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PubMed2003

Identification of Entamoeba histolytica thiol-specific antioxidant as a GalNAc lectin-associated protein.

Entamoeba histolytica is a human intestinal parasite that causes amebic dysentery. A cell surface amebic adhesin, the galactose and N-acetyl-D-galactosamine inhibitable (GalNAc) lectin mediates amebic adherence to and contact-dependent killing of host cells. Previous work has suggested that the GalNAc lectin transduces signals via protein interactions with its short cytoplasmic domain. We used a yeast two-hybrid system to screen an E. histolytica cDNA library for proteins that interact with the GalNAc lectin cytoplasmic domain. One isolate was the E. histolytica thiol-specific antioxidant (TSA). TSA is an enzyme that detoxifies hydrogen peroxide. TSA did not interact in yeast two-hybrid experiments with a mutant version of the lectin cytoplasmic domain, confirming the specificity of the lectin-TSA interaction. Furthermore, mutational analyses of the TSA isolate demonstrated that an in-frame five amino acid sequence introduced between amino acids 61-62 yielded a TSA mutant that did not interact with the lectin cytoplasmic domain upon expression in the yeast two-hybrid system. The association of TSA and GalNAc lectin was further supported by co-immunoaffinity purification. Confocal microscopy demonstrated co-localization of TSA and GalNAc lectin at sites of ameba:host cell contact. Recruitment of TSA by the GalNAc lectin suggests a novel mechanism of parasite defense against reactive oxygen intermediates generated by host peripheral mononuclear cells.

Acetylgalactosamine↗
PubMed2003

L10 ribosomal protein from Entamoeba histolytica share structural and functional homologies with QM/Jif-1: proteins with extraribosomal functions.

In the present work, the complete amino acid sequence of the Entamoeba histolytica ribosomal protein L10 (EhL10) is reported. cDNA of 630bp revealed an open reading frame that encodes a protein of 210 amino acids. Analysis of EhL10 ribosomal protein revealed 75% similarity and 57% identity with QM protein from Homo sapiens and 78 and 60%, respectively, with Arabidopsis thaliana. Western blot analysis of ribosomal proteins from E. histolytica showed that EhL10 protein is part of the ribosomal complex. Immunofluorescence analysis of EhL10 distribution in a transfected E. histolytica strain showed that EhL10 protein was mainly localized in the nucleus of trophozoites. Overexpression of EhL10 ribosomal protein in trophozoites transfected with the pExEhNeo/EhL10 vector exhibited a 60% reduction in cellular growth. DNA mobility-shift assays demonstrated that EhL10 ribosomal protein was able to destabilize the activating protein 1 (AP-1) complex binding specifically to the c-Jun-like protein. It is proposed in this study that the complex formation of EhL10 with c-Jun-like protein interferes with transcriptional activation of genes controlled by Jun (i.e. gene involved in cell growth). It is also being reported identification of a member of the AP-1 complex, the c-Jun-like protein, in nuclear extracts of E. histolytica using human-specific antibodies against this protein. The observations suggest that EhL10 may have an extraribosomal function in E. histolytica involved in suppression of cell proliferation in E. histolytica similar to the QM protein.

Amino Acid Sequence↗
PubMed1996

Upstream regulatory elements controlling expression of the Entamoeba histolytica lectin.

Entamoeba histolytica genomic organization and putative promoter elements appear to be distinct from both metazoan and better characterized protozoan organisms. The recent development of DNA-mediated transfection for E. histolytica enabled characterization of cis-acting promoter elements required for gene expression. A deletion and replacement analysis was conducted on the promoter of an E. histolytica gene encoding the heavy subunit of the N-acetyl-beta-D-galactosamine-specific adhesin (hgl5). Deletion of the DNA from -1000 bases to -272 bases upstream from the start of transcription of hgl5 did not decrease reporter gene expression. Subsequent nested deletions and 10-bp replacement mutagenesis identified four positive upstream regulatory elements between bases -219 to -200, -189 to -160, -69 to -60, and -49 to -40. A negative upstream regulatory element between bases -89 to -80 was conserved upstream of three other E. histolytica genes. Mutation of the previously unidentified 'GAAC' element conserved within the putative core promoter decreased reporter gene expression by 75%. Site directed mutagenesis of the putative TATA element decreased reporter gene expression by greater than 50%, while mutation of the putative initiator element resulted in a more modest decrease. This analysis suggests that E. histolytica promoters are unlike other protozoan promoters, with AT-rich upstream regulatory elements, a non-consensus TATA element, the "GAAC' element, and an unusual initiator element.

