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Background To understand the molecular basis of virulence variability in Entamoeba histolytica , this study presents results about differential gene expression induced by E. histolytica trophozoites in liver of hamsters in order to produce experimental amebic liver abscess (ALA) and consequently reactivate its virulence. Methods Amebic cultures were studied before (BALA) and after (AALA) inoculation in hamster peritoneal cavity. Markers of pathogenicity such as the rate of erythrophagocytosis, hemolytic activity, and cytotoxic effects on MDCK cell monolayers were evaluated in order to correlate these phenotypic characteristics to differential gene expression between virulent and non-virulent strains. Genotypic variability was determined by genetic polymorphism using the random-amplified polymorphic DNA (RAPD) technique, which defines the parasite genomic plasticity. mRNA differential display was used in order to identify variable transcripts levels. Results The rate of erythrophagocytosis and hemolytic activity were notably increased in AALA in comparison with BALA E. histolytica cultures, as well as the cytotoxic effect on MDCK cells. An increment in the transcription level of several mRNA was shown. Conclusions The RAPD technique allowed us to confirm differences in number and size of polymorphic markers bands between virulent and non-virulent stages, suggesting genomic adaptability in E. histolytica . Eight different genes (membrane-bound acid phosphatase, cysteine proteinase, two different ribosomal proteins, heat shock transcription factor, ribosomal RNA, aldehyde dehydrogenase-1 and patatin-like phospholipase) were sequenced and may be associated with a biological function related to the virulence of E. histolytica . Together these findings show genomic variability between virulent and non-virulent cultures of E. histolytica.

There are little data on the true prevalence and incidence of Entamoeba histolytica infection in Africa. This is due to the inability, historically, to differentiate Entamoeba histolytica from the more common, but non-pathogenic, Entamoeba dispar. In addition, newer studies have demonstrated that the previous gold standard, culture with zymodeme analysis, is insensitive in detecting the presence of infection, especially when compared to PCR. Recent published articles as well as data from the authors' previous work are reviewed and summarized to elucidate what is known about prevalence and incidence of Entamoeba histolytica in Africa. The majority of data on asymptomatic infection are published from South Africa, Egypt and Cote d'Ivoire. Egypt has high rates of asymptomatic infection detected in the stool (>21%), whereas South Africa and Cote d'Ivoire rates range between 0 and 2%. Seroprevalence estimates the rate of recent infection, because anti-amebic antibodies generally persist for

Classical descriptions of the pathology of amebiasis portray the parasite as the cause of tissue damage and destruction, and in recent years a number of amebic molecules have been identified as virulence factors. In this review we describe a series of experiments that suggest a more complex host-parasite relation, at least during the early stages of acute experimental amebic liver abscess in hamsters. The problems of extrapolating experiments in vitro to explain observations in vivo are discussed. The role of amebic cysteine proteases is examined and evidence presented to suggest that they are primarily related not to tissue damage but to amebic survival, which is required for the progression of the lesion. Inflammation is shown to be not only the major cause of tissue damage but also an absolute requirement for amebic survival in the liver, whereas complement and ischemia are not involved in the disappearance of the parasite in the absence of inflammation.

Pathogenesis in the parasite Entamoeba histolytica has been related to motility of the trophozoites. Motility is an important feature in amebas as they perform multiple motile functions during invasion of host tissues. As motility depends on the organization and regulation of the cytoskeleton elements, in particular of the actin cytoskeleton, the study of the molecular components of the machinery responsible for movement has been a key aspect to study in this parasite. Although many of the components have high homology in amino acid sequence and function to those characterized in higher eukaryotic cells, there are important differences to suggest that parasitic organisms may have developed adaptative differences that could be useful as targets to stop invasion. The purpose of this review is to evaluate current knowledge about the cytoskeleton of E. histolytica and the ways in which the parasite controls motility.

