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PubMed1992

Purification and biochemical properties of calmodulin in Entamoeba histolytica and its distribution during secretion of electron-dense granules.

1. Calmodulin (CaM) was detected during secretion of electron-dense granules by Entamoeba histolytica trophozoites with immunofluorescence. 2. It was purified to apparent homogeneity by chromatography with a yield of 2.26 micrograms of calmodulin/mg of protozoan protein. Purity was established by gel electrophoresis. 3. The parasite calmodulin has properties characteristic of calmodulin isolated from other eukaryotes: an apparent molecular weight of 19 or 17 kDa in presence of EGTA or CaCl2, respectively, activation in a calcium dependent manner of bovine heart cyclic nucleotide phosphodiesterase, and its UV spectrum.

Animals↗
PubMed1990

Characterisation of a repetitive DNA family from Entamoeba histolytica containing Saccharomyces cerevisiae ARS consensus sequences.

Several repetitive DNA families were identified in Entamoeba histolytica DNA digested with Sau3AI. Characterisation of one of these repetitive DNA families showed the presence of multiple copies of Saccharomyces cerevisiae autonomously replicating sequence (ARS) core consensus sequences. The E. histolytica ARS consensus sequences allowed a yeast-integrating plasmid, YIP5, to replicate autonomously in S. cerevisiae. A 'bent DNA' fragment was located in one member of this E. histolytica repetitive DNA family.

Animals↗
PubMed1993

Cloning of the Eh cdc2 gene from Entamoeba histolytica encoding a protein kinase p34cdc2 homologue.

To understand better growth regulation in the protozoan parasite, Entamoeba histolytica (Eh), a homologue of the cdc2 gene encoding the yeast cyclin-dependent protein kinase, p34cdc2, has been cloned and sequenced. This gene, called Eh cdc2, contains a 79-bp intron located in the same place as the second of four introns in the Schizosaccharomyces pombe cdc2 gene. The sequence of an Eh cdc2 cDNA confirms the conserved eukaryotic splice donor (GT) and acceptor (AG) sites and shows that Eh is able to splice mRNAs. The spliced Eh cdc2 open reading frame is 291 amino acids (aa) long, encoding an M(r) 33,806 protein. The primary sequence of Eh cdc2 is most like those of cdc2 homologues Eg1 of Xenopus laevis and CDK2 of man (52% aa identity with each) and codes for (i) the serine (Ser), threonine (Thr), and tyrosine residues phosphorylated in p34cdc2 proteins, (ii) 32 of 33 aa conserved in other Ser/Thr protein kinases, and (iii) the sequence PVTSVRE instead of PSTAIRE found in most p34cdc2 proteins. This is the first cell-division-cycle regulatory protein homologue, as well as the first intron identified from Eh.

Amino Acid Sequence↗
PubMed1994

Cloning and sequence determination of the gene coding for the pyruvate phosphate dikinase of Entamoeba histolytica.

We isolated three overlapping clones from a DNA genomic library of Entamoeba histolytica strain HM1:IMSS, whose translated nucleotide (nt) sequence shows similarities of 51, 48 and 47% with the amino acid (aa) sequences reported for the pyruvate phosphate dikinases from Bacteroides symbiosus, maize and Flaveria trinervia, respectively. The reading frame determined codes for a protein of 886 aa.

Amino Acid Sequence↗
PubMed1994

Cloning, genomic organization and transcription of the Entamoeba histolytica alpha-tubulin-encoding gene.

We have isolated and characterized an Entamoeba histolytica alpha-tubulin (alpha Tub)-encoding gene (Eh alpha tub). A 700-bp DNA fragment was amplified by PCR using primers derived from consensus alpha- and beta-Tub amino acid (aa) sequences from different organisms and E. histolytica DNA as the template. These PCR fragments were used to screen both genomic DNA and cDNA libraries in order to isolate an Eh alpha tub structural gene. Two overlapping clones containing the complete alpha tub ORF (1392 bp) were isolated from the genome and cDNA libraries. The deduced aa sequence of Eh alpha Tub has 55.5, 50 and 52% identity to Plasmodium falciparum alpha Tub 2, Saccharomyces cerevisiae alpha Tub 2 and human alpha Tub, respectively. Interestingly, the predicted Eh alpha Tub protein lacks a poly-acidic motif at its C terminus which is involved in Tub polymerization and microtubule-associated protein binding in other organisms. This fact may indicate a difference in tubule assembly in this organism and could provide a potential key for the development of therapeutic agents. According to Southern blot experiments and the sequences of several clones, at least two non-adjacent copies of alpha tub are present in the E. histolytica genome. A 1.5-kb transcript corresponding to the alpha tub mRNA was detected in mRNA from asynchronous E. histolytica trophozoites.

