. At the same time, trophozoites induce release of cytokines and apoptotic death of the hepatocytes close to them. PCHLS represent a new and suitable alternative model to study the pathogenesis of hepatic amoebiasis.
... The recombinant enzymes characterized to date are EhCP1 [77], EhCP2, EhCP3 [11], and EhCP5 [78]; all have similar substrate specificity, requiring arginine in the P2 position, as predicted by homology modeling [73]. In addition, they all can cleave a broad range of fundamental physiologic substrates, such as mucin [79], extracellular matrix proteins [80], inflammatory cytokines [70], and IgG [68]. ...
Proteases are successfully targeted in many human pathophysiologic and infectious diseases, exemplified by drugs against hypertension, osteoporosis, type II diabetes, and HIV/AIDS. Proteases that are indispensable or that function at critical bottlenecks in the parasite lifecycle are sought after, and several from parasitic protozoa impacting public health and animal welfare are currently being studied. Malaria alone infects 300-500 million people and causes approximately 3000 deaths daily, most of which represent children less than five years of age in sub-Saharan Africa. It is estimated that a total of 500 million humans are exposed to Trypanosoma brucei, Leishmania, and Trypansoma cruzi, the causative agents, respectively, of African sleeping sickness, leishmaniasis, and Chagas disease. Advances in genetic and selective chemical targeting of proteases in parasitic protozoa have shed light on the discrete biological roles that these enzymes play, and serve as a necessary tool in the target validation process. This chapter highlights the current status in the target validation process of all classes of proteases from parasitic protozoa of human clinical and veterinary significance.
... Several groups have reported enhancement of protease activity during tissue invasion by E. histolytica [11,21,22]. To further characterize the virulence proteins of this parasite, we report here the detection, purification and biochemical analysis of a novel E. histolytica cysteine protease, which is translocated from a cytoplasmic compartment to the plasma membrane during ingestion of red blood cells by the trophozoites. ...
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.
... CPs are considered to be an important virulence factor in the pathogenesis of amoebiasis and have been suggested to play a key role in tissue invasion and disruption of host tissues and modulation of cell-mediated immune response (Reed et al., 1993;Campbell and Chadee, 1997;Que and Reed, 1997). A direct correlation has been shown between the level of total CP activities of various strains, as determined by digestion of a chromogenic substrate, and the extent of cytopathic effect of trophozoites on tissue cultured monolayers of mammalian cells (De Meester et al., 1990;Keene et al., 1990). Good correlations have also been found between inhibition of CPs by the specific inhibitor L-transepoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and reduction of liver abscess formation (Stanley et al., 1995). ...
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.
... Cysteine proteinases have been shown to be important in parasite development and in pathogenesis (Berasain, Goni, McGonigle, Dowd, Dalton, Frangione & Carmona 1997;Coombs & Baxter 1984;Coombs, Hart & Capaldo 1982;Yamakami, Hamajima, Akao & Tadakuma 1995). The cysteine proteinase in Entamoeba histolytica has cytopathic effects on mammalian cells (Keene, Hidalgo, Orozco & McKerrow 1990) while that of Plasmodium falciparum degrades haemoglobin (Rosenthal 1995). ...
A monoclonal antibody (MAb-001), against a surface glycoprotein on Cryptobia salmositica inhibited the multiplication and oxygen consumption of both virulent and avirulent strains of the parasite. The classical cysteine proteinase inhibitor (E-64) and a cysteine proteinase activator (EDTA) affected the in vitro multiplication of C. salmositica. Concentrations of E-64 higher than 10 μM reduced the multiplication of C. salmositica while 5 mM of EDTA enhanced its multiplication. We propose that the cysteine proteinase is an important metabolic enzyme in C. salmositica and that binding of MAb-001 to the enzyme inhibited parasite multiplication and reduced oxygen consumption.
... A total of 19 bands ranged in molecular weight from 66 to 14 kDa were obtained using 12% resolution gel copolymerized with 0.2% gelatin. This result is comparable with other pathogenic protozoa where their proteinases have their apparent molecular weight in the range of 96–20 kDa (Keene et al., 1990; North et al., 1990; Robertson and Coombs, 1992). T. tenax isolates demonstrated a common as well as different proteinase expression patterns. ...
The role of Trichomonas tenax as a pathogen had been clearly implicated in various pathological processes that arise outside the boundaries of the mouth. Although a relationship between the increased occurrence of this protozoan and progression of periodontal disease has been demonstrated, the ability of T. tenax in causing oral infections and the precise mechanism of tissue damage is not well known. The present study aimed to investigate different isolates of T.tenax from individuals having oral infections. Plaques and/or calculi samples were collected from 70 individuals who were diagnosed as having periodontitis and/or gingivitis, then subjected to parasitological examination and culture on modified trypticase, yeast and iron medium (TYI-S-33). Isolates successfully maintained in culture were further subjected to analysis of protein profile of lysates by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and analysis of proteinases by non-denaturing gelatin-SDS-PAGE. Comparison of growth kinetics of seven T. tenax isolates showed a wide variability in the growth characteristics. Protein profiles of the seven isolates revealed a total 53 bands ranged in molecular weight (MW) from 5 to 95kDa using 12% resolution gel. Also, T. tenax isolates were found to possess 19 proteinase bands ranged in MW from 14 to 66kDa. The proteolytic bands were intensified by a cysteine proteinase activator and totally disappeared by treatment with a cysteine proteinase inhibitor suggesting that the proteinases were of cysteine proteinases type. The high frequency of T. tenax detected (28.6%) along with the variability in protein profiling and proteolytic activity of the isolates supports the possible pathogenicity of T. tenax and clarifies a conclusion that different strains with possibility of variable pathogenic potential may exist.
A book edited by J.Joseph Marr and Miklos Müller that sketches a picture of the state of the art of the biochemical research on parasites.
