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PubMed2006

Genetic diversity of glucose phosphate isomerase from Entamoeba histolytica.

To investigate the molecular basis of zymodeme analysis in the enteric protozoan parasite Entamoeba histolytica, genes encoding glucose phosphate isomerase (GPI) were isolated from four representative E. histolytica strains belonging to zymodeme II, IIalpha-, XIV, or XIX. Two alleles were obtained from each strain; six alleles with eight polymorphic nucleotide positions were identified among the four strains. Two of these eight polymorphic nucleotides resulted in non-conserved amino acid substitutions. Three GPI isoenzymes with distinct predicted isoelectric points were identified, which agrees well with the observed electrophoretic patterns of GPI from these strains. Amino acid comparisons of GPI from E. histolytica and other organisms revealed that all amino acid residues implicated for substrate binding and catalysis were conserved. Biochemical characterization of recombinant E. histolytica GPI confirmed that it possessed kinetic parameters similar to GPI from other organisms. The electrophoretic mobility of three GPI isoenzymes was examined by starch gel electrophoresis. Thus, we have established the molecular basis of the classical isoenzymes patterns that have been used for grouping E. histolytica isolates and for differentiation of E. histolytica from non-pathogenic Entamoeba dispar.

Amino Acid Sequence↗

PubMed2006

Entamoeba histolytica-associated diarrheal illness is negatively associated with the growth of preschool children: evidence from a prospective study.

The enteric protozoa, Cryptosporidium, Giardia and Entamoeba histolytica, cause diarrhea in children. We investigated the association of enteric protozoan-associated diarrheal illness with the nutritional status and growth of preschool children in Dhaka, Bangladesh. The subjects were 221 children aged 2-5 years who were followed prospectively for diarrheal illness for 3 years. The weight and height of the children were measured at entry and at 4-month intervals. Cryptosporidium and E. histolytica were diagnosed with commercially available stool antigen detection kits. Giardia was diagnosed by conventional microscopy. Cryptosporidium- and Giardia-associated diarrheal illness was not associated with the growth of the children. Children with E. histolytica-associated diarrheal illness had lower weight for age Z-score changes (-0.103+/-0.120 vs. 0.176+/-0.052, P=0.038). Similarly, the change in height for age Z-score was lower in children with E. histolytica-associated diarrheal illness (-0.348+/-0.186 vs. 0.142+/-0.08, P=0.018). Children with E. histolytica-associated diarrheal illness were 2.93 times (95% CI 1.01-8.52, P=0.047) more likely to be malnourished and 4.69 times (95% CI 1.55-14.18, P=0.006) more prone to be stunted. Entamoeba histolytica-associated diarrheal illness was negatively associated with the growth of preschool children.

Age Factors↗

PubMedDate not supplied

Nitric oxide synthase in Entamoeba histolytica: its effect on rat aortic rings.

NADPH-diaphorase activity has been considered as a nitric oxide synthase (NOS) marker. Therefore, the presence of NADPH-d activity in Entamoeba histolytica suggests that they have NOS activity. The aim of this work was to provide support for this contention. The amebic culture medium or amebic purified proteins induced relaxation of endothelium-denuded rat aortic rings pre-contracted with phenylephrine (10(-6) M), which was inhibited when the amebas were incubated with NG-monomethyl-L-arginine or aminoguanidine (NOS inhibitors), or by pretreatment of the aortic rings with methylene blue. L-Arginine reverted the L-NAME inhibitory effect. In addition, trophozoites produce NO in culture and they have proteins which were recognized by antibodies specific to NOS and show activity of NO synthase. In conclusion, our results provide evidence about the production of NO by trophozoites. This molecule may be responsible for the relaxation elicited by the amebic culture medium and may participate in the pathogenesis of the invasive amebiasis. Index Descriptors and Abbreviations: Entamoeba histolytica; NO, nitric oxide; NOS, nitric oxide synthase; iNOS, inducible nitric oxide synthase; ecNOS, endothelial nitric oxide synthase; NADPH-d, NADPH-diaphorase enzyme; beta-NADPH, beta-nicotinamide-adenine dinucleotide; L-NAME, N-omega-nitro-L-arginine methyl ester hydrochloride; NBT, nitobluetetrazolium; PBS, phosphate-buffered saline; EDTA, ethylenediaminetetraacetic acid; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Animals↗

PubMed2000

Studies on codon usage in Entamoeba histolytica.

