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PubMed2005

Preparation of Entamoeba histolytica antigens without enzymatic inhibitors.

The goal of this work is to report a novel assay that preserves antigenicity of extracts with high protease content without using enzymatic inhibitors. A great reduction of proteolytic activity in the insoluble chloroform/methanol and heated amoebic fraction (IC:MC) was obtained by this method, even in the presence of sodium dodecyl sulphate and 2-mercaptoethanol. The substrates azo-casein and the hide powder azure were used to determine the reduction of proteolytic activity of IC:MC compared with the activity of crude extract and crude extract with iodoacetamide. The IC:MC SDS-PAGE pattern shows a higher quantity of bands than extract with the inhibitor iodoacetamide or than crude extract. In addition, anti-Entamoeba histolytica antibodies from amoebic liver abscess patients recognized a richer antigenic Western blot pattern in the IC:MC fraction than in crude extract alone or with inhibitor. The described method has proved to be suitable to preserve amoebic antigens for its use in diagnostic tests and it can be used for immunological response studies against E. histolytica antigens. Furthermore we propose that this method to obtain the IC:MC fraction can be applied for the study of other microorganisms or cells with high enzymatic content.

Animals↗
PubMed2005

Enhanced pro-inflammatory chemokine/cytokine response triggered by pathogenic Entamoeba histolytica : basis of invasive disease.

The virulence of Entamoeba histolytica is governed by adhesion/colonization in the gut which is mediated by a galactose specific lectin. Two morphologically identical but distinct species i.e. pathogenic E. histolytica and non-pathogenic E. dispar, can be differentiated by distinct epitopes in the lectin. Both species bind to colonic epithelial cells, but only E. histolytica infection induces an inflammatory response and subsequent pathogenesis. Thus, comparing the responses of the intestinal cells to pathogenic and non-pathogenic lectins is a point of interest. The pathogenic lectin causes cytolysis of epithelial and immune-competent cells. Our data (both qualitative and mRNA quantitation) indicate that the epithelial cells responded to E. histolytica lectin with an increased expression of pro-inflammatory IL-2, IL-6, IL-8, MIP-1alpha, MCP-1, RANTES, GROalpha and GMCSF as compared to E. dispar lectin. The pathogenic LCM induced a significant increase in intracellular calcium concentration, proliferative response and chemotaxis of lymphocytes from ALA patients as compared to non-pathogenic LCM. High RANTES and IL-6 were induced in patients' lymphocytes by pathogenic LCM, along with their receptors CCR5 and CD126 as compared to NP-LCM. The local release of such a complex network of cytokines/chemokines could explain the histopathology of E. histolytica infection. The comparative low levels of these chemokines/pro-inflammatory cytokines and high levels of anti-inflammatory IL-10 in response to non-pathogenic E. dispar could explain the absence of an acute inflammatory response and the disease process. The cytokines and chemokines may provide a mechanism for initiation, amplification or containment of inflammation during disease state.

Agglutination Tests↗
PubMed2006

Subcellular distribution of the Entamoeba histolytica 140 kDa FN-binding molecule during host-parasite interaction.

Entamoeba histolytica trophozoites recovered from the host-parasite interface during abscess development obtain different stimuli compared with long-term cultured cells. In order to have a better understanding about the mechanisms in which the 140 kDa fibronectin (FN)-binding molecule (EhFNR) is involved during the invasive process, we decided to compare the regulation process of this molecule among long-term cultured trophozoites, FN-stimulated trophozoites, and trophozoites recently recovered from a liver abscess. A cDNA clone (5A) containing a fragment of the EhFNR that shows identity to the C-terminal region of the intermediate galactose lectin subunit Igl, was selected with a mAb (3C10). Identity of EhFNR with Igl was confirmed by immunoprecipitation with 3C10 and EH3015 (against the Gal/GalNAc intermediate subunit) mAbs. The 3C10 mAb was used as a tool to explore the modulation of the amoebic receptor (EhFNR). Our results showed specific regulation of the EhFNR in FN-interacted amoebas, as well as in trophozoites recovered at different stages of abscess development. This regulation involved mobilization of the receptor molecule from internal vesicles to the plasma membrane. Therefore, we suggest that in the host-parasite interface, the EhFNR (Igl) plays an important role in the adhesion process during abscess development.