Abstract Strongyloidiasis is a neglected tropical disease caused by the soil-transmitted nematode by Strongyloides stercoralis, that affects approximately 600 million people worldwide. In immunosuppressed individuals disseminated strongyloidiasis can rapidly lead to fatal outcomes. There is no gold standard for diagnosing strongyloidiasis, and infections are frequently misdiagnosed. A better understanding of the molecular biology of this parasite can be useful for example for the discovery of potential new biomarkers. Interestingly, recent evidence showed the presence of small RNAs in Strongyloididae, but no data was provided for S. stercoralis. In this study, we present the first identification of miRNAs of both L1 and iL3 larval stages of S. stercoralis. For our purpose, the aims were: (i) to analyse the miRNome of L1 and iL3 S. stercoralis and to identify potential miRNAs of this nematode, (ii) to obtain the mRNAs profiles in these two larval stages and (iii) to predict potential miRNA target sites in mRNA sequences. Total RNA was isolated from L1 and iL3 collected from the stool of 5 infected individuals. For the miRNAs analysis, we used miRDeep2 software and a pipeline of bio-informatic tools to construct a catalog of a total of 385 sequences. Among these, 53% were common to S. ratti, 19% to S. papillosus, 1% to Caenorhabditis elegans and 44% were novel. Using a differential analysis between the larval stages, we observed 6 suggestive modulated miRNAs (STR-MIR-34A-3P, STR-MIR-8397-3P, STR-MIR-34B-3P and STR-MIR-34C-3P expressed more in iL3, and STR-MIR-7880H-5P and STR-MIR-7880M-5P expressed more in L1). Along with this analysis, we obtained also the mRNAs profiles in the same samples of larvae. Multiple testing found 81 statistically significant mRNAs of the total 1553 obtained (FDR
All mucins are highly glycosylated and a key constituent of the mucus layer that is vigilant against pathogens in many organ systems of animals and humans. The viscous layer is organized in bilayers, i.e., an outer layer that is loosely arranged, variable in thickness, home to the commensal microbiota that grows in the complex environment, and an innermost layer that is stratified, non-aspirated, firmly adherent to the epithelial cells and devoid of any microorganisms. The O-glycosylation moiety represents the site of adhesion for pathogens and due to the increase of motility, mucolytic activity, and upregulation of virulence factors, some microorganisms can circumvent the component of the mucus layer and cause disruption in organ homeostasis. A dysbiotic microbiome, defective mucus barrier, and altered immune response often result in various diseases. In this review, paramount emphasis is given to the role played by the bacterial species directly or indirectly involved in mucin degradation, alteration in mucus secretion or its composition or mucin gene expression, which instigates many diseases in the digestive, respiratory, and other organ systems. A systematic view can help better understand the etiology of some complex disorders such as cystic fibrosis, ulcerative colitis and expand our knowledge about mucin degraders to develop new therapeutic approaches to correct ill effects caused by these mucin-dwelling pathogens.
Protozoan parasitic infections of the gastrointestinal (GI) tract are being increasingly recognized as an important public health problem in the United States. The intestinal protozoan pathogens include both parasites that are extracellular, such as Giardia lamblia, Blastocystis hominis, and Entamoeba histolytica, and the intracellular spore-forming parasites Cryptosporidium parvum, Cyclospora cayetanensis, and Isospora belli. The AIDS epidemic has played an important role in the recognition of intracellular spore-forming parasites as important gastrointestinal protozoan pathogens. Stool microscopic examinations may be performed in clinical laboratories only if specifically requested. In some cases in which infection is suspected on clinical grounds, stool examination may not be revealing, and endoscopy may be necessary to make the diagnosis. The recent rapid development of new molecular assays, will likely facilitate more rapid laboratory diagnoses of not only protozoan, but also viral and bacterial pathogens in the near future.
Entamoeba histolytica is the cause of high levels of morbidity and mortality throughout the developing world, having been ranked as the third most important parasitic agent of disease after malaria and schistosomiasis. In 1981, it was estimated that up to 500 million people are infected annually [265]. The majority of infections are asymptomatic and apparently non-pathogenic. In most of these cases, spontaneous eradication of the parasite is thought to occur within several months [28, 153]. Some 10%, however, go on to develop invasive amoebiasis [201] in which symptoms range from mild and transient diarrhoea to fulminating colitis characterized by the passage of bloody stools. In some cases dissemination via the bloodstream may result in the production of extraintestinal abscesses in other organs, principally the liver. Invasive amoebiasis is held to account for around 75 000 deaths per year [79].
In October 2000, The Institute of Genomic Research (TIGR) received funding from the National Institutes of Health to sequence approximately 99% of the Entamoeba histolytica genome. There are many questions regarding the pathogenicity and biology of E. histolytica that will be fully addressed by the genome project. Practical applications, such as new diagnostic agents, vaccine candidates, and potential new drug targets, may also be revealed. This chapter updates the latest knowledge regarding these aspects of this parasite and indicates those areas in which the genome project is likely to have a particular impact.
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.
Analysis of the cell-free supernatants of Perkinsus marinus cultures by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining revealed the presence of as many as 17 bands ranging in molecular weight from 239 to 32 kDa. These bands were not present in un-inoculated medium. Moreover, P. marinus produces extracellular proteins that possess proteolytic activities; the cell-free supernatants of P. marinus cultures could digest a variety of proteins including gelatin, casein, fibronectin and laminin. Oyster plasma was also digested by cell-free culture supernatants. The proteolytic activity in cell-free culture supernatants was detected 24 h post-inoculation, while no proteolytic activity could be detected in cell lysates. The proteolytic activities were characterized using substrate-impregnated sodium dodecylsulfate-polyacrylamide gels and had approximate molecular weights ranging from 55 to 35 kDa. The proteolytic activity of cell-free culture supernatants was inhibited by the serine protease inhibitors phenylmethylsulphonyl fluoride, 3,4-dichloroisocoumarin and soybean trypsin inhibitor. In contrast, inhibitors (i.e. trans-epoxysucciny-1-leucylamido(4-guanidino)-butane, 1,10-phenanthroline, captopril, ethylenediaminetetracetic acid, pepstatin A or diazoacetyl-DL-norleucine methyl ester) from the other three classes of proteases had no effect. It was concluded that the P. marinus proteases in cell-free culture supernatants are serine proteases.