Codon usage bias of Entamoeba histolytica, a protozoan parasite, was investigated using the available DNA sequence data. Entamoeba histolytica having AT rich genome, is expected to have A and/or T at the third position of codons. Overall codon usage data analysis indicates that A and/or T ending codons are strongly biased in the coding region of this organism. However, multivariate statistical analysis suggests that there is a single major trend in codon usage variation among the genes. The genes which are supposed to be highly expressed are clustered at one end, while the majority of the putatively lowly expressed genes are clustered at the other end. The codon usage pattern is distinctly different in these two sets of genes. C ending codons are significantly higher in the putatively highly expressed genes suggesting that C ending codons are translationally optimal in this organism. In the putatively lowly expressed genes A and/or T ending codons are predominant, which suggests that compositional constraints are playing the major role in shaping codon usage variation among the lowly expressed genes. These results suggest that both mutational bias and translational selection are operational in the codon usage variation in this organism.

Animals↗

PubMed2003

Molecular biology of the amitochondriate parasites, Giardia intestinalis, Entamoeba histolytica and Trichomonas vaginalis.

The amitochondriates are an assembly of unicellular protists that lack mitochondria, and often other typical eukaryotic organelles, such as peroxisomes. Relatively little research has been conducted on amitochondriates, even though marine waters are likely to be teeming with such organisms, representing yet unknown protist lineages. The three amitochondriates that have been studied in any detail are the three mucosal human parasites: Giardia, Trichomonas and Entamoeba. These pathogens have worldwide distribution and are the most commonly encountered parasites in North America and Europe. Despite significant differences in their lifecycles and pathogenic properties, Giardia, Trichomonas and Entamoeba are customarily grouped together based on their being microaerophilic, their anaerobic carbohydrate metabolism, their lack of mitochondria, and their placement on deep-branching lineages in eukaryotic phylogenetic trees. During the last decade, the development of functional tools has allowed molecular analyses of gene expression to be initiated on these divergent eukaryotes. The resulting data indicate significant differences between the organisation of genetic information and mechanisms of gene regulation in amitochondriates and other organisms. Promoter architecture, as well as the regulatory transcription factors required to mediate promoter activity, lacks the conservation observed for the transcriptional apparatuses of metazoa and even appear to be unique within the amitochondriates. Our knowledge of the molecular biology and gene expression in amitochondriates is still in its infancy and a discussion of the current status of research in this area is presented.

3' Untranslated Regions↗

PubMed2000

The genome of Entamoeba histolytica.

Estimation of genome size of Entamoeba histolytica by different methods has failed to give comparable values due to the inherent complexities of the organism, such as the uncertain level of ploidy, presence of multinucleated cells and a poorly demarcated cell division cycle. The genome of E. histolytica has a low G+C content (22.4%), and is composed of both linear chromosomes and a number of circular plasmid-like molecules. The rRNA genes are located exclusively on some of the circular DNAs. Karyotype analysis by pulsed field gel electrophoresis suggests the presence of 14 conserved linkage groups and an extensive size variation between homologous chromosomes from different isolates. Several repeat families have been identified, some of which have been shown to be present in all the electrophoretically separated chromosomes. The typical nucleosomal structure has not been demonstrated, though most of the histone genes have been identified. Most Entamoeba genes lack introns, have short 3' and 5' untranslated regions, and are tightly packed. Promoter analysis revealed the presence of three conserved motifs and several upstream regulatory elements. Unlike typical eukaryotes, the transcription of protein coding genes is alpha-amanitin resistant. Expressed Sequence Tag analysis has identified a group of highly abundant polyadenylated RNAs which are unlikely to be translated. The Expressed Sequence Tag approach has also helped identify several important genes which encode proteins that may be involved in different biochemical pathways, signal transduction mechanisms and organellar functions.

Animals↗

PubMedDate not supplied

Cyst production and transmission of Entamoeba and Endolimax.