One of the mechanisms that Entamoeba histolytica uses to evade host immune response is inhibition of the polymorphonuclear (PMN) leukocyte respiratory burst. In studies previously conducted in a model used in our laboratory, we observed that when treating trophozoites with different zinc concentrations certain amebic functions are inhibited while significantly limiting development of hepatic abscess in golden hamsters (Mesocricetus aureatus). We carried out an in vitro study using a chemoluminescent method to assess the effect zinc-treated amebic trophozoites exercise on respiratory burst in human PMNs. We measured response of PMNs incubated with E. histolytica trophozoites from cultures with TYI-S33 medium alone and with zinc. Zinc concentrations between 0.1 and 1.0 mM did not affect amebic trophozoite viability, and PMNs in contact with these in a zinc-free medium had an oxidative response similar to that obtained with zymosan and significantly greater (p

Background This study characterizes two long vesicle-associated membrane protein (VAMP) genes (SYBL-1 and SYBL-2) obtained from DNA of Entamoeba histolytica by PCR amplification. (Nucleotide sequences of the Entamoeba histolytica SYBL-1 and SYBL-2 genes appear in the GeneBank under accession numbers AY256852 and AY309014.) Methods The two cDNA products include one of 663 bp with sequence of 220 deduced amino acids, and a second of 693 bp with 230 deduced amino acid residues. Both products possess corresponding sequences for longin domain at N-terminus, a soluble N-ethylmaleimide-sensitive factor [NSF] attachment protein receptor (SNARE) coiled-coil region, a transmembrane domain (TM), and an intravesicular tail C-terminal, characteristics of all long VAMPs or longins. The current study identified presence of deduced peptide bonds in SYBL-1 and -2, which indicates that these two long VAMPs from E. histolytica could be appropriate substrates for zinc endopeptidases from tetanus and botulinum neurotoxins with specific activity toward neuronal synaptobrevins. Results Alignment by Basic Local Alignment Search Tool (BLAST) of deduced amino-acid sequence of long VAMPs genes SYBL-1 and -2 showed identity of between 20 and 40% with Caenorhabditis elegans , Dictyostelium discoideum , Drosophila melanogaster , Arabidopsis thaliana , Mus musculus , Homo sapiens, and Plasmodium falciparum . Conclusions It is possible that these two putative transport proteins are involved in endocytosis/exocytosis during a biological membrane fusion process, which may make them suitable candidates as targets for new drugs. According to published data, this is the first time that two such genes have been isolated from E. histolytica .

Interaction of Entamoeba histolytica trophozoites with extracellular matrix (ECM) proteins activates signaling pathways through G-protein-coupled receptors. Increments of adenylyl cyclase activity and cAMP produce a striking reorganization of actin into structures that apparently facilitate adhesive, locomotive, and secretory activities. The reorganization of actin is induced by phosphorylation of actin-associated proteins by diverse kinases activated during the signaling process. Although cAMP-dependent kinases have not yet been identified in this parasite, the activation of the adenylyl cyclase route and its effects on particular motility-related functions strongly suggest their presence. Phosphokinase A (PKA) was detected by phosphorylation of the specific substrate, kemptide, its further activation by cAMP, and its inhibition by H89. The catalytic subunit of the enzyme was identified by immunofluorescence microscopy and by immunoprecipitation. Adhesion and damage to cultured cells were monitored by FN-binding and cytotoxicity assays. A cAMP-dependent kinase activated by effectors and agonists of adenylyl cyclase and also during interaction of trophozoites with fibronectin (FN) was found. The enzyme is associated with small granules in the cytoplasm and upon activation, a fraction of its catalytic subunit with an Mr of 100 kDa was translocated to the nucleus, while another fraction was aggregated into big clusters. Activity and translocation were blocked by H89, a specific inhibitor of PKA. Trophozoites stimulated by dBcAMP or forskolin-formed lamellae and restructured actin, but no significant increase in their adhesion to FN was observed and only showed 10% stimulus in their capacity to damage target cells. Treatment with H89 decreased adhesion to 40% and caused 80% inhibition in cell damage. These amebas showed altered organization of the actin structures induced by dBcAMP or FN. Our results support previous suggestions concerning the participation of PKA in the response elicited by the interaction of E. histolytica trophozoites with ECM proteins. They also indicate that adhesion and secretion in conjunction with motile activities are related to invasion processes.