A 30-kDa cysteine proteinase was purified from extracts of axenically grown trophozoites of Entamoeba histolytica strain HM1:IMSS. The purification procedure involved two consecutive chromato,oraphic steps. Sequence analysis revealed high similarity with histolysin and with other 27-kDa cysteine proteinase. Western-blot analysis using F(ab')(2) fragments of a polyclonal antibody raised against the purified enzyme revealed that when the amebic extract was prepared in the absence of proteinase inhibitors there were many positive bands ranging in relative molecular weight from 115 to 12.5 kDa, but when the extract was prepared in the presence of proteinase inhibitors there was only a single 30-kDa positive band. Similar results were obtained with immunoprecipitates. This phenomenon would suggest the formation of multimer aggregates of the 30-kDa cysteine proteinase after partial proteolysis.
Publisher Summary
This chapter discusses the mechanisms of invasion of a parasite into host tissue or host cells. Single cell protozoan parasites evade the host immune response by residing within host cells and enter the host cell with minimal trauma to ensure that they preserve an environment suitable for their replication and do not trigger a lethal host immune response. This intracellular invasion paradigm is also shared by a helminth parasite, Trichinella spiralis. Often an invasive larval form penetrates skin or the mucosa of the gastrointestinal tract to enter into the host.. The chapter states that the invasion phenomenon itself can be divided into three successive steps: recognition and attachment, internalization and vacuole development, vacuole maturation. Entamoeba histolytica, which is a protozoan, does not invade intra cellularly at any stage of its life cycle unlike other protozoas. The process of its invasion (extracellularly) and its properties are also elaborated in the chapter. .To complete the invasion process, helminth parasites have evolved sensory organs for finding and navigating within the host, attachment mechanisms utilizing specialized mouth structures or glue-like secretions, and hydrolytic enzymes to digest macromolecular barriers in the extracellular space
The most important feature of the pathology of human amebiasis is the greatly destructive nature of the anatomical lesions produced by the protozoan Entamoeba histolytica. Recent advances on the knowledge of biochemistry, immunology, cellular and molecular biology, and genetics of this parasite, added to the use of different in vitro, in vivo, and ex vivo models to analyze host–parasite interactions or the production of intestinal and extraintestinal amebic lesions, all have given a better perception of the mechanisms of pathogenesis in amebiasis. The present chapter is divided into three parts: First, a general review of the pathology of human amebiasis; second, a short review of the mechanisms of invasion and production of damage in the host, and third, a review of the different in vivo experimental models currently available to study the mechanisms involved in amebic infection. In reference to pathogenesis, each factor, molecule or gene, or mechanism of target cell damage is reviewed individually in other chapters of this section on “Pathogenesis and Immunity” in the present book. Therefore, the meticulous or probing aspects of the studies are mentioned by each responsible and expert group of researchers. In this review, we mention in general each of the different factors of pathogenesis in amebiasis. The contributions obtained using different techniques and methodologies of experimental models are emphasized, and the subjects that still need to be unraveled to understand how this microscopic parasite has earned its well-deserved “histolytic” name are discussed.
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.
Publisher Summary
This chapter explains the metabolism of amino acid and protein. Many parasites can utilize amino acids as energy sources during their life cycle and most amino acids can be used by at least one group of parasites. The chapter focuses on recent findings and pathways that appear to be especially active in parasites or have unique features, which distinguish the parasite's metabolism.The ways in which parasite and host amino acid metabolism differ include the extent of alanine formation in many protozoan parasites and aspects of sulfur amino acid metabolism in anaerobic protozoa and helminths. Protein degradation provides a source of amino acids for many parasites. Many of the proteolytic enzymes likely to be involved are well characterized and the roles played by some of these in specific proteolytic processes are being identified. In many respects the basic features of amino acid and protein metabolism of parasitic protozoa and helminths resemble those of their mammalian hosts. Proteins are broken down extracellularly or within lysosomes, and amino acids taken up and used for biosynthesis or energy metabolism. The chapter summarizes that although, many of the features of parasite amino acid and protein metabolism resemble those of the hosts, there are marked differences with respect to the properties of the enzymes involved, the relative importance of common pathways and the presence of parasite-specific pathways.
Perkinsus marinus causes devastating losses in populations of the eastern oyster (Crassostrea virginica). Our studies have demonstrated that P. marinus secretes extracellular serine proteases which enhance parasite propagation and compromise host defences. Crassostrea virginica, however, possesses several inhibitors of these proteases. The Pacific oyster (C. gigas) is resistant to P. marinus and possesses protease inhibitors with significantly higher specific activities than those in C. virginica. Interestingly, Crassostrea spp. themselves, elaborate metalloprotease activities which can be detected in their plasma, and are increased during P. marinus infections. Together our work suggests that there may be a broad spectrum of humoral host defences that is brought to bear on P. marinus infections by these two Crassostrea species.
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.
Lysosomes are acidic intracellular vacuoles of heterogeneous shape, size, and content. Lysosomes contain hydrolytic enzymes that degrade proteins, lipids, carbohydrates, and nucleic acids derived from intracellular (through autophagy) and extracellular (through heterophagy) sources. Lysosomal degradation regulates several physiological cell functions. These include turnover of cellular organelles and extracellular constituents; amino acid and glucose homeostasis; processing of proteins; lipid metabolism; cell growth, differentiation, and involution; host defenses against microorganisms and other pathogens; and removal of necrotic and foreign material from the circulation and from tissues.
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.
Cysteine peptidases (CPs) of Entamoeba histolytica are considered to be important pathogenicity factors. Previous studies have found that under standard axenic culture conditions, only four (ehcp-a1, ehcp-a2, ehcp-a5, and ehcp-a7) out of 35 papain-like ehcp genes present in the E. histolytica genome are expressed at high levels. Little is known about the expression of CPs in E. histolytica during amoebic liver abscess (ALA) formation. In the current study, a quantitative real-time PCR assay was developed to determine the expression of the various ehcp genes during ALA formation in animal models. Increased expression of four ehcp genes (ehcp-a3, -a4, -a10, and -c13) was detected in the gerbil and mouse models. Increased expression of another three ehcp genes (ehcp-a5, -a6, and -a7) was detected in the mouse model only, and two other ehcp genes (ehcp-b8 and -b9) showed increased expression in the gerbil model only. Trophozoites of the nonpathogenic E. histolytica HM-1:IMSS clone A1, which was unable to induce ALAs, were transfected with vectors enabling overexpression of those CPs that are expressed at high levels under culture conditions or during ALA formation. Interestingly, overexpression of ehcp-b8, -b9, and -c13 restored the pathogenic phenotype of the nonpathogenic clone A1 whereas overexpression of various other peptidase genes had no effect on the pathogenicity of this clone.