Amino Acid Sequence↗
PubMed1994

Characterization of two distinct gene transcripts for ribosomal protein L21 from pathogenic and nonpathogenic strains of Entamoeba histolytica.

A second gene (rp-L21) copy, clone g34, coding for ribosomal (r-) protein L21, was isolated from the pathogenic (P) strain HM-1:IMSS cl6 of the intestinal parasite Entamoeba histolytica (Eh). The gene was compared to the previously isolated copy, gLE3 [Petter et al., Mol. Biochem. Parasitol. 56 (1992) 329-334], with respect to its primary structure, mRNA levels and binding to the r-complex during translation. Unlike the gLE3 gene copy [Petter et al., Mol. Biochem. Parasitol. 56 (1992) 329-334], g34 was found not to be physically connected to an actin gene copy. Homologous copies of the two rp-L21 genes were also characterized from the nonpathogenic (NP) strain SAW1734R clAR, as well as from its P derivative. Sequence comparison of the coding regions of the two rp-L21 revealed almost full identity. Significant differences were found, however, within their 3' and 5' flanking regions. Using the 3' rapid amplification of cDNA ends (3' RACE) method [Frohman et al., Proc. Natl. Acad. Sci. USA 85 (1988) 8998-9002], as well as Northern and slot blot hybridizations, it was demonstrated that both rp-L21 mRNAs are found in similar amounts. However, as was shown by differential hybridization, the relative binding of each transcript to the r-complex varied somewhat between P and NP strains. This finding suggests that the control of expression of rp-L21 in Eh may involve regulation at the post-transcriptional level.

Amino Acid Sequence↗
PubMed1995

Increase in mRNA of multiple Eh pgp genes encoding P-glycoprotein homologues in emetine-resistant Entamoeba histolytica parasites.

With the goal of understanding possible mechanisms of drug resistance by the protozoan parasite Entamoeba histolytica (Eh), two novel Eh P-glycoprotein (Pgp) genes (Eh pgp5 and Eh pgp6) were sequenced, and the expression of four Eh pgp genes determined in wild-type (wt) clone A and emetine-resistant (EmR) clone C2 amebae. The Eh pgp5 gene encodes a 1301-amino acid (aa) protein that is similar to those of Eh pgp1 (64% aa identity), Eh pgp2 (61%), Eh pgp6 (39%) and Homo sapiens MDR (multidrug-resistance-encoding)(Hs MDR1; 38%) genes. The 1282-aa Eh pgp6 open reading frame (ORF), which is 19-28 aa shorter than those encoded by other Eh pgp, is also similar to those of Eh pgp1 (46% aa identity), Eh pgp2 (38%), and Hs MDR1 (39%). Both Eh pgp5 and Eh pgp6 ORF predict two ATP-binding cassettes and twelve hydrophobic alpha-helices, which form the putative transmembrane channel. EmR clone C2 amebae, growing at all concentrations of drug, show increased amounts of Eh pgp1 and Eh pgp6 mRNA when compared to wt clone A amebae. In contrast, only clone C2 amebae selected for growth at the highest concentrations of emetine (100-200 micrograms/ml) show increased Eh pgp5 mRNA, while mRNA of both clone C2 and clone A Eh amebae fail to bind an Eh pgp2-specific probe. It appears then that multiple Pgp may contribute to amebic Em resistance in vitro.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

A novel transcribed repeat element from Entamoeba histolytica.

We have identified an unusual 0.55-kb DNA repeat element specific to Entamoeba histolytica (Eh) which we call interspersed element (IE). The IE is a common feature in independently isolated genomic and cDNA fragments. Hybridization of labeled IE sequences to trophozoite DNA, RNA and first-strand cDNA prepared from poly(A)-enriched mRNA indicate that the IE are reiterated about 500 times per Eh trophozoite and that one or more can be found as RNA transcripts. These features and the degree of conservation of IE suggest a possible role for these sequences.

Animals↗
PubMed1996

Heterogeneity of Entamoeba histolytica rac genes encoding p21rac homologues.

Entamoeba histolytica (Eh), the parasite that causes amebic dysentery, is the only protozoan that phagocytoses bacteria, epithelial cells and red blood cells. Numerous low-molecular weight GTP-binding proteins, called p21rac, are implicated in signal transduction and actin polymerization during phagocytosis by macrophages and Dictyostelium discoideum (Dd). Here, molecular cloning techniques were used to obtain four Eh rac genes that encoded putative p21rac, as well as segments of two Eh rac pseudogenes. The predicted Eh p21rac, which share 55-81% amino acid (aa) identities with each other, include one that closely resembles the p21rac1 of man, Dd, Drosophila melanogaster and Caenorhabditis elegans; two that resemble the p21racC of Dd; and one that is unique. An alignment of the Eh rac ORF with other rac family proteins reveals multiple aa that distinguish p21rac1, p21racC and p21cdc42. We conclude that the Eh genes encoding amebic p21rac, which are the first identified from a protozoan parasite, are numerous and heterogeneous.