Entamoeba histolytica is responsible for human invasive amebiasis. This disease has an endemic behavior in specific geographic areas throughout the world, and is commonly associated with population with low socioeconomic and educational conditions (1). It is in these endemic areas where the evaluation of prevalence of infection and disease through the detection of antiameba antibodies in serum and secretions is particularly valuable. Although there is an increasing number of new strategies to make available better antibody and antigen detection tests (2), the complexity of data analysis obtained in epidemiologic trials in highly endemic areas justifies the search for new antigens of E. histolytica with biological or molecular characteristics that may optimize antibody detection tests for use in field studies. In the present article, we describe the purification of three proteases of E. histolytica HM-1:IMSS that previously showed to be highly recognized (91.4%) by serum antibodies from patients with amebic dysentery (3), and in more than 95% of amebic liver abscess patients. Preliminary data suggest that antibody levels to these proteins decrease significantly after 6 months of treatment, which makes them attractive for the detection of new cases of invasive amebic disease.

Background Entamoeba histolytica forms cyst-like structures (CLS) in PEHPS but not in TYS-33 medium. Sodium dodecyl sulfate [(SDS (0.1%)] dissolves most of them in 10 min, but not natural cysts. Chitin is responsible mainly for cyst wall resistance. Its synthesis depends on Mg 2 +, Mn 2 +, or Co 2 +, whose action is interactive. With the aid of the Simplex method, we analyzed the effect of 20 blends of these cations to find the one that, when added to PEHPS, produced the highest proportion of CLS resistant to 1% SDS (RCLS). Methods The concentration of Mg 2 +, Mn 2 +, and Co 2 + was determined in PEHPS and TYI-S-33 with a flame atomic absorption spectrometer. The proportion of RCLS produced in PEHPS with each ion blend was tested. The CLS and RCLS affinity to fluorescein wheat germ agglutinin (WGA/FITC), which binds chitin, was determined. Results PEHPS contained a similar concentration of Co 2 + (0.52 μM) and 3.4 and 1.6 times more Mg 2 + (798 μM) and Mn 2 + (3.15 μM) than TYI-S-33, respectively. The proportion of RCLS increased gradually in PEHPS until reaching 3.6 ± 1.43% with MgCl 2 1.22 mM, MnCl 2 14.44 mM, and CoCl 2 19.44 mM (ion blend No. 20). Both CLS and RCLS bound WGA/FITC. The RCLS formed in the presence of ion blend No. 20 appeared wrinkled. Conclusions Mg 2 +, Mn 2 +, and Co 2 + enhanced the ability of PEHPS to form RCLS, possibly because these ions stimulated their chitin synthesis. Although ion blend No. 20 produced the highest proportion of RCLS, this high ion concentration may be toxic for encysting amebas.