Analysis of the cell-free supernatants of Perkinsus marinus cultures by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining revealed the presence of as many as 17 bands ranging in molecular weight from 239 to 32 kDa. These bands were not present in un-inoculated medium. Moreover, P. marinus produces extracellular proteins that possess proteolytic activities; the cell-free supernatants of P. marinus cultures could digest a variety of proteins including gelatin, casein, fibronectin and laminin. Oyster plasma was also digested by cell-free culture supernatants. The proteolytic activity in cell-free culture supernatants was detected 24 h post-inoculation, while no proteolytic activity could be detected in cell lysates. The proteolytic activities were characterized using substrate-impregnated sodium dodecylsulfate-polyacrylamide gels and had approximate molecular weights ranging from 55 to 35 kDa. The proteolytic activity of cell-free culture supernatants was inhibited by the serine protease inhibitors phenylmethylsulphonyl fluoride, 3,4-dichloroisocoumarin and soybean trypsin inhibitor. In contrast, inhibitors (i.e. trans-epoxysuccinyll-leucylamido(4-guanidino)-butane, 1, 10-phenanthroline, captopril, ethylenediaminetetracetic acid, pepstatin A or diazoacetyl-DL-norleucine methyl ester) from the other three classes of proteases had no effect. It was concluded that the P. marinus proteases in cell-free culture supernatants are serine proteases.
AmebiasisBlastocystis hominisGiardiasisDientamoeba fragilisBalantidiasisCoccidia (Cryptosporidium, Isospora, and Cyclospora)
Cysteine proteinases of protozoan and helminth parasites are considered to have a high potential as targets for novel antiparasite agents. This has stimulated research on the enzymes and a large body of data has now been accumulated. This Perspective provides an overview of the current situation, with recent advances being highlighted. Emphasis is given to the Type I cysteine proteinases of trypanosomatid protozoa, which are atypical in having an extra C-terminal domain, and the asparaginyl endopeptidase ofSchistosoma, for which the only homologues known are those in plants. The locations of parasite cysteine proteinases are described, with emphasis on the extralysosomal sites, and the putative roles of the enzymes in host-parasite interactions, including parasite nutrition and host invasion, are discussed. The major advances being made in elucidating cysteine proteinase function in protozoan parasites through genetic manipulation, including targeted gene deletion, are presented, with the Type I cysteine proteinases ofLeishmania being used as an example. The importance of this approach in the validation of the enzymes as drug targets is discussed. The current status of attempts to exploit parasite cysteine proteinases with drugs is presented, and future prospects are outlined.
The recent development of axenic culture for E. dispar allowed us to examine this ameba's ability to bind and lyse target cells and compare it to E. histolytica which has been axenically cultured for years. We found that under axenic conditions, E. dispar's adherence to target cells, ligand binding, and cytotoxicity were less than that of E. histolytica. These events were Gal/GalNAc-inhibitable supporting the idea that E. dispar expresses a lectin similar to E. histolytica. Genetic analysis showed that E. dispar had at least two members of the lectin heavy subunit family and four members of the lectin light subunit family that hybridized to ehhgl and ehlgl gene probes. A library screen produced clones which were isolated and sequenced. Derived amino acid sequences showed that the E. dispar heavy and light subunit clones were 86% and 79% identical, respectively, to their E. histolytica counterparts. In particular, the region which contains the epitopes for two adherence-enhancing monoclonal antibodies and a complement-sensitizing monoclonal antibody (amino acids 882–959 of the lectin heavy subunit) were conserved between the species.
In this paper, we attempted to define the role of phagocytosis in the virulence of Entamoeba histolytica. We have isolated, from a highly phagocytic and virulent strain, a clone deficient in phagocytosis. Trophozoites of wild-type strain HM1:IMSS were fed with Escherichia coli strain CR34-Thy- grown on 5-bromo,2'-deoxyuridine. The trophozoites that had incorporated the base analog through phagocytosis of the bacteria were killed by irradiation with 310 nm light. The survivors, presumably trophozoites defective in phagocytosis, were grown until log phase and submitted two more times to the selection procedure. Clone L-6, isolated from a subpopulation resulting from this selection procedure, showed 75-85% less erythrophagocytic activity than the wild-type strain. The virulence of clone L-6 and strain HM1:IMSS was measured. The inoculum required to induce liver abscesses in 50% of the newborn hamsters inoculated (AD50) of HM1:IMSS was 1.5 X 10(4) trophozoites. Clone L-6 trophozoites failed to induce liver abscesses in newborn hamsters even with inocula of 5 X 10(5) trophozoites. Virulence revertants were obtained by successive passage of L-6 trophozoites through the liver of young hamsters. The trophozoites that recovered the ability to produce liver abscesses simultaneously recuperate high erythrophagocytic rates. These results show that phagocytosis is involved in the aggressive mechanisms of E. histolytica.
The major excreted protein (MEP) of mouse fibroblast cells is the 39,000 Mr precursor to a lysosomal acid protease (cathepsin L) induced by malignant transformation, growth factors, and tumor promoters. We have cloned and characterized the gene for MEP from NIH-3T3 cells. This cosmid clone (pcosMMEP), containing the unique 12,000-base pair mouse MEP gene, has been transfected into monkey kidney (CV-1) cells and human epidermoid carcinoma (A431) cells. The stable A4MEP transfectants produce mouse MEP that is an active cathepsin which is secreted, glycosylated, and processed intracellularly to lower molecular weight forms as in the wild-type NIH-3T3 cells. The CVMEP cells (nontransformed phenotype) produce quantities of mouse MEP similar to that found in NIH-3T3 cells, whereas the A4MEP cells (transformed phenotype) produce greater amounts of MEP similar to the levels seen in Kirsten virus-transformed NIH-3T3 cells. The MEP mRNAs from both mouse cells and stably transfected human cells are the same size and have the same single major site for initiation of transcription, indicating that the cloned mouse MEP promoter is active in transfected cells.