We studied the cyst production of 340 asymptomatic carriers with single or mixed infections of Entamoeba histolytica/Entamoeba dispar, Endolimax nana and Entamoeba coli. It was found that the main source of transmission was a small group of 37 (10.9%) carriers who produced 70.2% of all cysts. Gender was an important factor for cyst production of Ent. histolytica/Ent. dispar, since males produced 6-fold more cysts than females. Females produced 'large' (13-15 microns) Ent. histolytica/Ent. dispar cysts and males small (10-12 microns) cysts when concentration was > 2000 cysts/g. When concentration was < 2000 cysts/g, cysts were the same size for both sexes (11.6 +/- 0.3 microns). In Ent. coli infections, an inverse relationship between the number and size of cyst was found for both sexes. Cysts of End. nana were not affected by gender or cyst concentration. Following cyst production by a carrier infected with Ent. coli and Ent. histolytica for 28 days, we identified synchronized cycles of cyst production for both species. Altogether, these data suggest that cyst-mediated transmission is a highly regulated process in which synchronization among different species and gender of the host have an important role in transmission.

Amebiasis↗

PubMedDate not supplied

Evaluation of newer diagnostic methods for the detection and differentiation of Entamoeba histolytica in an endemic area.

We evaluated the colorimetric polymerase chain reaction (PCR)-based method for the detection and differentiation of Entamoeba spp. and compared the efficacy of E. histolytica-specific antigen detection in faeces with the detection of specific antibodies to E. histolytica-specific antigen in faeces, by enzyme-linked immunosorbent assay. Faecal samples were obtained from patients attending hospital in Chandigarh, India, from March 2001 to February 2002. The PCR-based colorimetric method was found to be the most sensitive (100%) and it could differentiate between pathogenic and non-pathogenic Entamoeba spp. The present study also emphasized that the antigen detection system may prove to be a better diagnostic tool than the antibody detection system in endemic areas.

Animals↗

PubMedDate not supplied

Overdiagnosis of amoebiasis in the absence of Entamoeba histolytica among patients presenting with diarrhoea in Wonji and Akaki, Ethiopia.

To confirm the high reported incidence of intestinal amoebiasis among study participants at 2 cohort sites in Ethiopia where an HIV/AIDS study is taking place, stool samples of 232 patients with complaints of diarrhoea were examined for the presence of Entamoeba histolytica and E. dispar DNA between April and December 2001. By microscopy, 91 (39%) of the study participants were reported to harbour Entamoeba trophozoites and/or four-nucleated cysts. Using specific E. histolytica and E. dispar DNA amplification and detection, none of the study participants were found to be infected with E. histolytica and only 21 (9%) with E. dispar. The consequences of the overdiagnosis of E. histolytica are briefly discussed.

Adolescent↗

PubMed2001

The cell cycle of Entamoeba invadens during vegetative growth and differentiation.

The cell division cycle of Entamoeba invadens was studied during vegetative growth of trophozoites and during their differentiation into cysts. During vegetative growth of trophozoites, it was observed that DNA synthesis typically continued after one genome content had been duplicated. During encystation, DNA synthesis was arrested after 4n genome content had been synthesised. Using multi-parameter flow cytometry, the light scattering properties of cysts and trophozoites were studied. The cytoplasmic granularity, reflected by the side scatter of light, was proportional to DNA content of trophozoites, whereas cysts with similar DNA contents showed heterogeneity in their cytoplasmic granularity. Dynamic changes in the intracellular calcium pools were observed during differentiation of trophozoites to cysts. Comparison of E. invadens and Entamoeba histolytica cell cycles suggest that both organisms may have similar regulatory processes during cell division and differentiation. Since E. histolytica cannot be induced to encyst in axenic culture, analysis of the E. invadens cell cycle during encystation may be useful for identifying homologous processes in E.histolytica.

Animals↗

PubMed2003

Molecular analysis of repetitive DNA elements from Entamoeba histolytica, which encode small RNAs and contain matrix/scaffold attachment recognition sequences.

We have isolated two DNA elements-Eh MRS1 and Eh MRS2-from Entamoeba histolytica, which contain the eukaryotic consensus Scaffold/Matrix Attachment Region (S/MAR) bipartite recognition sequences. Both these sequences bind to high salt extractable nuclear proteins and insoluble nuclear matrix proteins in E. histolytica HM1:IMSS, suggesting that the predicted S/MAR recognition sequences may indeed function as scaffold attachment regions in E. histolytica. Sequence analysis shows that Eh MRS1 and Eh MRS2 contain internal tandem repeats ranging from units of 8-11bp and are themselves present as independent arrays of tandemly repeating units of approximately 1100bp each. Eh MRS1 and Eh MRS2 are localised on different chromosomes in E. histolytica HM1:IMSS. Both Eh MRS1 and Eh MRS2 also code for small molecular weight RNAs of unknown function. Thus, two unique sequences-Eh MRS1 and Eh MRS2-demonstrate very similar properties, suggesting that they belong to a superfamily of genomic elements, which may function as scaffold or matrix attachment sites in Entamoeba.