Background. Cysteine-proteinases are thought to play an important role in E. histolytica pathogenicity. Although effective blockage of proteolytic activities can be obtained with several known inhibitors, the high cellular toxicity of most of the inhibitors precludes experimentation with live cells or animal models. Specific cysteine-proteinase inhibitors that could be utilized in studies of virulence are of great need in the field of amebiasis. Methods. Cysteine-proteinase activities were determined in trophozoite lysates by azocasein degradation and after PAGE and gelatin zymograms. Inhibition of the activities was assessed in the presence of 0.01‐2.5 mM concentrations of divalent cations of the IIB and VIII series such as Zn, Cd, Hg, Ni, and Co. Reversibility was induced with 25 mM L-cysteine or 50 mM L-histidine and by metal chelation with 5 mM phenantroline. The inhibitory effect of ZnCl 2 was tested with live cells in fibronectin-binding and cytotoxicity assays. Results. ZnCl 2 specifically inhibited cysteine-proteinase activities in trophozoite lysates in a concentration-dependent manner. Additionally, 1.0‐2.5 mM ZnCl 2 blocked proteolysis in more than 70%. This inhibition was completely reverted by L-cysteine, L-histidine, or phenantroline. Similar results were obtained by analyzing individual cysteine-proteinase activities separated in gelatin zymograms. At these concentrations, ZnCl 2 significantly interfered with trophozoite adhesion, thus making amebas deficient in substrate degradation and cell damage. Conclusions. ZnCl 2 is an effective inhibitor of amebic cysteine-proteinases. Its low toxicity at relatively high concentrations, high solubility, and low cost make it adequate for live cell experimentation and animal models of amebic virulence. © 1999 IMSS. Published by Elsevier Science Inc.

Mice have been considered resistant to develop intestinal and extraintestinal amebic lesions. The immune system of this animal has been amply studied; however, previous studies performed in normal, SCID (1), and other strains of mice have not been conclusive to determine the participation of the immune cells in the development or rejection of the amebic liver infection. The objective of this work was to determine the presence and topographical localization of neutrophils and macrophages in two strains of mice, based on the identification of specific molecules, and also to analyze the activation status of liver sinusoidal endothelial cells by expression of intercellular adhesion molecules (ICAM-1) during the development of the amebic lesion.

We have previously demonstrated that intraperitoneal (i.p.) immunization of mice with glutaraldehyde-fixed trophozoites (GFT) of Entamoeba histolytica elicites strong local and systemic immune responses (1). One of the most common drugs used against a wide variety of anaerobic protozoan parasites and anaerobic bacteria is metronidazole, whose toxicity has been reviewed. The main side effects of metronidazole are headache, nausea, dry mouth, and metallic taste. Other more severe symptoms are experienced only occasionally (2). Although related chemicals have caused blood dyscrasias, only temporary neutropenia, reversible after discontinuation of therapy, occurs with metronidazole (3). Mebendazole is a versatile antihelminthic agent. Benzimidazoles produce many biochemical changes in susceptible nematodes; there is strong evidence, however, that the primary action of these drugs is to inhibit microtubule polymerization by binding to b -tubulin. Mebendazole does not cause significant systemic toxicity in routine clinical use, this probably resulting from its low systemic bioavailability. Rare side effects in patients treated with high doses of mebendazole include allergic reactions, alopecia, reversible neutropenia, agranulocytosis, and hypospermia (4). In a previous work (5), we analyzed by ELISA the antiamebic antibody response in the sera and intestinal samples of metronidazole–mebendazole pretreated, and nontreated GFT-immunized mice. We found that the GFT-immunized mice pretreated with metronidazole and mebendazole showed a significantly lower IgM and IgG serum response and a significant IgM and IgA response in intestinal fluids and supernatants of lamina propia lymphocytes from the large intestine than nontreated GFT i.p.-immunized mice. Moreover, we found that sera of metronidazole–mebendazole pretreated mice showed very low recognition of amebic proteins by Western blot. These data suggest that metronidazole and mebendazole treatment suppresses both the mucosal and systemic humoral immune response. The purpose of this work was to determine the effect of a 7-day 2.5 mg/per dose metronidazole and mebendazole (1:1) pretreatment in Balb/c mice immmunized i.p. with GFT by analyzing lamina propia lymphocyte supernatants from the small and large intestine and the systemic antiamebic immune response.