Amino Acid Sequence↗
PubMed1995

The antiamoebic effect of a crude drug formulation of herbal extracts against Entamoeba histolytica in vitro and in vivo.

The antiamoebic effect of a crude drug formulation against Entamoeba histolytica was studied. In the traditional system of medicine in India, the formulation has been prescribed for intestinal disorders. It comprises of five medicinal herbs, namely, Boerhavia diffusa, Berberis aristata, Tinospora cordifolia, Terminalia chebula and Zingiber officinale. The dried and pulverized plants were extracted in ethanol together and individually. In vitro amoebicidal activity was studied to determine the minimal inhibitory concentration (MIC) values of all the constituent extracts as well as the whole formulation. The formulation had a MIC of 1000 micrograms/ml as compared with 10 micrograms/ml for metronidazole. In experimental caecal amoebiasis in rats the formulation had a curative rate of 89% with the average degree of infection (ADI) reduced to 0.4 in a group dosed with 500 mg/kg per day as compared with ADI of 3.8 for the sham-treated control group of rats. Metronidazole had a cure rate of 89% (ADI = 0.4) at a dose of 100 mg/kg per day and cured the infection completely (ADI = 0) when the dosage was doubled to 200 mg/kg per day. There were varying degrees of inhibition of the following enzyme activities of crude extracts of axenically cultured amoebae: DNase, RNase, aldolase, alkaline phosphatase, acid phosphatase, alpha-amylase and protease.

Acid Phosphatase↗
PubMed1995

Evaluation of an enzyme-linked immunoassay for the detection in serum of antibodies to Entamoeba histolytica.

A commercially available enzyme-linked immunoassay (ELISA) was compared to an indirect hemagglutination test (IHA) for the detection of antibodies to Entamoeba histolytica on 225 patients' serums. All of the 107 serums that had IHA titers of < 32 (interpreted as excluding the presence of invasive amebiasis) were ELISA negative. Sixty-three of 68 (93%) serums that had IHA titers of > or = 128 (interpreted as indicative of the presence of active or recent infection) were ELISA positive. Fifty serums had IHA titers of 32 or 64 that were considered "equivocal," and the ELISA results for these were: six positive, 35 negative, and nine "intermediate" (optical density values between the arbitrary positive cutoff and the low positive control). It was concluded that "intermediate" (and negative) ELISA results should be interpreted as excluding the presence of invasive amebiasis. Using these criteria, the results obtained with this ELISA appear to compare favorably with those of the "gold standard" IHA. Therefore, this ELISA provides a reliable alternative to the IHA for the serologic diagnosis of amebiasis, which may be advantageous for some laboratories in terms of lower cost, shorter test time, and improved efficiency.

Animals↗
PubMed1987

Development of inflammation and augmented chemotactic responsiveness of murine peritoneal macrophages following treatment with Entamoeba histolytica trophozoites.

The accumulation of inflammatory cells in the peritoneal cavity of C57BL/6 mice was examined following intraperitoneal injection of Entamoeba histolytica trophozoites. Two different strains of E. histolytica were used: a virulent strain (IP:0682:1) and a non-virulent strain (DKB). Injection of 10(6) trophozoites of either strain resulted in significant increases in the numbers of total peritoneal cells, macrophages and polymorphonuclear cells as compared to either saline-injected control mice or mice injected with 10-fold lower doses of trophozoites. The in vitro chemotactic response of macrophages from amoebae-induced exudates was also examined. Macrophages from mice treated with strain IP:0682:1 or DKB strain trophozoites were more responsive to complement-derived chemotactic factors than macrophages from saline-injected mice. This increase was significant on day 2 and persisted at enhanced levels until day 20 when the experiment was terminated. In addition, it was found that trophozoites activated normal mouse serum resulting in the production of serum-derived chemotactic activity.

Amebiasis↗
PubMed1987

Immunosuppression and splenomegaly in Entamoeba histolytica infection in mice.