Animals↗

PubMed1999

L-myo-Inositol 1-phosphate synthase from Entamoeba histolytica.

L-myo-Inositol 1-phosphate synthase (I-1-P synthase) catalyses the primary reaction for the synthesis of inositol in a variety of prokaryotes, eukaryotes and in the chloroplasts of algae and higher plants. Inositol is a precursor of essential macromolecules like membrane phospholipids, GPI anchor proteins and lipophosphoglycans, which play a determinant role in the pathogenesis of protozoan parasites such as Leishmania and Entamoeba. However, there is no report of I-1-P synthase or its gene from these organisms. The gene INO1 coding for this enzyme was first cloned from Saccharomyces cerevisiae and subsequently from several plants. Using molecular cloning techniques we have isolated and characterised the INO1 gene coding for the enzyme I-1-P synthase from Entamoeba histolytica. Simultaneously, we have purified and characterised the native enzyme from E. histolytica trophozoites and the cloned gene product from Escherichia coli. The gene product and the purified enzyme were both shown to be recognised by a heterologous anti-I-1-P synthase antibody from the phytoflagellate Euglena gracilis. Phylogenetic analysis of I-1-P synthase sequences from different eukaryotes suggest that it is highly conserved across species and the origin of this enzyme precedes the evolutionary divergence of modern eukaryotes.

Amino Acid Sequence↗

PubMed2000

Differences in substrate specificity and kinetic properties of the recombinant hexokinases HXK1 and HXK2 from Entamoeba histolytica.

Entamoeba histolytica is responsible for amoebic colitis and liver abscess in humans. Entamoeba dispar is a closely related, morphologically indistinguishable nonpathogenic species. The hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) isoenzyme patterns distinguish the pathogenic and nonpathogenic species. Both species possess two hexokinases with very similar molecular mass and different isoelectric points. In order to understand the role of the two different isoenzymes from E. histolytica, we purified the recombinant hexokinases HXK1 and HXK2 and examined substrate spectrum and kinetic properties. The two enzymes displayed similar temperature and pH optima, they were inhibited strongly by AMP and ADP, not by glucose 6-phosphate. Both enzymes phosphorylated glucose well and were unable to phosphorylate fructose or galactose. We also detected significant differences. HXK1 was more sensitive to inhibition by AMP and ADP. Mannose was phosphorylated well by HXK1, but at a much lower rate by HXK2. We attempted to expand the substrate spectrum of E. histolytica HXK1 by modifying its active site to become similar to the active site of the fructose phosphorylating yeast hexokinase PII. None of the nine mutants gained any fructokinase activity, but all of them retained at least some glucokinase and mannokinase activity. Mannokinase activity was decreased drastically by two single amino acid exchanges, both of which contributed significantly to this effect. The data indicate that a complex interaction of a number of amino acid residues is necessary for the ability to phosphorylate a given hexose.

Animals↗

PubMed1989

Interaction of different strains of Entamoeba histolytica with target cells: characterization of electrophysiological and morphological features.

Two strains of Entamoeba histolytica with pathogenic zymodemes (SFL3, HK9), one strain with non-pathogenic zymodeme ("Bru") and one non-pathogenic Entamoeba sp. strain ("cold strain"), were investigated with respect to their interaction with target cells. Three test systems were used: 1) direct microscopical observation and qualitative as well as quantitative evaluation of contact and binding events with MDCK cells as targets, 2) kinetics of cytotoxic activity as measured by means of chromium release from 51Cr-labelled K562 cells, and 3) electrophysiological observations with freshly prepared mouse liver cells. We observed that the non-pathogenic cold strain interacted only shortly with target cells (statistical events, interaction type "I"), but did not induce morphological changes, chromium release or depolarization of targets. Non-pathogenic and avirulent strain "Bru" showed, apart from type "I"-binding, the ability to establish tight (type "II") and long-lasting contact (type "III") with targets, but again without cytotoxic effects. The pathogenic but avirulent strain HK9 tightly interacted (type "II") and sometimes long-lasting with target cells, but morphological changes and chromium release were of a moderate degree during the first 20 min, and depolarization was only a rare event. In contrast, strain SFL3 produced tight and long-lasting contacts (type "III" binding), leading to cell death in 83% (type "IV" interaction) within 20 min, substantial chromium release within 10 min and rapid depolarization ("electric collapse") of target cells.

