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PubMedDate not supplied

Entamoeba histolytica: a beta 1 integrin-like fibronectin receptor assembles a signaling complex similar to those of mammalian cells.

During tissue invasion, Entamoeba histolytica trophozoites interact with endothelial cells and extracellular matrix (ECM) proteins such as fibronectin (FN), collagen, and laminin. It has been demonstrated that trophozoites interact with FN through a beta1 integrin-like FN receptor (beta 1EhFNR), activating tyrosine kinases. In order to characterize the signaling process triggered by the amoebic receptor, activation, and association of tyrosine kinases and structural proteins were determined. As a result of FN binding by the beta 1EhFNR, the receptor itself, FAK, and paxillin were phosphorylated in tyrosine. Co-immunoprecipitation experiments showed that a multimolecular signaling complex was formed by the amoebic FN receptor, FAK, paxillin, and vinculin. These results strongly suggest that a signaling pathway, similar to the one used in mammalian cells, is activated when E. histolytica trophozoites adhere to FN.

Animals↗
PubMedDate not supplied

Entamoeba invadens: inhibition of excystation and metacystic development by aphidicolin.

The effect of aphidicolin, a specific inhibitor of the replicative DNA polymerases, on the excystation and metacystic development of Entamoeba invadens was examined. The protein profile of metacystic amoebae and their immunogenicity in the presence and absence of aphidicolin were also examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Excystation, which was assessed by counting the number of metacystic amoebae after the induction of excystation, was inhibited by aphidicolin in a concentration-dependent manner during incubation compared to the controls. Metacystic development, when determined by the number of nuclei in amoeba, was also inhibited by aphidicolin, because the percentage of 4-nucleate amoebae in cultures with aphidicolin during incubation was higher than that in cultures without the drug. The addition of aphidicolin to cultures at day 1 of incubation reduced the number of metacystic amoebae thereafter compared to cultures without the drug. The inhibitory effect of aphidicolin on excystation and metacystic development was reversed by removal of the drug. Pretreatment of cysts with aphidicolin before transfer to a growth medium containing the drug had no further effect on the excystation and metacystic development. Cellular proteins of metacystic amoebae with 4 nuclei, which were predominant even at day 3 in the cultures with aphidicolin, reacted strongly with rabbit anticyst serum absorbed with trophozoite proteins. In contrast, those of metacystic amoebae with 1 nucleus, which were predominant at day 3 in cultures without aphidicolin, no longer reacted with the absorbed anticyst serum, suggesting change in the expression of proteins during metacystic development.

Animals↗
PubMedDate not supplied

Interaction between chondroitin-6-sulfate and Entamoeba histolytica as revealed by force spectroscopy.

We have observed by atomic force microscopy (AFM) the amoeba surface and probed the interaction force between Entamoeba histolytica and chondroitin-6-sulphate (C6S). We have used several substrates to adhere trophozoites. The best reproducibility in sample preparation was obtained with fibronectin-coated coverslips and when the cells were fixed with paraformaldehyde. The images obtained with the AFM showed that the trophozoite exhibits an irregular surface. Pseudopods and waving adhesion plaques could be observed. Force spectroscopy analysis showed that the trophozoite surface strongly interacts with C6S-functionalized tips. During cantilever retraction, attractive force peaks were observed at distances up to 1.3 microm above the trophozoite surface. Statistical analysis of the force distributions collected for five samples shown a reproducible 2.2 nN mean adhesion force. We observed a reduction of the adhesion force and of the interaction distance after addition of galactose to the buffer solution suggesting that the observed interaction is also Gal/GalNAc-lectin-mediated.

Animals↗
PubMed1985

Entamoeba histolytica: impedance measurements and cytotoxicity in the presence of bepridil, verapamil, and cytochalasin D.