Animals↗
PubMed1996

Detection, purification and partial characterization of beta-amylase from trophozoites of Entamoeba invadens.

Trophozoites of Entamoeba invadens were able to ingest glucopolysaccharides and metabolize them. An activity capable of degrading these substrates was purified to apparent homogeneity. The enzyme was identified as beta-amylase (alpha-1,4-D-glucan maltohydrolase EC 3.2.1.2): It was active against glycogen, amylose and amylopectin in a ratio of 100:198:133 and was also able to attack linear alpha-1,4-glucooligosaccharides with more than three glucose moieties. All degradation experiments yielded maltose as reaction product, and no free glucose could be detected. While amylose was completely degraded, amylolysis of glycogen and amylopectin yielded limit dextrins besides maltose. The enzyme was completely inactive against cyclohexaamylose, cycloheptaamylose and pullulan, indicating its lack of endo-glucosidase specificity. Hydrolysis of 4-nitrophenyl-maltoheptaoside resulted in the successive removal of maltose units from the non-reducing end yielding 4-nitrophenyl-maltopentaoside, -trioside and -glucoside. No 4-nitrophenyl-glycosides with even numbered glucose moieties were formed from this substrate. The enzyme exhibited a relative molecular mass of M(r) = 45,000 +/- 5% by gel filtration and sodium dodecyl sulfate (SDS) gel electrophoresis and the N-terminal sequence 1 VEVNVMLPL 9. Optimal hydrolysis was observed at pH 5.5 and a temperature of 42 degrees C. On the basis of inhibition by mercury ions and p-chloro-mercurybenzoate and abrogation of this effect by thiol reagents beta-amylase was identified as sulfhydryl-enzyme.

Animals↗
PubMed1997

Tetracycline-controlled gene expression in Entamoeba histolytica.

To provide further tools for functional genetics of the protozoan parasite Entamoeba histolytica, we have tested the suitability of the bacterial TN10-encoded tet-repressor/tet-operator system for gene regulation in ameba trophozoites. Expression of the tet-repressor within the ameba was driven by the wild-type endogenous lectin gene promoter from episomal transfected plasmids. Tetracycline-inducible expression of a reporter gene driven by a modified tet-operator-bearing lectin gene promotor was monitored by transient and episomal transfection. Promotor activity was dependent on the position of the tet-operator insertion. Under appropriate conditions, expression of the reporter gene in tet-repressor expressing cells revealed only background levels but was inducible up to 240-fold by the addition of non-toxic amounts of tetracycline reaching full activity within 36 to 48 h. Because of the tight and rapid control by tetracycline, the tet-repressor controlled lectin gene promotor should be a usefull tool for reverse genetic approaches in E. histolytica as well as for recombinant protein expression within this anaerobic organism.

Animals↗
PubMed1997

Characterization of EhCaBP, a calcium-binding protein of Entamoeba histolytica and its binding proteins.