A peptidyl fluoromethyl ketone (Z-Phe-Ala CH2F) was found to be an effective compound in a time dependent inactivation of cathepsin B isozymes from a number of tissues including human tumors. The effect was visualized by employing an activity-specific fluorescent print technique preceded by isoelectric focusing. The technique could yield additional information of selective inhibition of isozymes as observed with rat pancreas. The fluoromethyl ketone is 30-fold more potent than the known inhibitor of cathepsin B, Z-Phe-AlaCHN2 in parallel evaluation. Furthermore, the fluoromethyl ketone may have in vivo potential in the inhibition of cathepsin B, in view of the results of toxicological studies. The findings demonstrate that the application of enzyme-directed overlay membranes, impregnated with specific substrates, following isoelectric focusing could be very useful in the study of proteases and their involvement in the oncogenic process.
The tumor-induced marrow and red blood cell cytolysis assays have been used to explore the mechanism of cancer cell destruction of normal cells. Previously, we suggested that tumor-induced cytolysis was caused by tumor cell membrane-bound serine proteases. In this study, we have shown that concentrations of the broad-spectrum serine protease inhibitor diisopropylfluorophosphate that did not inhibit tumor cell DNA and protein synthesis completely abrogated tumor-induced red blood cell cytolysis. In addition, tumor cell membranes isolated by differential and sucrose density gradient centrifugation and characterized by electron microscopy and enzyme marker analysis were cytolytic for rat 59Fe-labeled red blood cells. The specific activity expressed as release index (%) per microgram of protein was 1.620 for the tumor cell membrane preparations as compared to 0.002 for intact Walker 256 tumor cells. Tumor cell membranes solubilized in Triton X-100 had activity in the p-toluenesulfonyl-L-arginine methyl ester assay for trypsin-like enzymes and the N-benzoyl-L-tyrosine ethyl ester assay for chymotrypsin-like enzymes. The enzyme activities demonstrated in these assays could be inhibited by N-alpha-p-tosyl-L-lysine chloromethyl ketone HCl and L-1-tosylamide-alpha-phenyl-ethyl chloromethyl ketone, respectively. Using [3H]diisopropylfluorophosphate affinity labeling of the tumor cell membrane proteins followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we have identified membrane-bound serine protease(s) that appear to be responsible for tumor-induced marrow and red blood cell cytolysis.
FPLC anion-exchange and chromatofocusing chromatography were used to purify the major neutral proteinase from secretions of axenically cultured Entamoeba histolytica trophozoites. HM-1 strain trophozoites, which were more proteolytically active than the less virulent HK-9 strain, were used for purification of the enzyme. It is a thiol proteinase with a subunit Mr of approximately 56,000, a neutral pH optimum, and a pI of 6. The importance of this enzyme in extraintestinal amoebiasis is suggested by its ability to degrade a model of connective tissue extracellular matrix as well as purified fibronectin, laminin, and type I collagen. The enzyme caused a loss of adhesion of mammalian cells in culture, probably because of its ability to degrade anchoring proteins. Experiments with a peptide substrate and inhibitors indicated that the proteinase preferentially binds peptides with arginine at P-1. It is also a plasminogen activator, and could thus potentiate host proteinase systems.
A cytolytic pore-forming protein (PFP, perforin) was purified from isolated granules of cloned NK-like cytolytic cells, which showed an apparent Mr of 70–75 kd (reduced) and 62–66 kd (nonreduced). Cytolysis produced by this protein occurred only in the presence of Ca2+ and was accompanied by the formation of membrane lesions of 160 Å diameter. The purified protein depolarized cells and made lipid vesicles leaky to monovalent and divalent ions. This protein formed large, voltage insensitive and nonselective ion channels in planar bilayers that remained preferentially in the open state. The channels were heterogeneous in size distribution averaging 400 pS/U in 0.1 M NaCl. The membrane lesions formed by PFP were morphologically and functionally similar to those formed by intact NK-like cells and their granules. This PFP could be released from granules during cell killing, followed by its polymerization on target membranes to form large transmembrane pores.
The enteric pathogen, Entamoeba histolytica, appears to cause disease by adhering to and then destroying mucosal barriers. Using an in vitro method of studying the interaction of E. histolytica with target cells (Chinese hamster ovary [CHO] and human erythrocytes [RBC]), we examined the mechanism of amebic adherence and its role in lysis of target cells. Killing and phagocytosis of target cells by amebas ceases at 4°C, allowing observation of adherence. Amebas adhere to CHO cells at 4°C, 78.9% formed rosettes (amebas with ≥3 adherent CHO cells each) at 2 h. At 37°C, cytochalasins B and D inhibit adherence of amebas to CHO cells (P < 0.0005). Amebas adhere to and kill CHO cells in media with <0.1 μM calcium and magnesium plus 10 mM EDTA, indicating that divalent cations are not required in the medium. Adherence of amebas to human RBC was not ABO blood group specific and showed greater adherence to human than bovine or sheep RBC (P < 0.005). Neither Fc nor complement receptors were found on amebas by standard rosette studies. The amebic adherence receptor is not trypsin (0.125%) sensitive nor inhibited by trypan blue (1 mM). N-acetyl-d-galactosamine (GALNAc) inhibited the adherence of amebas to CHO cells and human RBC (0.1 g/100 ml or 4.5 mM GALNAc, P < 0.005) by binding to a receptor on the amebic surface. GALNAc abolishes amebic cytolysis of target CHO cells (determined by 111Indium oxine release from CHO cells, P < 0.001) but not amebic phagocytosis of CHO cells. By suspending ameba-CHO cells rosettes in dextran, we found that GALNAc (1%) reversibly inhibits amebic adherence (P < 0.0005) and that cytochalasins decrease amebic killing of adherent CHO cells (P < 0.025).
These findings indicate that the adherence of E. histolytica to target cells requires microfilament function, is via a specific amebic receptor that has affinity for GALNAc, and is required to lyse cells. Inhibition of the adherence of E. histolytica may alter the pathogenicity of this organism.