Entamoeba histolytica , a protozoan parasite and causative agent of human amebiasis, has unusual cellular and biochemical features. It lacks mitochondria and peroxisomes. The presence of a structured tubovesicular network such as endoplasmic reticulum (ER) and Golgi complex has not been clearly established, raising the question of which cellular and molecular mechanisms of protein traffic exist in this organism. Despite this, secretion of several amebic proteins to the extracellular environment is well documented. Furthermore, E. histolytica glycosylates some of its proteins. Glycosylation is a classic activity marker for transit of proteins through the eukaryotic secretory pathway. Two enzymes (GlcNAc-1-P transferase and GlcNAc transferase) involved in early steps leading to N-glycosylation have been characterized at the biochemical level in E. histolytica (1). However, the functional compartments associated to these activities are still to be identified. To provide molecular evidence supporting the existence of an ER-like functional compartment in this protozoan, we searched for evolutionary conserved genes coding for eukaryotic ER resident proteins. A good candidate was the STT3 gene, a subunit of the oligosaccharyltransferase complex. An entamebal STT3-expressed sequence tag (EST) has been reported (2). N-linked glycosylation of proteins is carried out in the lumen of the ER. The first step involves the en bloc transfer of the precursor Glc 3 Man 9 GlcNAc 2 from the dolichol diphosphate-linked core-oligosaccharide assembled at the membrane of the ER to the appropriate Asn residues (AsnX-Ser/Thr) of nascent polypeptide chains. The reaction is catalyzed by the transmembranal ER lumen-oriented enzyme complex oligosaccharyltransferase (OTase). In yeast, the STT3 gene is essential for growth and protein glycosylation. Sc Stt3p regulates the substrate specificity and assembly of the OTase complex (3). Depletion of the Stt3 protein results in loss of transferase activity in vivo and a deficiency in the assembly of the OTase complex. The 78 kDa Sc Stt3 protein has an N-terminal hydrophobic domain with 12 predicted membrane-spanning segments and a carboxy-terminal hydrophilic domain that is located in the lumen of the ER. Homologues of the STT3 yeast gene have been isolated from C. elegans (4), mouse, and human (5), among others. In this report, we describe the cloning of the E. histolytica STT3 gene, a molecular probe for the presence of a functional compartment equivalent to the eukaryotic ER in this parasite.

Entamoeba histolytica is an enteric protozoan parasite of humans and the causative agent of amebiasis. Physiopathological mechanisms related to the production of liver damage during amebic infection are poorly understood. However, a deficient parasite-specific cell-mediated immunity has been reported in human amebiasis, which gradually recovers following antiamebic therapy. The potential contribution of cytokines for this suppression remains to be elucidated. Cytokine responses to infectious agents tend to segregate into two different patterns. Th-1 responses are characterized by interleukin (IL) 2, interferon-gamma (IFNg ), and tumor necrosis factor-alpha (TNFa ) production; Th-2 responses are marked by the production of IL-4, 5, 6, 9, and 10. Protozoan infections tend to be susceptible to Th1 responses, while helminth infections are usually controlled by Th-2 responses. TNFa , a cytokine produced by macrophages and monocytes, is a potent mediator of inflammatory and immunological reactions. In combination with IFNg , TNFa has been shown to endow murine macrophages and human neutrophils with the capacity to kill E. histolytica in vitro , although large quantities of these cytokines are required to produce this effect (1). However, macrophages isolated from amebic liver abscess are functionally deficient, because they are unresponsive to IFNg and lipopolysaccharide (LPS)-activating signals for production of TNFa and cytotoxicity against amebas, and are also deficient in their ability to develop a respiratory burst (2). As macrophages are potent cells for amebicidal activity, a Th-1 cytokine response would be central in controlling invasive amebiasis, whereas production of macrophage downregulating cytokines, such as IL-4 and IL-10, could inhibit the cellular immune response to E. histolytica (3). Although there are many in vitro studies, information on the production of cytokines during the in vivo amebic infection is lacking. The purpose of the present study was to identify TNFa in the amebic liver abscess in hamsters, because this cytokine may play an important role in the pathogenesis of this protozoan infection.