Entamoeba histolytica, and invasive enteric protozoa, kills mammalian target cells by sequential adherence and cytolytic events. Using platinum plate electrodes with an alternating current source placed in a Wheatstone bridge circuit, the impedance (resistance to ion flow) of a cell suspension of axenic amebae (strain HM1-IMSS) was measured. The impedance of the amebic cell suspension, expressed as resistivity (in ohm-cm), was significantly greater than the test solution and increased with decreasing temperature or greater cell packing (P less than 0.01), indicating that the resistivity measurements reflected the impedance of the amebic surface membrane. Cytochalasin D (10 micrograms/ml), a microfilament inhibitor which inhibited amebic in vitro adherence and cytolysis of target Chinese hamster ovary (CHO) cells (P less than 0.001), also increased resistivity of the amebic suspension (P less than 0.01). Exposure of amebae to bepridil (10(5) M), a slow-channel blocker, inhibited amebic killing of target cells (P less than 0.01) and also increased the resistivity of the amebic suspension (P less than 0.01), but both to a lesser degree than cytochalasin D (P less than 0.001). In contrast, exposure of amebae to verapamil followed by washing had no effect on amebic killing of target cells or resistivity of the amebic suspension. The increased resistivity measured in cytochalasin D or following exposure to bepridil was not due to a change in cell density of the amebic suspension. These studies indicate that changes in impedance of the amebic surface membrane are produced by bepridil and cytochalasin D. The effect of these agents on membrane impedance may contribute directly to the concurrent observed alteration in amebic cytopathogenic capacity or may serve as a parallel marker for the cell membrane alterations induced by such pharmacologic agents which inhibit amebic microfilament function or calcium flux.

Adhesiveness↗
PubMed2000

Two novel calcium-binding proteins from cytoplasmic granules of the protozoan parasite Entamoeba histolytica.

We report on the molecular characterisation of two novel granule proteins of the protozoon and human pathogen Entamoeba histolytica. The proteins, which were named grainin 1 and 2, show a considerable structural similarity to calcium-binding proteins, particularly within EF-hand motifs. Each grainin possesses three of these putative calcium-binding sites. Based on careful inspection of known structures of protein families containing EF-hands, a domain of grainin 1 covering two EF-hand motifs was modeled by homology. Calcium-binding activity of grainins was demonstrated by two independent methods. These granule proteins may be implicated in functions vital for the primitive phagocyte and destructive parasite such as control of endocytotic pathways and granule discharge.

Amino Acid Motifs↗
PubMed2001

The unusual transhydrogenase of Entamoeba histolytica.

We have expressed and purified a protein fragment from Entamoeba histolytica. It catalyses transhydrogenation between analogues of NAD(H) and NADP(H). The characteristics of this reaction resemble those of the reaction catalysed by a complex of the NAD(H)- and NADP(H)-binding subunits of proton-translocating transhydrogenases from bacteria and mammals. It is concluded that the complete En. histolytica protein, which, along with similar proteins from other protozoan parasites, has an unusual subunit organisation, is also a proton-translocating transhydrogenase. The function of the transhydrogenase, thought to be located in organelles which do not have the enzymes of oxidative phosphorylation, is not clear.

Animals↗
PubMed2004

Membrane lipid composition protects Entamoeba histolytica from self-destruction by its pore-forming toxins.

The protozoan parasite and human pathogen Entamoeba histolytica is protected against killing by its own lytic effector proteins. Amoebae withstand doses of amoebapores, their pore-forming polypeptides, that readily kill human Jurkat T cells. Moreover, the polypeptides do not bind to the amoebic surface membrane as evidenced by using fluorescently labelled amoebapores and confocal laser microscopy. Experiments employing liposomes as a minimalistic membrane system and the major isoform amoebapore A revealed that the lipid composition of amoebic membranes prevents binding of the cytolytic molecule and that both the phospholipid ingredients and the high content of cholesterol contributes to the protection of the toxin-producing cell.

Adenosine Triphosphate↗
PubMed1998

Molecular characterization of an exceptionally acidic lysozyme-like protein from the protozoon Entamoeba histolytica.

The protozoan parasite Entamoeba histolytica contains a second antibacterial protein with lysozyme-like properties. The newly recognized bacteriolytic protein was purified from extracts of amoebic trophozoites to allow amino-terminal sequencing. Subsequent molecular cloning revealed that it is an isoform of the amoeba lysozyme described previously but also demonstrated a substantial sequence divergence of the two forms. As lysozymes typically are basic proteins, the novel amoebic protein differs markedly in having a pI of 4.5. There is no significant similarity of both amoeba lysozymes with any bacteriolytic protein of other organisms reported so far; however, striking sequence identity is found with predicted gene products of unknown function derived from the bacteria-feeding nematode Caenorhabditis elegans.