The ability of tumor cells to invade into and through normal tissue during the metastatic cascade has been attributed to tumor-associated degradative enzymes including proteinases of the metallo, serine and cysteine classes. Work from several laboratories has established that the cysteine proteinases cathepsins L and B are released from tumor cells, primarily as latent precursor forms. In addition, a cathepsin B-like cysteine proteinase has been shown to be associated with the plasma membrane fraction of several animal and human tumors. This form of the enzyme retains activity under physiologic (or pathologic) conditions including at neutral pH and in the presence of low Mr inhibitors. Since we have established that cathepsin B can degrade the basement membrane attachment glycoprotein laminin, we speculate that plasma membrane-associated cathepsin B may participate in focal dissolution of the basement membrane during tumor cell extravasation.
The cellular bases of the powerful cytolytic activity of the human protozoan parasite Entamoeba histolytica were explored by studying the effect of the virulent strain HM1:IMSS on epithelial monolayers of MDCK cells using a combination of time-lapse microcinematography and transmission and scanning electron microscopy. Early alterations of the epithelial cell membranes were detected by measuring changes in the transepithelial electrical resistance of MOCK monolayers mounted in Ussing chambers. The aggressive mechanism of E. histolytica trophozoites was found to be a complex, multifactorial phenomenon that included hit-and-run damage to the plasma membrane of effector cells mediated through contact, phagocytosis of lysed or apparently intact, but detached, MDCK cells, and inlracellular degradation of ingested cells. Following contact with amebas, the epithelial monolayers showed a pronounced lowering of transepithelial resistance, opening of tight junctions, distortion of microvilli, surface blebbing, and the presence of minute focal discontinuities in the plasma membrane. There was no evidence of amebic exocytosis, membrane fusion, or junction formation between the parasite and host plasma membranes. Although modifications in the epithelial cell membranes usually preceded lysis, the cytolytic activity of the parasite did not exclusively involve damage to the plasma membrane of the cultured host cells but also was mediated by avid phagocytosis, the displacement and separation of neighboring cells by means of pseudopodial activity, and the “pinching-off” of the peripheral cytoplasm of epithelial cells.
A rapid and simple assay for cytopathogenicity of axenically cultivated Entamoeba histolytica has been developed employing baby hamster kidney (BHK) or mouse 3T3 cells conventionally tissue cultured. Three of the twelve amebal strains tested produced total destruction of the BHK cell monolayer in 2--3 hours, and these three strains are the three most virulent strains for the newborn hamster liver. Two additional strains were of moderate cytopathogenicity in vitro and of moderate virulence in vivo. Seven strains were of low cytopathogenicity and virulence. Within these three major groupings, however, the cytopathogenicity ranking was not entirely reproducible. The general correspondence of cytopathogenicity in vitro and virulence in vivo and the possibility of obtaining data within a few hours suggest such an assay may prove a useful tool in amebiasis research.
Entamoeba histolytica kills cells by contact dependent cytolysis. The mechanism underlying this process must be of rapid onset because target cells round up and show marked zeiosis within 15 min of contact. In earlier work, we identified a remarkable ion-channel forming protein which we named amoebapore, that may contribute to the amoeba-induced target cell killing. Within the amoeba it exists as part of a supramolecular aggregate together with other proteins of unknown function. In this work we report the purification of a solubilized form of the amoebapore. Amoebapore was found to exist as an apparent dimer of the previously reported protein whose molecular weight had been determined under denaturing conditions. Two isoforms of this dimer, with pI values of 6.8 and 5.3 present at a ratio of 7 to 1, were identified and purified. Both isoforms demonstrate ion-channel forming activity in planar lipid membranes. These channels show a unit conductance of 5-20 pS and remain open for less than 1 s. Upon lateral aggregation, opening becomes concerted to a greater degree with channel conductance are observed. The isolated particulate form of amoebapore depolarizes cells.
The major secreted proteinase of Entamoeba histolytica, a 56-kDa neutral cysteine proteinase, activates C by cleaving C3. The action of the proteinase is similar to C-derived C3 convertases because it produces a single cleavage of the alpha-chain in a dose- and time-dependent manner and cleaves C3 between residues 78 and 79, only one amino acid residue distal to the natural site acted on by the C3 convertases. C3a generation was detected by RIA. The 105-kDa fragment produced by the cleavage of the alpha-chain was structurally and functionally equivalent to the alpha'-chain of C3b, as demonstrated by susceptibility to the action of factors I and H and participation in the activation of the alternative pathway of C. Activation of C by the 56-kDa neutral cysteine proteinase may play a role in the early inflammatory response in amebic lesions and thus contribute to the pathogenesis of invasive amebiasis.
A cytotoxic cysteine proteinase with a molecular weight of 16,000 was isolated from axenically grown trophozoites of Entamoeba histolytica. The enzyme was purified from frozen-thawed strain HM-1 by ion-exchange chromatography on DEAE-cellulose, organomercurial agarose affinity chromatography, and size-exclusion chromatography. The purified enzyme had proteinase activity that could be demonstrated on azocasein (pH 5), hemoglobin (pH 5), or carbobenzoxy-L-arginyl--L-arginyl-7-amino-4-trifluoromethylcoumarin++ + (Z-arg-arg-AFC), a substrate specific for cathepsin B. Enzyme activity was stable to high pH, but not to 40 C for 1 hr or 56 C for 0.5 hr. As typical of cysteine proteinases, inhibition of activity on Z-arg-arg-AFC by p-chloromercuribenzoate or mercury was reversed by free sulfhydryl groups. Both the proteinase and cytotoxic activities of the purified amoebal cathepsin B were inhibited by leupeptin and serum and activated by free sulfhydryl groups, supporting the hypothesis that both activities are characteristics of amoebal cathepsin B. Virulent strains of E. histolytica (HM-1 and Rahman) had significantly more cathepsin B activity per milligram protein than less virulent strains (HK-9, Laredo, and Huff). The correlation between higher levels of cathepsin B activity in strains with greater virulence could indicate a role for amoebal cathepsin B in the pathogenesis of amoebiasis.