A novel calcium-binding protein (EhCaBP) has been recently identified and characterized from the protozoan parasite Entamoeba histolytica. In order to decipher the function of this protein, a few basic properties were investigated and compared with the ubiquitous Ca(2+)-signal transducing protein calmodulin (CaM). Indirect immunofluorescence and immunoprecipitation analyses using specific antibodies against EhCaBP suggest that it is a soluble cytoplasmic protein with no major post-translational modification. EhCaBP did not stimulate cAMP-phosphodiesterase activity, differentiating it from all known CaMs. Affinity chromatography of [35S]methionine-labelled proteins of E. histolytica trophozoites using EhCaBP-sepharose column showed Ca(2+)-dependent binding of a group of proteins. Radiolabelled proteins from the same extract also bound to CaM-sepharose. However, the proteins bound to the two columns were different as revealed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. At least one of the EhCaBP-binding proteins became phosphorylated as revealed by in vivo phosphorylation analysis. The binding-proteins could not be detected in E. invadens (a species that is pathogenic in reptiles) and E. moshkovskii (which is found in the human gut but is not pathogenic), two species in which EhCaBP-like protein has not been found. Two distinct Ca(2+)-dependent protein kinases, which get activated by EhCaBP and CaM respectively, were detected in E. histolytica. These kinases require different levels of Ca2+ for their maximal activities. Affinity chromatography also showed the binding of protein kinase(s) to EhCaBP in a Ca(2+)-dependent manner. Our data suggest that there may be novel Ca(2+)-signal transduction pathway in E. histolytica mediated by EhCaBP.

Animals↗
PubMed1997

A tetracycline-inducible gene expression system in Entamoeba histolytica.

We have developed an episomal inducible gene expression system in Entamoeba histolytica based on the TetR repressor. The tetR gene was placed under control of 5' and 3' ferredoxin (fdx) regulatory sequences on a plasmid encoding the hygromycin resistance gene directed by 5' and 3' hgl sequences. The reporter luciferase constructs were introduced on a second episome bearing the neomycin resistance gene controlled by 5' and 3' actin sequences. The reporter constructs were driven by the hgl5 promoter in which the tetO sequence was introduced. We found that the optimal tetO location for induction by tetracycline was +4 from the start of transcription. The efficiency of repression and the induction ratio could be improved by increasing hygromycin levels, presumably by increasing tetR plasmid levels. Under these conditions, maximal induction of reporter luciferase could be effected with 5 micrograms/ml tetracycline in 18 h. This system permits regulated expression of the reporter gene over two orders of magnitude and should be useful in the analysis of gene function.

Animals↗
PubMed1997

Entamoeba histolytica contains a gene encoding a homologue to the 54 kDa subunit of the signal recognition particle.

We have determined the nucleotide sequence and predicted amino acid sequence of the 54 kDa subunit of the signal recognition particle (SRP54) from the amitochondrial protist Entamoeba histolytica. The SRP54 gene was isolated from a genomic library using a polymerase chain reaction (PCR) probe. Nucleotide sequence analysis of a 2.3 kb fragment, derived from a 7 kb genomic clone, revealed an open reading frame encoding a protein of 487 amino acids (MW 53.8 kDa). The identities of the predicted amino acid sequence with its homologues from other species were between 24 and 47%. Functional domains previously defined for the SRP54-type proteins were present in the entamoebal sequence, such as the amino-terminal GTP binding domain (G domain) and the carboxy-terminal methionine rich domain (M domain). SRP54 mRNA contains an extra G residue at the 5' end, suggesting that capping of poly-A(+) transcripts is present in E. histolytica. Evolutionary analysis of the SRP54 based on phylogenetic inference placed the E. histolytica sequence as an early divergence of the eukaryotic tree. Although the function of the entamoebal homologue remains to be elucidated, the identification of the SRP54 gene constitutes the first evidence for SRP related proteins in protozoans.

Amino Acid Sequence↗
PubMed1997

Unusual genome organisation in Entamoeba histolytica leads to two overlapping transcripts.