Animals↗

PubMedDate not supplied

Expression of the serine rich Entamoeba histolytica protein (SREHP) in the avirulent vaccine strain Salmonella typhi TY2 chi 4297 (delta cya delta crp delta asd): safety and immunogenicity in mice.

Infection by the intestinal protozoan parasite Entamoeba histolytica remains a significant threat to health in much of the world. Here we describe the successful expression of the serine rich Entamoeba histolytica protein (SREHP), a protective antigen of ameba, in an attenuated vaccine strain Salmonella typhi TY2 chi 4297 (delta cya delta crp delta asd). The attenuation of S. typhi TY2 chi 4297 was not altered by expression of the SREHP-maltose binding protein (MBP) fusion protein and mice parenterally vaccinated with S. typhi TY2 chi 4297 expressing SREHP-MBP developed serum anti-amebic and anti-LPS antibodies. S. typhi TY2 chi 4297 expressing SREHP-MBP represents a prototype combination vaccine designed to prevent both amebiasis and typhoid fever.

Animals↗

PubMed2000

Entamoeba sp. (Sarcomastigophora: Endamoebidae) from ostriches (Struthio camelus) (Aves: Struthionidae).

The first case of Entamoeba of the 1-nucleate mature cyst group in birds is described. Trophozoites and cysts of Entamoeba have been found in ostriches (Struthio camelus) from farms located all over Spain. The cysts are large (13.47microm mean diameter); they possess one nucleus when mature, with a large endosome and peripheral chromatine arranged in small granules; chromatoid bodies, when present, are large and elongated. Trophozoites are large (19. 88microm mean diameter), with a clear differentiation between ecto- and endoplasm, this containing numerous vacuoles; the nucleus is large and diffuse. The characteristics of this amoeba resembles but do not completely fulfill those of E. suis and E. chattoni; also, these species are from mammals.

Animal Husbandry↗

PubMed1994

Isolation and characterization of a species-specific multicopy DNA sequence from Entamoeba histolytica.

A genomic library of Entamoeba histolytica (pathogenic strain HM-1:IMSS) was screened to detect repetitive DNA clones other than those from the highly abundant ribosomal DNA (rDNA). One such clone (HMc) had a 2.3 kb insert which hybridized with the main genome and not the rDNA circle. Southern hybridization of E. histolytica genomic DNA, digested with EcoR I and probed with HMc, showed multiple bands. The banding pattern was identical in all axenic pathogenic strains tested. Differences, however, existed when the banding pattern of a pathogenic strain was compared with that of a non-pathogenic strain. HMc was present in about 25-30 copies per genome in strain HM-1:IMSS. Nucleotide sequence analysis of HMc revealed a partial open reading frame which hybridized with a 1.35 kb poly A+ transcript in Northern blots. The deduced amino acid sequence did not, however, show significant homology with known proteins. The HMc sequence was found only in E. histolytica as it hybridized with 5 different axenic strains of E. histolytica but did not recognize other closely related species of Entamoeba. It has thus the potential to be used as a species-specific DNA probe.

Amino Acid Sequence↗

PubMed1995

Prevalence of antibodies against Entamoeba histolytica in Mexico measured by ELISA.

The prevalence of antibodies against Entamoeba histolytica was studied in the Mexican population using an immunoenzyme assay in solid phase (ELISA) and semiautomatic equipment. The antigen was a mixture of membrane proteins obtained by Triton X-100 extraction from an axenic culture of Entamoeba histolytica HM1-IMSS. The method was standardized by comparing serum samples from amoebic liver abscess patients with healthy volunteers. From the 60,538 samples supplied by the National Seroepidemiology Survey, antibodies were found in 4.49% (4.32-4.65% at 95% confidence limit). More significant titres occurred in the central region of the country. The ratio female to male was 1.25:1. The population living in metropolitan areas had probably been infected at a younger age than those living in the country. Important differences were found in the seroprevalence obtained by ELISA compared with a study which used indirect haemagglutination (IHA) in the same sample frame.

Adolescent↗