We have used chemical mutagenesis to isolate 10 independent phagocytosis-deficient mutants of Entamoeba histolytica from a highly virulent clone (strain HM1:IMSS). Phagocytosis-deficient mutants were characterized by several biologic properties
related to amoebic pathogenic mechanisms: virulence, adhesion to red blood cells, cytopathic effect of live trophozoites on
cell culture monolayers, ability of amoebic extracts to destroy cell culture monolayers, and growth in semisolid agar. All
of these mutants were also deficient in in vivo virulence and in vitro cytopathic effect. Three were also deficient in adhesion,
and the extracts of three others were unable to destroy cell culture monolayers. These findings suggest a strong correlation
between phagocytosis and in vivo and in vitro cytopathogenicity. Both adherence of trophozoites to target cells and toxin
activity of crude extracts of amoebae seem to be necessary factors for the expression of the virulence of this parasite. Traits
responsible for growth in semisolid agar do not seem to be involved in virulence.
The cysteine proteinase of Entamoeba histolytica was shown to hydrolyze the cyanogen bromide peptide alpha 1-CB2 of calf skin collagen type I yielding two split products. Amino acid analyses of the formed peptides and estimation of the amino-terminal residues revealed that the alpha 1-CB2 peptide was exclusively cleaved between Gly10 and Leu11 within the sequence -Arg9-Gly10-Leu11-yielding two peptides with 7 and 29 amino acids, respectively. Under identical conditions the ratio of hydrolysis of the Gly10-Leu11 bond in the alpha 1-CB2 peptide to the Gly23-Phe24 bond within the internal sequence -Arg22-Gly23-Phe24- of bovine insulin B-chain was 100:65. The enzyme was found to split both benzyloxycarbonyl-Arg-Arg-methoxy-2-naphthylamide and benzyloxycarbonyl-Arg-Gly-2-naphthylamide. The ratio of hydrolysis of these substances was 100:11.6. Benzyloxycarbonyl-Gly-Arg-2-naphthylamide was a very poor substrate for the enzyme.
We report a one-step method for the purification to homogeneity of a cysteine proteinase of Entamoeba histolytica (histolysin) by affinity chromatography of the soluble extract of the parasite on immobilized phenylalanyl(2-phenyl)aminoacetaldehyde semicarbazone. The enzyme has an apparent Mr of 26,000 by SDS/polyacrylamide-gel electrophoresis and 29,000 by gel chromatography. Its pH optimum varies widely, from 5.5 with azocasein to approx. 7 with other protein substrates and benzyloxycarbonylphenylalanyl-L-citrullylaminomethylcourmarin++ + (Z-Phe-Cit-NHMec), and to 9.5 with benzyloxycarbonylphenylalanylarginylaminomethylcoumarin (Z-Phe-Arg-NHMec) and benzyloxycarbonylarginylarginylaminomethylcourmarin (Z-Arg-Arg-NHMec). Values of Km, kcat. and kcat/Km are 1.5 microM, 130 s-1 and 87 X 10(6) M-1.s-1 for Z-Arg-Arg-NHMec, and 32 microM, 0.4 s-1 and 0.012 x 10(6) M-1.s-1 for Z-Phe-Arg-NHMec, respectively, at pH 7.5 and 37 degrees C. The enzyme is inhibited by leupeptin and such inhibitors of cysteine proteinases as L-transepoxysuccinyl-L-leucylamido-4-(guanidino)butane, peptidyldiazomethanes, iodoacetic acid and chicken cystatin. The tentative N-terminal amino acid sequence of the enzyme closely resembles that of papain. Histolysin does not degrade type I collagen or elastin, but it is active against cartilage proteoglycan and kidney glomerular basement-membrane collagen. It also detaches cells from their substratum in vitro, and could well play a role in tissue invasion.
A cysteine proteinase has been isolated from the virulent strain HM1:IMSS of Entamoeba histolytica by two gel chromatography steps, ion exchange chromatography on DEAE-cellulose and affinity chromatography on organomercurial-Sepharose. The purified proteinase was homogeneous, as judged by polyacrylamide gel electrophoresis in the presence and in the absence of sodium dodecyl sulphate, and was a monomeric protein with a relative molecular mass of Mr 27000 +/- 2000 possessing an N-terminal alanine. The enzyme was inhibited by natural and synthetic inhibitors against cysteine proteinases. Split experiments employing blocked and unblocked peptide analogues with -2-naphthylamide moieties indicate that the cleavability was enhanced by the presence of basic residues, such as arginine or lysine, near the acyl end of the substrate. With unblocked tetrapeptides as substrates, exopeptidase activity of the amebic enzyme required an arginine at the P2-position, lysine could not substitute for arginine. The protease was able to digest native collagen type I, with an initial attack at the alpha 2-chain of the molecule.
Perforin is one of the cytolytic factors present in the cytoplasmic granules of mouse cytotoxic T lymphocytes and natural killer cells. We have determined the sequence of the N-terminal amino acids of perforin purified from a mouse natural killer cell line, and, by using oligonucleotide probes corresponding to the amino acid residues, we have identified a complementary DNA encoding perforin from the cDNA library of a mouse cytotoxic T lymphocyte clone. As predicted from the functional similarities between perforin and the ninth component of the serum cytolytic system, complement (C9) (refs 4-8), the deduced primary structure of perforin has homology with C9 at their respective functionally conserved regions. We find that perforin is only expressed in killer cell lines, and not in helper T lymphocytes or other tumour cells tested. Thus we have provided direct molecular evidence that a killer-cell-specific protein evolutionally linked to C9 is involved in cell-mediated cytolysis.
Evidence is presented that Giardia lamblia and Entamoeba histolytica, phylogenetically unrelated aerotolerant anaerobes, have crucial thiol groups on or easily accessible to their external surface. Both parasites were killed by three structurally unrelated thiol-blocking reagents which penetrate intact cells poorly or not at all. The parasites were protected from p-chloromercuribenzenesulfonic acid (10-100 microM) by cysteine or by reduced glutathione. Killing was arrested with identical kinetics by addition of either cysteine (which quickly penetrates the cells) or bovine serum albumin (which does not penetrate intact cells) at various times after p-chloromercuribenzenesulfonic acid, indicating that the reactive site may be on the outer surface of the cell. Proteins lacking cysteine did not protect. Sensitivity of three other protozoa to p-chloromercuribenzenesulfonic acid was also tested. Trichomonas vaginalis (anaerobic) was at least as sensitive as E. histolytica and G. lamblia, while Crithidia fasciculata and Paramecium tetraurelia (both aerobic) were less sensitive. Thiol groups on the G. lamblia surface were demonstrated directly by fluorescence-activated cell sorter analysis of trophozoites which had been modified with a thiol-specific hapten, N-iodoacetyl-N'-(5-sulfonic-1-naphthyl)ethylenediamine and reacted with fluorescent antibody to this hapten.