We have isolated homologs of the mini chromosome maintenance (MCM) gene family from the parasitic protozoan Entamoeba histolytica. The full length genomic and cDNA clones for the Eh MCM3 gene have been characterised. The Eh MCM3 gene is much smaller than the Saccharomyces cerevisiae MCM3 gene and other eukaryotic homologs of the MCM3/P1 family. The predicted Eh Mcm3 protein was 597 amino acids long and showed 37 and 46% positional identity with the Sc Mcm3 and the mouse P1 homologs respectively. While proceeding along the chromosome from the Eh MCM3 gene, we have identified a homolog (Eh PAK) of the murine p21 activated kinase (Rn KPAK), or S. cerevisiae STE20. Eh PAK lies 126 bp upstream of the Eh MCM3 gene. The predicted Eh p21 activated kinase protein was 459 amino-acids long and showed 33% positional identity with the murine p21 activated kinase and its yeast homolog Ste20. Analysis of cDNA and genomic sequences shows that the 3' untranslated region (UTR) of the Eh PAK mRNA and the 5' UTR of the Eh MCM3 mRNA are transcribed from a common 40 bp genomic segment. This is the first report of an amoeba gene being physically linked to a second gene such that their transcripts are overlapping and there is no non-transcribed intergenic region between the two genes. Primer extension studies have confirmed that unlike most E. histolytica genes, which have short 5' UTRs, the Eh MCM3 mRNA has a 126 bp long 5' UTR and the Eh PAK mRNA has a 265 bp long 5' UTR.

Amino Acid Sequence↗
PubMed1997

Inhibition of expression of the lysine-rich 30 kDa surface antigen of Entamoeba dispar by the transcription of its antisense RNA.

The gene coding for the 30 kDa lysine rich surface antigen (Ed-Ag) that is present on membrane surfaces of Entamoeba dispar trophozoites has been characterized. A specific monoclonal antibody MAb 318-28 prepared against this antigen reacts with all E. dispar strains tested, but not with any of the antigens of E. histolytica. In order to understand the function of this antigen, we constructed two plasmids, pEdA-9 and pEdA-Rev, in which the antigen-coding sequence was introduced into the pEhAct-Neo shuttle vector in the direct and opposite orientation, respectively. When E. dispar trophozoites were transfected with pEdA-9, only a slight increase was observed in the expression of the antigen. However, when E. dispar trophozoites were transfected with pEdA-Rev, the expression of the native 30 kDa antigen was significantly inhibited. This inhibition was proportional to the level of resistance of the E. dispar culture to the neomycin derivative G418. Cytopathic assays detected only a slight difference between untransfected, pEdA-9 transfected and pEdA-Rev transfected trophozoites.

Amebicides↗
PubMed1997

Heterogeneity of DNA content and expression of cell cycle genes in axenically growing Entamoeba histolytica HM1:IMSS clone A.

The cell division cycle of Entamoeba histolytica was studied using multi-parametric flow cytometry in asynchronous and partially synchronised cells. Dynamic changes in the DNA synthesis and DNA content of axenically growing trophozoites were observed by using 5-bromo-2'-deoxyuridine (BrdU) uptake and DNA specific fluorochromes. It was observed that DNA synthesis in these cells continues beyond the typical S-phase stop point when DNA duplication is complete. Asynchronously growing E. histolytica cells could be synchronised by serum starvation followed by serum re-addition. BrdU incorporation in synchronised cells showed that cell synchrony is maintained for at least one generation time, in which the G1 phase lasts for 2-3 h and the S-phase lasts for 5-6 h. Analysis of our results revealed that E. histolytica trophozoites, growing in axenic medium, are made up of a heterogenous population of euploid and polyploid cells. The number of polyploid cells increases with age of the cells in culture. Expression of putative cell cycle and signal transduction markers was studied using specific antibodies and changes in their expression levels have been correlated with changes in the DNA content. Based upon our results we could identify G1, S and G2 phases of the cell cycle of E. histolytica and also predict the mechanism underlying the generation of polyploidy in these cells, which may have significant effects on its biology and pathogenesis.

Animals↗
PubMed1997

Electron probe analysis and biochemical characterization of electron-dense granules secreted by Entamoeba histolytica.