Amino Acid Sequence↗
PubMed2006

Proteomic analysis of Entamoeba histolytica.

In this study, the proteome of axenically grown Entamoeba histolytica parasites was explored by two-dimensional gel electrophoresis (2-DE), employing a practical and effective procedure for the solubilization of E. histolytica proteins. Approximately 900 protein species in the pH range between 4 and 7 were detected by Coomassie Blue staining. Ninety-five spots were excised, trypsinated and subjected to mass spectrometry. The resultant data from peptide mass fingerprints were compared with those available in the E. histolytica genome and the (non-redundant) National Center for Biotechnology Information (NCBI) databases for the identification and categorization of proteins. Sixty-three of the proteins identified were predicted to relate to the cytoskeleton, surface, glycolysis, RNA/DNA metabolism, the ubiquitin-proteasome system, vesicular trafficking and signal transduction. The present study demonstrates, for the first time, that corresponding genes are indeed expressed in E. histolytica and provides a foundation for further proteomic studies of this parasite.

Animals↗
PubMed2004

Present characteristics of symptomatic Entamoeba histolytica infection in the big cities of Japan.

Medical records, for 2000 and 2001, of symptomatic amoebic patients who were treated at our hospitals in Tokyo, Yokohama and Osaka were studied retrospectively for the purpose of gathering epidemiological data on symptomatic Entamoeba histolytica infection. A total of 58 patients were treated. Fifty-five of them were male, and 96% of the male patients were Japanese. The mean age of patients was 44.9 years old, and 91% of patients contracted the disease in Japan. Fifty-six per cent of the male patients indicated that they were practising homosexuals, and 44% of the male patients denied these practices or left the question unanswered. The serum Treponema pallidum haemagglutination test was positive in 45% of the patients, and antibody to human immunodeficiency virus (HIV) was positive in 45%. Our study revealed that recent symptomatic E. histolytica infection almost exclusively afflicted middle-aged males in the big cities of Japan, that a majority of the patients were probably exposed to the causative organism during homosexual activity, and that an increasing number of patients will be co-infected with HIV.

AIDS Serodiagnosis↗
PubMed1997

Present characteristics of symptomatic amebiasis due to Entamoeba histolytica in the east-southeast area of Tokyo.

Admitted medical records, from January 1988 to December 1995, of 28 symptomatic amebic patients who lived in the east-southeast area of Tokyo were studied retrospectively, in order to find the present characteristics of symptomatic amebiasis due to Entamoeba histolytica in this area. Categorized by disease, there were 14 cases of colitis, 9 cases of liver abscess, 4 cases of colitis with liver abscess, and 1 case of liver abscess with brain abscess. Patients consisted of 26 Japanese males, 0 Japanese females, 1 non-Japanese male and 1 non-Japanese female. The mean age of colitis patients and liver abscess patients was 55.4 years old and 41.3 years old, respectively. The presumed place of contraction was Japan in 64% of the patients. Forty-eight percent of male patients indicated that they engaged in homosexual or bisexual practices, and 36% of male patients who denied such sexual practices or did not answer the question had no history of marriage. Positive rate of serum titre for Treponema pallidum hemagglutination test (TPHA) and human immunodeficiency virus antibody was 42.9% and 0%, respectively. Sixty-seven percent of TPHA-positive patients indicated that they engaged in male homosexual or bisexual practices. Zymodeme patterns of E. histolytica isolated from 4 colitis patients were XIV in 1 case and II in 3 cases. Symptomatic amebiasis in the east-southeast area of Tokyo is a disease which predominantly afflicts males, especially those in their middle age, and most patients contract the disease in Japan. The high rates of patients who engaged in male homosexual or bisexual practices and the high rates of patients with positive TPHA suggest that amebiasis is likely to be sexually transmitted disease in homosexual and bisexual men in the east-southeast area of Tokyo, and zymodeme II may be the predominant type in symptomatic amebic colitis in this area.