Animals↗
PubMed1998

Induction of a spectroscopically defined transition by guanidinium hydrochloride on a recombinant calcium binding protein from Entamoeba histolytica.

Sequence analysis and metal ion binding studies reported earlier have established that the calcium binding protein (CaBP) from the parasitic ameboid Entamoeba histolytica protein has four canonical EF hand motifs which bind calcium. Equilibrium denaturation studies on both the apo and the holo forms of this protein indicate the presence of stable transition intermediates at low denaturant concentrations as revealed by the binding of the non-specific hydrophobic dye ANS. Fast reaction kinetics shows that the binding of the Gdn+ ions at or near the Ca2+ sites in the N-terminal domain influences metal ion binding to the sites in the C-terminal domain. Isothermal calorimetric titrations performed using low GdnHCl concentrations reveal the presence of two binding sites of low affinity, both being endothermic in nature. Thus the stabilization of CaBP observed at low GdnHCl concentration represents a native-like intermediate, with the Gdn+ ions mimicking Ca2+ binding at the N-terminal domain of this protein.

Animals↗
PubMed1999

Secondary structure of a calcium binding protein (CaBP) from Entamoeba histolytica.

A calcium binding protein from Entamoeba histolytica, (EhCaBP, M(r) approximately 15 kDa) is the causative agent for amoebiosis and has a very low sequence homology (approximately 30%) with other known CaBPs. Almost complete sequence specific resonance assignments for (1)H, (13)C and (15)N spins in EhCaBP were obtained using double and triple resonance NMR experiments. Qualitative interpretation of the nuclear Overhauser enhancements, chemical shift indices and of hydrogen exchange rates threw valuable light upon the secondary structure of this protein. CaBP is found to have two globular domains each of which consists of two pairs of helix-loop-helix motifs. Though this protein has a very small sequence homology with calmodulins, the topological arrangement of the alpha-helices and beta-strands in EhCaBP resemble them.

Amino Acid Sequence↗
PubMed1996

Interaction of LS174T human colon cancer cell mucins with Entamoeba histolytica: an in vitro model for colonic disease.

BACKGROUND & AIMS: Colonic mucins secreted by goblet cells protect the colon by preventing the attachment of enteric pathogens to the epithelium. Entamoeba histolytica overcomes this protective barrier and causes ulcerations, allowing the parasite to disseminate to the liver and form abscesses. An in vitro model is used to study the interaction between E. histolytica and colonic mucins. METHODS: Secretory mucins from the colonic adenocarcinoma cell line LS174T were collected and their functions assessed by their ability to inhibit amebic adherence to target cells and killing. The cytoprotective effect of mucus against E. histolytica cytolysis of LS174T monolayers was studied at 37 degrees C. RESULTS: Sepharose 4B column chromatography, metabolic labeling with [3H]glucosamine, cesium chloride density gradient centrifugation, and amino acid and carbohydrate compositional analysis revealed that LS174T cell mucins were typical of native colonic mucins. Mucin O-linked oligosaccharides bound to and inhibited the adherence of amebae to Chinese hamster ovary cells. E. histolytica killing of Chinese hamster ovary cell monolayers occurred rapidly, whereas killing of LS174T monolayers with an intact mucus layer was significantly retarded. CONCLUSIONS: Our results show that colonic mucins serve as the first line of host defense against amebic invasion and provide a useful model to study pathogen-mucin interactions.

Animals↗
PubMed1997

Entamoeba histolytica stimulates interleukin 8 from human colonic epithelial cells without parasite-enterocyte contact.