The interaction of Entamoeba histolytica with collagen induces the intracellular formation and release of electron-dense granules (EDGs) containing collagenase activity which are important in the pathogenicity of this parasite. Purified EDGs contain at least 25 polypeptides with acidic pIs, nine gelatinase activities, small molecules, including inorganic phosphate (Pi), pyrophosphate (PP) and other elements, including Na, Mg, S, Cl, K, Ca and Fe as measured by scanning transmission electron microscopy. Six of these polypeptides with apparent molecular weights of 108, 106, 104, 97, 68 and 59 kDa and two protease activities with apparent molecular weights of 40 and 85 kDa were detected exclusively in the EDGs and were not observed in total trophozoite extracts. Actin was also detected in the EDGs. Therefore, EDGs are a complex of mainly cationic proteins, which contains numerous proteolytic activities, actin and small molecules such as P(i), PP and cations.

Actins↗
PubMed1998

Molecular cloning and characterization of the genes encoding two isoforms of cysteine synthase in the enteric protozoan parasite Entamoeba histolytica.

The enteric protozoan parasite Entamoeba histolytica was shown to possess cysteine synthase (CS) activity. The cDNA and genomic clones that encode two isoforms of the E. histolytica CS were isolated and characterized from a clonal strain of E. histolytica by genetic complementation of the cysteine-auxotrophic Escherichia coli NK3 with an E. histolytica cDNA library. The two types of the E. histolytica CS genes differed from each other by three nucleotides, two of which resulted in amino acid substitution. Deduced amino acid sequences of the E. histolytica CS, with a calculated molecular mass of 36721 Da and an isoelectric point of 6.39, exhibited 38-48% identity with CS of bacterial and plant origins. The absence of the amino-terminal transit peptide in the deduced protein sequences and the presence of the CS protein mainly in the supernatant fraction of the amoebic lysate after cellular fractionation suggested that the identified E. histolytica CS genes encoded cytosolic isoforms. Substrate specificity of the recombinant E. histolytica CS was similar to that of plant CS. Phylogenetic analysis indicates that the amoebic CS, first described in Protozoa, does not belong to any families of the CS superfamily, and represents a new family.

Amino Acid Sequence↗
PubMed1999

The electrophoretic karyotype of Entamoeba histolytica.

Although knowledge about gene organization and transcription control in the protozoan parasite Entamoeba histolytica has increased substantially during the last few years, questions remain open about ploidy, organization and number of chromosomes in this human pathogen. To get insight into these questions conditions were elaborated to consistently separate E. histolytica chromosomes using pulsed-field gel electrophoresis. Southern blot analyses indicated variations in number and size of homologous chromosomes between various E. histolytica isolates, but only minor differences were observed between clones of a given isolate. Depending on the isolate used 31-35 chromosomes were identified ranging in size from 0.3 to 2.2 megabases. The assignment of 68 independent cDNA probes to the chromosomes of three axenically cultured E. histolytica isolates identified 14 linkage groups, which suggested a haploid genome-size of < or =20 megabases. As single copy probes bound to as many as four chromosome-sized bands, it is most likely that E. histolytica has a functional ploidity of at least 4.

Animals↗
PubMed1999

Pantropic retroviral vectors mediate gene transfer and expression in Entamoeba histolytica.

Transformation of Entamoeba histolytica has been previously reported, but the foreign genes have all been replicated episomally. Pantropic retroviral vectors based on the Moloney murine leukemia virus with the envelope glycoprotein of vesicular stomatitis virus (VSV-G) have an extremely broad host range and can be concentrated to high titer. To investigate whether these pseudotyped, pantropic vectors can mediate gene transfer and expression in E. histolytica, we constructed a retroviral vector, in which a hygromycin phosphotransferase is expressed from the E. histolytica actin promoter. Data confirm the infection, integration, and expression of a foreign gene mediated by the provirus. To our knowledge, this is the most evolutionarily distant example of successful integration and expression of a mammalian retrovirus. Pantropic retroviral vectors may thus facilitate genetic analysis in species lacking transformation systems.

Animals↗
PubMed1999

Regulation of Entamoeba invadens encystation and gene expression with galactose and N-acetylglucosamine.