Adult↗
PubMed2005

Cellular and molecular mechanisms that underlie Entamoeba histolytica pathogenesis: prospects for intervention.

The protozoan parasite Entamoeba histolytica is the causative agent of amoebic dysentery. It is prevalent in developing countries that cannot prevent its fecal-oral spread and ranks second in worldwide causes of morbidity by parasitic infection. Improvements in sanitation would help curb disease spread. However, a lack of significant progress in this area has resulted in the need for a better understanding of the molecular and cellular biology of pathogenesis in order to design novel methods of disease treatment and prevention. Recent insight into the cellular mechanisms regulating virulence of E. histolytica has indicated that processes such as endocytosis, secretion, host cell adhesion and encystation play major roles in the infectious process. This review focuses on components of the molecular machinery that govern these cellular processes and their role in virulence, and discusses how an understanding of this might reveal opportunities to interfere with E. histolytica infection.

Animals↗
PubMed2001

NMR derived solution structure of an EF-hand calcium-binding protein from Entamoeba Histolytica.

We present the three-dimensional (3D) solution structure of a calcium-binding protein from Entamoeba histolytica (EhCaBP), an etiologic agent of amoebiasis affecting millions worldwide. EhCaBP is a 14.7 kDa (134 residues) monomeric protein thought to play a role in the pathogenesis of amoebiasis. The 3D structure of Ca(2+)-bound EhCaBP has been derived using multidimensional nuclear magnetic resonance (NMR) spectroscopic techniques. The study reveals the presence of two globular domains connected by a flexible linker region spanning 8 amino acid residues. Each domain consists of a pair of helix-loop-helix motifs similar to the canonical EF-hand motif of calcium-binding proteins. EhCaBP binds to four Ca(2+) with high affinity (two in each domain), and it is structurally related to calmodulin (CaM) and troponin C (TnC) despite its low sequence homology ( approximately 29%) with these proteins. NMR-derived structures of EhCaBP converge within each domain with low RMSDs and angular order-parameters for backbone torsion angles close to 1.0. However, the presence of a highly flexible central linker region results in an ill-defined orientation of the two domains relative to one other. These findings are supported by backbone (15)N relaxation rate measurements and deuterium exchange studies, which reveal low structural order parameters for residues in the central linker region. Earlier, biochemical studies showed that EhCaBP is involved in a novel signal transduction mechanism, distinct from CaM. A possible reason for such a functional diversity is revealed by a detailed comparison of the 3D structure of EhCaBP with that of CaM and TnC. The studies indicate a more open C-terminal domain for EhCaBP with larger water exposed total hydrophobic surface area as compared to CaM and TnC. Further dissimilarities between the structures include the presence of two Gly residues (G63 and G67) in the central linker region of EhCaBP, which seem to impart it a greater flexibility compared to CaM and TnC and also play crucial role in its biological function. Thus, unlike in CaM and TnC, wherein the length and/or composition of the central linker have been found to be crucial for their function, in EhCaBP, both flexibility as well as amino acid composition is required for the function of the protein.

Amino Acid Sequence↗
PubMed2005

Structural characterization of the apo form of a calcium binding protein from Entamoeba histolytica by hydrogen exchange and its folding to the holo state.