Intestinal amoebiasis caused by Entamoeba histolytica trophozoites in mice is accompanied by a depression in the ability of this host to develop an immune response to sheep red blood cells. The number of splenic plaque-forming cells was reduced in mice inoculated intracecally with 2.5 x 10(5) trophozoites at 15, 25, 40, 65 and 75 days after infection when compared with non-infected mice. It was found that there was no significant difference between the spleen weight of the infected and non-infected control animals at 5 and 10 days following infection. However, a significant increase in spleen weight was observed by 15 days of infection and the spleens remained enlarged until termination of the experiment at 75 days. Thus, there was an inverse correlation between the PFC response and the spleen weight of infected animals.

Amebiasis↗
PubMed1988

Activation of macrophages by Entamoeba histolytica extracts in mice.

The effect of Entamoeba histolytica extracts on the production of inflammatory macrophages and the release of hydrogen peroxide (H2O2) and superoxide (O2-) from these cells was examined in C57BL/6 mice. Two different strains of E. histolytica, either virulent (IP:0682:1) or nonvirulent (DKB), were used in this study. The number of macrophages recovered from the peritoneal cavity of mice treated 5 days previously with 150 micrograms of either strain of amoebic extracts was significantly higher than in the saline-treated groups. Macrophages from mice treated with 150 micrograms of the IP:0682:1 strain of amoebic extracts exhibited a powerful burst of oxidative metabolis when triggered with phorbol myristate acetate (PMA). Extract from the non-virulent strain was much less effective in activating macrophages for the production of oxidative metabolites. Thus, a crude extract preparation from E. histolytica, particularly the virulent strain, induces a strong macrophage inflammatory response in which the cells also produce high levels of H2O2 and O2-.

Animals↗
PubMed1992

Activated mouse macrophages kill Entamoeba histolytica trophozoites by releasing reactive nitrogen intermediates.

Mouse macrophages activated by gamma interferon (IFN-gamma) and bacterial lipopolysaccharide (LPS) are highly cytotoxic for the enteric protozoan parasite Entamoeba histolytica. Herein, we show that this killing by activated macrophages is L-arginine dependent, inasmuch as it was blocked by exogenous arginase or NG-monomethyl-L-arginine. These two inhibitors had no effect on E. histolytica cytolytic activity against L929 fibroblasts. Also, macrophage killing of E. histolytica always correlated with nitrite presence in the supernatant fluids. Finally, it was shown that addition of excess iron or the reductant sodium dithionite to activated macrophages blocked their ability to kill E. histolytica. Overall, this suggests that killing of E. histolytica by activated macrophages depends on the production of reactive nitrogen intermediates which leads to critical iron loss and protozoan parasite death.

Animals↗
PubMed1992

A model of interaction between Entamoeba histolytica and Shigella flexneri.

The establishment of a relationship between Entamoeba histolytica and certain bacteria may contribute to the expression and/or enhancement of the pathogenicity of this parasite. Recent experiments have shown that bacteria expressing mannose-binding lectins on their surface could attach to mannose-containing molecules on the surface of amoebae. In this study, we established a model of interaction between. E. histolytica and Shigella flexneri. Using well-characterized mutants of S. flexneri, we studied the role of type I pili expression and the invasive phenotype of S. flexneri in the interaction between amoebae and the bacteria. Type I pili expression allowed attachment and subsequent internalization of S. flexneri by amoebae, these events were not observed in isogenic strains that did not express type I pili. Invasive as well as non-invasive variants of S. flexneri expressing type I pili were slowly digested by amoebae following internalization. Morphological studies showed that the specific features of the interaction depend on the dynamics of the distribution of mannose residues on the amoebic membrane during the interaction.

Animals↗
PubMed1996

Role of signalling and cytoskeletal rearrangements in the pathogenesis of Entamoeba histolytica.

Virulence of Entamoeba histolytica is characterized by intestinal tissue invasion, engulfment of host cells and the formation of liver abscesses. The actin-rich cytoskeleton of the amoeba allows rapid changes in morphology in response to signals from external stimuli. Cellular and molecular studies have described some of the proteins that participate in signalling and in cytoskeletal changes.

Animals↗
PubMed2005

Genetic characterization of Entamoeba dispar isolates in Northeast Brazil.

The genetic variability of Entamoeba dispar strains was investigated in 39 positive isolates on a survey of 1783 individuals from two different cities of Northeast Brazil (Recife and Macaparana) using two polymorphic species-specific loci (loci 1-2 and 5-6). A combinatory clustering analysis revealed no geographical correlation and remarkable genetic polymorphism among all the isolates examined. Nevertheless, a comparison of the frequency of eight individual PCR products, shared by both Recife and Macaparana populations, for the two loci, showed that only one product of locus 5-6 was significantly different between the two cities. These results suggested that the Macaparana population is infected by similar strains and that locus 5-6 shows potential in assaying questions related to the molecular epidemiology of this region.

Adolescent↗