Encystation of Entamoeba invadens parasites is prevented by the presence of free galactose or N-acetylglucosamine in the encystation medium. Galactose prevents the formation of amoeba cellular aggregates which develop during the early phase of encystation, suggesting the presence of functional cell surface galactose-binding molecules, whereas N-acetylglucosamine allows aggregation to occur and prevents cyst formation at a later point. While studying sugar inhibition of amoeba encystation, it was found that high efficiency encystation required the inclusion in encystation medium of precise amounts of polyvalent galactose-terminated molecules, and these molecules could be supplied by serum or by defined glycoconjugates, including mucin. Addition of free galactose to encystation medium prevented the accumulation of three transcripts which are normally upregulated during encystation, and N-acetylglucosamine prevented accumulation of one of the transcripts. These results suggest the presence of distinct sugar-sensitive pathways that regulate differentiation of the amoeba trophozoite into infectious cysts.

Acetylglucosamine↗
PubMed1999

Entamoeba histolytica lacks trypanothione metabolism.

Entamoeba histolytica lacks glutathione reductase activity and the ability to synthesise glutathione de novo. However, a recent report suggested that exogenous glutathione can be taken up and conjugated to spermidine to form trypanothione, a metabolite found so far only in trypanosomatids. Given the therapeutic implications of this observation, we have carefully analysed E. histolytica for evidence of trypanothione metabolism. Using a sensitive fluorescence-based HPLC detection system we could confirm previous reports that cysteine and hydrogen sulphide are the principal low molecular mass thiols. However, we were unable to detect trypanothione or its precursor N1-glutathionylspermidine [ < 0.01 nmol (10(6) cells)(-1) or < 1.7 microM]. In contrast, Trypanosoma cruzi epimastigotes (grown in a polyamine-supplemented medium) and Leishmania donovani promastigotes contained intracellular concentrations of trypanothione two to three orders of magnitude greater than the limits of detection. Likewise, trypanothione reductase activity was not detectable in E. histolytica [ < 0.003 U (mg protein)(-1)] and therefore at least 100-fold less than trypanosomatids. Moreover, although E. histolytica were found to contain trace amounts of glutathione (approximately 20 microM), glutathione reductase activity was below the limits of detection [ < 0.005 U (mg protein)(-1)]. These findings argue against the existence of trypanothione metabolism in E. histolytica.

Animals↗
PubMed1999

Early and late endosomal compartments of Entamoeba histolytica are enriched in cysteine proteases, acid phosphatase and several Ras-related Rab GTPases.

Pure populations of early and late endosomes of Entamoeba histolytica were isolated by magnetic fractionation and characterized. It was shown that these vesicles were enriched in acid phosphatase and cysteine protease activities. An important virulence factor, a 27-kDa cysteine protease, was also enriched in early and late endosomes of E. histolytica. These data suggest that E. histolytica hydrolases reside in compartments that are part of or communicate with the endosomal pathway. To begin to identify the role of Rab GTPases in E. histolytica, an oligonucleotide approach was employed to screen an E. histolytica cDNA library for genes encoding Rab-like proteins. cDNAs encoding a Rab11-like protein (EhRab11) and a novel Rab protein (EhRabA) were isolated and characterized. The EhRab11 cDNA predicts a polypeptide of at least 206 amino acids with a molecular mass of at least 23.2 kDa. Phylogenetic analysis and alignment of EhRab11 with other Rab proteins demonstrated that EhRab11 shared significant homology at the amino acid level with Rab11-like proteins from a number of other eukaryotes, suggesting that EhRab11 is a Rab11 homolog for E. histolytica. The EhRabA clone predicts a polypeptide of 219 amino acids with a molecular mass of at least 24.5 kDa. EhRabA shared only limited homology at the amino acid level with other Rab proteins, suggesting that it is a novel member of this family of GTP-binding proteins. Finally, Western blot analysis demonstrated that EhRab11 and a previously described Rab7-like GTPase from E. histolytica was enriched in magnetically purified endosomal compartments of this organism.

Acid Phosphatase↗