One of the calcium binding proteins from Entamoeba histolytica (EhCaBP) is a 134 amino acid residue long (M(r) approximately 14.9 kDa) double domain EF-hand protein containing four Ca(2+) binding sites. CD and NMR studies reveal that the Ca(2+)-free form (apo-EhCaBP) exists in a partially collapsed form compared to the Ca(2+)-bound (holo) form, which has an ordered structure (PDB ID ). Deuterium exchange studies on the partially structured apo-EhCaBP reveal that the C-terminal domain is better structured than the N-terminal domain. The protein can be reversibly folded and unfolded upon addition of Ca(2+) and EGTA, respectively. Titration shows a slow initial folding of the apo form with increasing Ca(2+) concentration, followed by a highly cooperative folding to its final state at a certain threshold of Ca(2+). Ca(2+) and the EGTA titration taken together show that site II in the N-terminal domain has the highest affinity for Ca(2+) contrary to earlier studies. Further, this study has thrown light on the relative Ca(2+) binding affinity and specificity of each site in the intact protein. A structural model for the partially collapsed form of apo-EhCaBP and its equilibrium folding to its completely folded holo state has been suggested. Large conformational changes seen in transforming from the apo to holo form of EhCaBP suggest that this protein should be functioning as a sensor protein and might have a significant role in host-parasite recognition.

Amino Acid Sequence↗
PubMed1997

Thermodynamics of metal ion binding and denaturation of a calcium binding protein from Entamoeba histolytica.

The thermodynamics of the binding of calcium and magnesium ions to a calcium binding protein from Entamoeba histolytica was investigated by isothermal titration calorimetry (ITC) in 20 mM MOPS buffer (pH 7.0) at 20 degrees C. Enthalpy titration curves of calcium show the presence of four Ca2+ binding sites. There exist two low-affinity sites for Ca2+, both of which are exothermic in nature and with positive cooperative interaction between them. Two other high affinity sites for Ca2+ exist of which one is endothermic and the other exothermic, again with positive cooperative interaction. The binding constants for Ca2+ at the four sites have been verified by a competitive binding assay, where CaBP competes with a chromophoric chelator 5,5'-Br2 BAPTA to bind Ca2+ and a Ca2+ titration employing intrinsic tyrosine fluorescence of the protein. The enthalpy of titration of magnesium in the absence of calcium is single site and endothermic in nature. In the case of the titrations performed using protein presaturated with magnesium, the amount of heat produced is altered. Further, the interaction between the high-affinity sites changes to negative cooperativity. No exchange of heat was observed throughout the addition of magnesium in the presence of 1 mM calcium. Titrations performed on a cleaved peptide comprising the N-terminus and the central linker show the existence of two Ca2+ specific sites. These results indicate that this CaBP has one high-affinity Ca-Mg site, one high-affinity Ca-specific site, and two low-affinity Ca-specific sites. The thermodynamic parameters of the binding of these metal ions were used to elucidate the energetics at the individual site(s) and the interactions involved therein at various concentrations of the denaturant, guanidine hydrochloride, ranging from 0.05 to 6.5 M. Unfolding of the protein was also monitored by titration calorimetry as a function of the concentration of the denaturant. These data show that at a GdnHCl concentration of 0.25 M the binding affinity for the Mg2+ ion is lost and there are only two sites which can bind to Ca2+, with substantial loss of cooperativity. At concentrations beyond 2.5 M GdnHCl, at which the unfolding of the tertiary structure of this protein is observed by near UV CD spectroscopy, the binding of Ca2+ ions is lost. We thus show that the domain containing the two low-affinity sites is the first to unfold in the presence of GdnHCl. Control experiments with change in ionic strength by addition of KCl in the range 0.25-1 M show the existence of four sites with altered ion binding parameters.

Amino Acid Sequence↗
PubMed2004

Effects of bisphosphonates on the growth of Entamoeba histolytica and Plasmodium species in vitro and in vivo.