BACKGROUND & AIMS: The mechanisms involved in the initiation of host mucosal inflammation in amebiasis are not fully understood. This study characterized the effect of Entamoeba histolytica components on interleukin 8 (IL-8) gene expression in human colonic cells. METHODS: Colonic cells were stimulated with amebic proteins, secretory components, or live trophozoites (separated with 0.45-microm pores), and the levels of IL-8 messenger RNA (mRNA) and protein were detected. RESULTS: Live amebae or their components enhanced IL-8 mRNA levels in the colonic cells (T84, LS174T, and Caco-2). In T84 cells, the accumulation of IL-8 mRNA induced by amebic components occurred in a dose- and time-dependent fashion. Increased secretion of IL-8 was noted after 12-hour stimulation; neutralizing antibodies against IL-15 or tumor necrosis factor alpha did not inhibit IL-8 production. Nuclear run-on assays showed that amebae-induced IL-8 gene occurred by a posttranscriptional mechanism. Cycloheximide treatment resulted in superinduction of IL-8 mRNA; however, dexamethasone inhibited E. histolytica-induced IL-8 gene expression. CONCLUSIONS: E. histolytica can directly stimulate the induction of IL-8 by colonic cells in the absence of cell-cell contact or injury.

Animals↗
PubMed2000

Purification and properties of an acid phosphatase from Entamoeba histolytica HM-1:IMSS.

Entamoeba histolytica contains and secretes acid phosphatase, which has been proposed as a virulence factor in some pathogenic microorganisms. In this work, we purified and characterised a membrane-bound acid phosphatase (MAP) from E. histolytica HM-1:IMSS and studied the effect of different chemical compounds on the secreted acid phosphatase and MAP activities. MAP purification was accomplished by detergent solubilisation, and affinity and ion exchange chromatographies. The enzyme showed a pI of 5.5-6.2, an optimum pH of 5.5, and a Km value of 1.14 mM with p-nitrophenyl phosphate.

Acid Phosphatase↗
PubMed2003

An ecto-protein tyrosine phosphatase of Entamoeba histolytica induces cellular detachment by disruption of actin filaments in HeLa cells.

Actin cytoskeleton disruption in host cells has been demonstrated for PTPases from pathogenic microorganisms. In this work, we analysed whether the secreted acid phosphatase from Entamoeba histolytica has phosphotyrosine phosphatase activity and the possibility that this activity may participate in damaging host cells. The secreted acid phosphatase of E. histolytica, which catalyses p-nitrophenyl phosphate hydrolysis at acid pH values, was found to have phosphotyrosine phosphatase activity. The enzymatic properties of phosphotyrosine phosphatase and acid phosphatase were virtually identical and included: Km values of 10 x 10(-4) M, no requirement for divalent cations, and sensitivity to molybdate, vanadate, and tungstate. The phosphotyrosyl phosphatase activity caused significant levels of cell rounding and detachment correlating with disruption of the actin stress fibres in HeLa cells. Thus, our data suggest that secreted phosphotyrosine phosphatase could play a cytotoxic role during amoebic infection.

Actin Cytoskeleton↗
PubMed2003

A virulence attenuated amoebapore-less mutant of Entamoeba histolytica and its interaction with host cells.

Entamoeba histolytica, the protozoan parasite which causes amoebiasis, is an exclusively human pathogen so developing a vaccine could effectively impact the spread of the disease. Recently we developed a genetically modified avirulent strain, termed G3, from the virulent E. histolytica strain HM-1:IMSS. The new strain lacks the important virulence factor, the amoebapore-A. The objective of our current study was to investigate the avirulence of the attenuated strain as well as to examine the antigenic and immunogenic responses of these trophozoites as potential candidates for a live vaccine. Functional assays were conducted to characterise the virulent behaviour of the G3 strain. This behaviour was compared to the virulent strain HM-1:IMSS and the non-virulent strain Rahman. Western blots were conducted to confirm the lack of amoebapore-A in the E. histolytica G3 strain and to demonstrate that it had no influence on the presence of other virulence factors. Results of these two sets of tests proved the G3 strain to be phenotypically similar to the avirulent Rahman strain while antigenically identical to the virulent HM-1:IMSS, apart from the lack of the amoebapore-A protein. Intraperitoneal immunisation of hamsters with G3 trophozoites compared to sham immunised hamsters resulted in IgG anti-HM-1:IMSS antibodies. The level of humoral response was variable and further testing has to take place before introducing this new strain as a vaccine.

Animals↗
PubMed1998

Identification and characterisation of early reactions of asparagine-linked oligosaccharide assembly in Entamoeba histolytica.