The lesions induced in man by Entamoeba histolytica are characterized by massive tissue injury in the absence of major local signs of a host immune response. The amoeba damages surrounding cells preferentially by contact-mediated cytolysis. Recently, a presumptive aetiological factor underlying this process has been identified. It is a protein, amoebapore, capable of spontaneous incorporation into host cell membranes. Therein it induces high conductance ion-channels which rapidly collapse the cellular transmembrane potential and lead to a prelytic state. Amoebapore is present within the amoeba in a highly aggregated state in a small, dense particle. It is shed into the medium in a particulate form by a stimulus-mediated process. Release is enhanced by addition of concanavalin A, lipopolysaccharide or the calcium ionophore A23187. Surface-labelling of intact amoeba, followed by fractionation of the homogenate in self-generating Percoll gradients, identified two labelled fractions, the plasma membrane and a particulate fraction sedimenting in the region of intracellular particulate amoebapore. This latter fraction appears to be material in the process of exocytosis. A highly immunogenic surface lipid has been identified and shown to be involved in the rapid surface redistribution of immune complexes, their shedding and endocytosis. The relevance of these findings to the immunoprophylaxis of amoebiasis is discussed.
A heat-labile cytotoxin was isolated from virulent strains of axenically cultivated Entamoeba thistolytica. Strains of E. histolytica representing a spectrum of virulence as determined in animal and in vitro models of disease were examined for cytotoxic activity.
Extracts of virulent strain HMI possessed marked cytotoxic activity, those of moderately virulent strain 200 showed intermediate
activity, and those of avirulent strains 303 and Rahman showed no activity. The cytotoxin was partially purified from the
cell-free supernatant of sonicated E. histolytica HMI trophozoites by ammonium sulfate precipitation and gel filtration. Cytotoxic activity was stable in a narrow pH range
(6–7.2) and in 1 m NaCI, urea, and guanidine. Specific immune rabbit and human antiserum as wellas the protease inhibitors aprotinin, pepstatin,
and leupeptin inhibited cytotoxicity. The partially purified cytotoxin did not have any detectable degradative enzymatic activities.
Thus, virulent strains of E. histolytica possess an immunogenic cytotoxic protein which may be important in the pathophysiology of amoebiasis.
Intact trophozoites of the virulent Entamoeba histolytica strain HM-1:IMSS (HM-1) destroyed a monolayer of baby hamster kidney (BHK) cells at a higher rate and efficiency than trophozoites of the nonvirulent strain HK-9. The destructive effect could be partially attributed to the proteolytic activity of the amoeba, since quantitative differences were found in the enzymatic activity of the two strains tested. Crude extracts or secreted enzymes of HM-1 trophozoites digested Azocoll, as well as the bovine cold-insoluble globulin fraction, at a much higher rate than the corresponding preparations from HK-9. This proteolytic activity was found to be activated by free sulfhydryl groups. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the BHK cell proteins of pre- and postamoebic activities showed patterns similar to the trypsin effect on the same target cells. These enzymes were found to digest the proteins participating in the attachment of the target cells to the substrate and, consequently, cause detachment of these cells.
The proteinase activity of the low molecular weight cytotoxin of Entamoeba histolytica was correlated with its cytotoxicity. Gel-filtered amebal toxin (mol wt 10-30,000) proteinase activities could be assayed on azocasein at pH 6 or on hemoglobin at pH 4.5. Proteinase activity was inhibited by serum fractions, thiol reagents, heavy metals, leupeptin, and antipain. The cytotoxic activity of gel-filtered amebal toxin was inhibited by serum fractions, leupeptin, and antipain. Increased proteinase and cytotoxic activity was produced by treatment with cysteine. These data support the action of a thiol proteinase in the production of cytopathic effects by gel-filtered amebal toxin in vitro. The cytotoxic and proteinase activities were further purified using a combination of column chromatography and preparative isoelectric focusing. Two low molecular weight cytotoxins with proteinase activity on both substrates were isolated. The major cytotoxin had an isoelectric point of 4.5 and a molecular weight of 22,000; the other cytotoxin had a basic isoelectric point. These substances may be cathepsin B-like proteinase and elastase or cathepsin G-like proteinases of E. histolytica. The major proteinase activity in the high molecular weight fraction was not cytotoxic. The isoelectric points of the high molecular weight proteinase activities corresponded to that of mammalian cathepsin D. The major cell rounding cytotoxic activity of E. histolytica extracts in vitro is probably due to the activity of a thiol-containing cathepsin B-like proteinase.
Amoebiasis, the infection of humans with Entamoeba histolytica, has a worldwide distribution; humans are the main reservoir and source of infection(1), although some other primates can also be infected. The motile trophozoite of E. histolytica (Fig. 1) lives in the lumen of the large intestine where it multiplies and eventually differentiates into cysts which are shed in the faeces and are responsible for transmission of infection. Two forms of amoebiasis are recognized: luminal amoebiasis where no clinical signs or symptoms are apparent, and invasive amoebiasis where the trophozoites invade the intestinal mucosa to produce dysentery or amoeboma, and can spread in blood to give extraintestinal lesions such as liver abscess. Isoenzyme markers for pathogenic and non-pathogenic types of E. histolytica are well documented, but there is some debate (see Parasitology Today, vol. 3, 37-43) about whether the two types represent completely separate entities or if they can change from one type to the other under certain circumstances (Box 1). Nonpathogenic types produce no apparent symptoms; in this article Adolfo Martínez-Palomo discusses the pathology associated with pathogenic types.
Entamoeba his-tolytica: correlation between virulence and content of proteolytic enzymes Thiol groups on the surface of anaerobic parasitic protozoa
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Parasite proteases. Experi-mental Parasitology 68, 11 l-l 15 Entamoeba histolytica phagocytosis as a virulence factor
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The pathogenesis of amoebiasis Structural bases of the cytolytic mechanisms of Entamoeba histolytica Experimental amebiasis. III. A rapid in vitro assay for virulence of Entamoeba histolytica
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