The effects of a series of 102 bisphosphonates on the inhibition of growth of Entamoeba histolytica and Plasmodium falciparum in vitro have been determined, and selected compounds were further investigated for their in vivo activity. Forty-seven compounds tested were active (IC(50) < 200 microM) versus E. histolytica growth in vitro. The most active compounds (IC(50) approximately 4-9 microM) were nitrogen-containing bisphosphonates with relatively large aromatic side chains. Simple n-alkyl-1-hydroxy-1,1-bisphosphonates, known inhibitors of the enzyme farnesylpyrophosphate (FPP) synthase, were also active, with optimal activity being found with C9-C10 side chains. However, numerous other nitrogen-containing bisphosphonates known to be potent FPP synthase inhibitors, such as risedronate or pamidronate, had little or no activity. Several pyridine-derived bisphosphonates were quite active (IC(50) approximately 10-20 microM), and this activity was shown to correlate with the basicity of the aromatic group, with activity decreasing with increasing pK(a) values. The activities of all compounds were tested versus a human nasopharyngeal carcinoma (KB) cell line to enable an estimate of the therapeutic index (TI). Five bisphosphonates were selected and then screened for their ability to delay the development of amebic liver abscess formation in an E. histolytica infected hamster model. Two compounds were found to decrease liver abscess formation at 10 mg/kg ip with little or no effect on normal liver mass. With P. falciparum, 35 compounds had IC(50) values <200 microM in an in vitro assay. The most active compounds were also simple n-alkyl-1-hydroxy-1,1-bisphosphonates, having IC(50) values around 1 microM. Five compounds were again selected for in vivo investigation in a Plasmodium berghei ANKA BALB/c mouse suppressive test. The most active compound, a C9 n-alkyl side chain containing bisphosphonate, caused an 80% reduction in parasitemia with no overt toxicity. Taken together, these results show that bisphosphonates appear to be useful lead compounds for the development of novel antiamebic and antimalarial drugs.

Animals↗
PubMed2003

The cell cycle of Entamoeba histolytica.

Entamoeba histolytica, is a microaerophilic protist, which causes amoebic dysentery in humans. This unicellular organism proliferates in the human intestine as the motile trophozoite and survives the hostile environment outside the human host as the dormant quadri-nucleate cyst. Lack of organelles--such as mitochondria and Golgi bodies--and an unequal mode of cell division, led to the popular belief, that this organism preceded other eukaryotes during evolution. However, data from several laboratories have shown that, contrary to this belief, E. histolytica is remarkable in its divergence from other eukaryotes. This uniqueness is witnessed in many aspects of its biochemical pathways, cellular biology and genetic diversity. In this context, I have analysed the cell division cycle of this organism and compared it to that of other eukaryotes. Studies on E. histolytica, suggest that in its proliferative phase, this organism may accumulate polyploid cells. Thus 'checkpoints' regulating alternation of genome duplication and cell division appear to be absent in this unicellular protist. Sequence homologs of several cell cycle regulating proteins have been identified in amoeba, but their structural divergence suggests that they may not have equivalent function in this organism. The regulation of cell proliferation in E. histolytica, may be ideally suited to survival of a parasite in a complex host. Analysis of these molecular details may offer solutions for eradicating the pathogen by hitherto unknown methods.

Animals↗
PubMed2001

Inhibition of gene expression in Entamoeba histolytica with antisense peptide nucleic acid oligomers.

Peptide nucleic acids (PNAs) may be a potent tool for gene function studies in medically important parasitic organisms, especially those that have not before been accessible to molecular genetic knockout approaches. One such organism is Entamoeba histolytica, the causative agent of amebiasis, which infects about 500 million people and is the cause of clinical disease in over 40 million each year, mainly in the tropical and subtropical world. We used PNA antisense oligomers to inhibit expression of an episomally expressed gene (neomycin phosphorotransferase, NPT) and a chromosomal gene (EhErd2, a homolog of Erd2, a marker of the Golgi system in eukaryotic cells) in axenically cultured trophozoites of E. histolytica. Measurement of NPT enzyme activity and EhErd2 protein levels, as well as measurement of cellular proliferation, revealed specific decreases in expression of the target genes, and concomitant inhibition of cell growth, in trophozoites treated with micromolar concentrations of unmodified antisense PNA oligomers.