Sequential incubation of a mixed membrane fraction isolated from Entamoeba histolytica trophozoites with the nonionic detergents Brij 35 and Igepal CA-630 rendered a soluble fraction with the ability to transfer N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to dolichol phosphate to form a lipid saccharide that was identified as a mixture of dolichol-P-P-GlcNAc and dolichol-P-P-(GlcNAc)2 as follows. (a) The reaction occurred only in the presence of exogenously added dolichol phosphate and was strongly inhibited by tunicamycin and amphomycin; (b) Over 90% of the aminosugar moiety of the lipid saccharide was released by mild acid hydrolysis and was identified as a mixture of GlcNAc and diacetylchitobiose [(GlcNAc)2]; (c) Time course experiments revealed that dolichol-P-P-(GlcNAc)2 accumulated at the expense of a parallel decrease in dolichol-P-P-GlcNAc revealing the tandem operation of UDPGlcNAc:dolichol-P GlcNAc-1-P transferase and UDPGlcNAc:dolichol-P GlcNAc transferase. Mg2+ and to a lower extent Mn2+ were required for catalytic activity and were optimal at 2.5 mM and 1.25 mM, respectively. Common phospholipids with different head groups failed to increase catalytic activity and phosphatidylglycerol was inhibitory. At low concentration, nucleotides such as ATP, GMP and GTP brought about stimulations of 24-54% but higher concentrations were inhibitory. Others were inhibitory at all concentrations the strongest being those containing a uridine base.

Acetylglucosamine↗
PubMed1999

Lack of a chromosomal copy of the circular rDNA plasmid of Entamoeba histolytica.

A number of small circular DNAs constitute a part of the genome of Entamoeba histolytica. Among them, the 24.5 kb circular DNA encoding rRNA (EhR1) is the most abundant. Pulsed field gel electrophoresis was used to determine if a chromosomal copy of EhR1 exists and what fraction of the total genome is circular. The results show that the chromosomes of E. histolytica are linear, and that no copy of EhR1 could be detected in any of the linear chromosomes.

Animals↗
PubMed2002

Development of a diagnostic test for Entamoeba histolytica using idiotype expression in human.

The protozoan parasite Entamoeba histolytica is the etiological agent of human amebiasis. The pathology of the disease starts with the cytolysis of the host target cells by amoebae. It is initiated by the adhesion of trophozoites to the host cells, through surface lectin via specific receptors. These adherence lectins have been demonstrated to be highly conserved, and can be recognised by serum antibodies from patients with invasive amebiasis. Some of these molecules have been used as antigens in serologic studies, which has been very helpful in the diagnosis of invasive intestinal amebiasis. However, false-positive serologic reactivity can occur using E. histolytica extracts and purified antigens. Additional problems are because the extracts display a great enzymatic activity. Several diagnostic methods, using different molecules and techniques, have been described. However, the problem still remains since these tests are not capable of differentiating between amoebic liver abscess (ALA) and intestinal amebiasis.Here, the research has been addressed to the 66-kDa antigen, which is a part of the outer membrane proteins from the E. histolytica strain HM1-IMSS trophozoites. First of all, we characterized the 66-kDa antigen in order to prove the relevance. We found that the 66-kDa antigen is a part of the plasma membranes and is distributed rather homogeneously on the cell surface of trophozoites. Apparently, the 66-kDa antigen is a glycoprotein. Using a monoclonal antibody (MAb), we found 25% of inhibition in the erythrophagocytosis by the trophozoites. Starting form one monoclonal antibody, we prepared an anti-idiotype (anti-Id) antibody reagent, with the purpose of searching for the different expressions of the idiotype between the sera from ALA and the intestinal amebiasis patients. Moreover, we produced the antibody Ab3 that is capable of recognising the 66-kDa antigen; it means that the Ab2 displays the internal image of the antigen. We found that 91.6% of the serum from ALA patients displayed the expression of the Id. In contrast, 15.7% of the E. histolytica asymtomatic cyst carriers displayed the Id expression, 6.6% of the patients with another parasite infection, and 11% of the negative controls (serum from umbilical cords of newborn babies). Our results showed that the expression of the Id could be differentiated among the AHA patients from the other groups with a 91.6% sensibility and 88.3% specificity.

Animals↗