Proteases of Entamoeba histolytica are considered to be an important virulence factor in the pathogenesis of amebiasis. Cysteine proteinases have been suggested to play a key role in tissue invasion and modulation of cell-mediated immune response. Several cysteine proteinases with molecular masses in the range of 16–96 kDa have been observed in extracts of E. histolytica (1). Up to six different cysteine proteinase genes have been identified, and inhibition of their expression by antisense RNA has been shown to affect amebic virulence (2,3). The presence of a collagenase in E. histolytica trophozoites has been reported (4). Very little is known, however, about serine and metallo proteinases in this parasite, and no function has been attributed to such proteases. The present study was undertaken to investigate the presence and characterization of serine and metallo proteinases of E. histolytica.

Introduction Entamoeba histolytica , a human invasive parasite, adheresto extracellular matrix (ECM) components, particularly tofibronectin (FN) and collagen (COL), eliciting structural(rearrangement of the cytoskeleton) and metabolic (releaseof proteases) responses, suggesting active participation ofkinases in these processes, as described in other eukaryoticcells (1). Although much remains to be learned about thebiochemical events of trophozoite activation, it is clear thatprotein phosphorylation is fundamental to all activationpathways. The activation of trophozoites is regulated in partby engagement of specific receptors on the cell surface withligands present on the host cell surface or ligands present inthe ECM. Previously, we have described the existence of anamebic b1 integrin-like receptor (2) that associates with ty-rosine kinases as a result of FN stimulation (3). In this work,we have identified by renaturation assays the induction of a55-kDa kinase, which is autophosphorylated on tyrosine inresponse to amebic cell adhesion to FN and COL.Materials and MethodsInteraction of amebas with extracellular matrix compo-nents. E. histolytica HM-1:IMSS was grown as described(2). Plastic dishes precoated with FN (100 mg/mL) and COLI (300 mg/mL) were incubated with trophozoites at differenttimes. After incubation at 378C, nonadherent cells were re-moved and adhered parasites were lysed in the presence of10 mM Tris-HCl, pH 7.5, 150 mM NaCl, and a cocktail ofprotease and phosphatase inhibitors to obtain total extracts.Cells adhered to plastic were used as control.Renaturation assays. The kinase renaturation assay is amodification of Ferrel and Martin (4). Briefly, 50 mg of to-tal extracts from nonstimulated or FN- or COL-stimulatedtrophozoites were subjected to 10% SDS-PAGE and trans-ferred to PVDF membranes using a semidry transfer unit.Blotted samples were incubated for 1 h in 7 M guanidine-HCl, 2 mM EDTA, 50 mM DTT, and 50 mM Tris, pH 8.3.Membranes were washed in renaturation buffer containing100 mM NaCl, 2 mM DTT, 2 mM EDTA, 1% BSA, 0.1%NP-40, and 50 mM Tris-HCl, pH 7.5 and then incubated at48C in the same buffer for 16 h. Blots were blocked for 1 hwith 5% BSA (120 mM NaCl, 30 mM Tris-HCl, pH 7.5).Kinases were assayed incubating the blots with 50 mCi/mL[g

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It is now generally accepted that what was earlier known as Entamoeba histolytica actually is comprised of two genetically distinct but morphologically indistinguishable species— Entamoeba histolytica and Entamoeba dispar. The confirmation of these two distinct species of Entamoeba is perhaps the major accomplishment in the field of amebiasis research during the last decade. It is believed that only E. histolytica , sensu stricto , can cause intestinal and extraintestinal disease or produce asymptomatic colonization, while E. dispar cannot invade tissues. The role of antiamebic IgA response in immune defense against E. histolytica infection is not fully understood. A number of studies have reported that antiamebic IgA was present in the saliva of subjects with invasive amebiasis and asymptomatic cyst passers of E. histolytica or E. dispar (1–4). However, it is controversial whether antiamebic salivary IgA response occurs during asymptomatic E. histolytica infection (2,4). The galactoseinhibitable adherence lectin of E. histolytica is a well-characterized protein of E. histolytica. The present work analyzes the IgA antibody response against this amebic lectin in saliva of preschool children in Bangladesh.