Animals↗
PubMed1984

Immunogenicity of RNA-protein fraction isolated from axenic Entamoeba histolytica (NIH:200).

The immunogenicity of Entamoeba histolytica RNA (EhRNA) was studied in guinea-pigs. Animals immunized with a single dose of EhRNA and Freund's complete adjuvant (FCA) showed a high level of cell-mediated immune response (CMIR) as assessed by percent leucocyte migration inhibition (74.8 +/- 2.9) after 1 week of immunization. This response faded away gradually and by week 5 after immunization the percent leucocyte migration inhibition was 27.1 +/- 8.27, which is quite close to control values. The immunogenic activity of EhRNA was found to be specific, since yeast RNA failed to elicit immunological responses, and was RNase-sensitive. The CMIR could be maintained at its high level by giving boosters or multiple doses. Anti-EhRNA antibodies (haemagglutinins or precipitins) could not be demonstrated at any stage.

Animals↗
PubMed1985

Interactions between trophozoites of Entamoeba histolytica and cells of the immune system.

The interaction between Entamoeba histolytica trophozoites and cells of the immune system was studied in vitro. Both immune as well as non-immune mononuclear cells (MNC) to some extent were capable of exerting a cytotoxic effect on E. histolytica trophozoites. Incorporation of immune serum directed against the trophozoites in the reaction mixture resulted in a marked increase in the killing capacity of MNC. Highest killing was observed (96.7 +/- 1.04%) with MNC obtained from animals immunized with fraction-I(F-I) of the crude amoebic extract in association with anti-F-I serum. This cytotoxic event was found to be independent of complement involvement and required very low antibody concentrations. This antibody-dependent cellular cytotoxicity against E. histolytica in vitro was found to correlate well with the results of experiments reported earlier by us and others where a high degree of protection has been shown after vaccination of animals with F-I followed by challenge with highly virulent strains of E. histolytica.

Animals↗
PubMed1987

Elicitation of protective immunity to Entamoeba histolytica--an experimental study.

Immunization of hamsters with plasma membrane (PM) antigens of virulent subline of axenic Entamoeba histolytica (NIH: 200 V) entrapped in multilamellar phosphatidyl choline liposomes conferred 100% protection to a subsequent intrahepatic amoebic challenge. In contrast, vaccination of hamsters with live amoebic trophozoites injected intradermally failed to protect any of the animals. The protected animals had significantly high levels of anti-PM anti-amoebic antibodies, cellular sensitization and macrophage-mediated antibody-dependent cytotoxicity against amoebic trophozoites. However, none of the intradermally immunized animals had anti-PM anti-amoebic antibodies at the time of challenge. Such animals also had significantly low macrophage-mediated antibody-dependent cellular cytotoxicity. The data indicate the potential prophylactic use of PM antigens against hepatic amoebic infection.

Amebiasis↗
PubMed2005

The genome of the protist parasite Entamoeba histolytica.

Entamoeba histolytica is an intestinal parasite and the causative agent of amoebiasis, which is a significant source of morbidity and mortality in developing countries. Here we present the genome of E. histolytica, which reveals a variety of metabolic adaptations shared with two other amitochondrial protist pathogens: Giardia lamblia and Trichomonas vaginalis. These adaptations include reduction or elimination of most mitochondrial metabolic pathways and the use of oxidative stress enzymes generally associated with anaerobic prokaryotes. Phylogenomic analysis identifies evidence for lateral gene transfer of bacterial genes into the E. histolytica genome, the effects of which centre on expanding aspects of E. histolytica's metabolic repertoire. The presence of these genes and the potential for novel metabolic pathways in E. histolytica may allow for the development of new chemotherapeutic agents. The genome encodes a large number of novel receptor kinases and contains expansions of a variety of gene families, including those associated with virulence. Additional genome features include an abundance of tandemly repeated transfer-RNA-containing arrays, which may have a structural function in the genome. Analysis of the genome provides new insights into the workings and genome evolution of a major human pathogen.

Animals↗