Histolytica Protein Research Articles

Molecular-Genotyping Detection of Entamoeba histolytica in Diarrheic Patients.

Amebiasis is caused by Entamoeba histolytica, a protozoan that is found worldwide. The degree of pathogenesis of clinical isolates varies greatly. This study was aimed to molecular identification of E. histolytica in children using the nested polymerase chain reaction (nPCR), and then, a genotyping of positive E. histolytica isolates using the quantitative PCR (qPCR) assay through targeting serine-rich E. histolytica protein (SREHP) gene. For this purpose, a total of 50 bloody diarrheic stool samples were collected from the children attended to the Al-Zahraa' Teaching Hospital and Alkut Hospital for Gynecology, Obstetric and Pediatrics (Alkut, Wasit, Iraq) were subjected to the present study from September to December 2021. Firstly, the extracted DNAs that amplified using specific primers through targeting 18S rRNA gene and tested using nPCR assay were revealed an overall 48% (24/50) positive samples for E. histolytica. For genotyping, our results were detected an existence of four different genotypes (I, II, III and IV) with a significant prevalence of Genotype-II (54.17%) when compared to Genotype-I (20.83%), Genotype-III (12.5%) and Genotype- IV (12.5%). In addition, results of melting temperature of targeted genotypes were 84ºC, 83 - 83.5ºC, 82.5ºC and 81ºC for Genotype-I, II, III and IV, respectively. In conclusion, molecular amplification of 18S rRNA gene was revealed the large prevalence of E. histolytica among bloody diarrheic children of study areas; while, amplification of SREHP gene was reflected the widespread phenotypic variation of the Genotype-II suggesting the high ability of this genotype to spread infection in children. In different endemic areas as Iraq, the utilization of high-resolution genotyping methods showed the extremely polymorphic genetic structure of this parasite.

Protein Sumoylation Is Crucial for Phagocytosis in Entamoeba histolytica Trophozoites.

Posttranslational modifications provide Entamoeba histolytica proteins the timing and signaling to intervene during different processes, such as phagocytosis. However, SUMOylation has not been studied in E. histolytica yet. Here, we characterized the E. histolytica SUMO gene, its product (EhSUMO), and the relevance of SUMOylation in phagocytosis. Our results indicated that EhSUMO has an extended N-terminus that differentiates SUMO from ubiquitin. It also presents the GG residues at the C-terminus and the ΨKXE/D binding motif, both involved in target protein contact. Additionally, the E. histolytica genome possesses the enzymes belonging to the SUMOylation-deSUMOylation machinery. Confocal microscopy assays disclosed a remarkable EhSUMO membrane activity with convoluted and changing structures in trophozoites during erythrophagocytosis. SUMOylated proteins appeared in pseudopodia, phagocytic channels, and around the adhered and ingested erythrocytes. Docking analysis predicted interaction of EhSUMO with EhADH (an ALIX family protein), and immunoprecipitation and immunofluorescence assays revealed that the association increased during phagocytosis; whereas the EhVps32 (a protein of the ESCRT-III complex)-EhSUMO interaction appeared stronger since basal conditions. In EhSUMO knocked-down trophozoites, the bizarre membranous structures disappeared, and EhSUMO interaction with EhADH and EhVps32 diminished. Our results evidenced the presence of a SUMO gene in E. histolytica and the SUMOylation relevance during phagocytosis. This is supported by bioinformatics screening of many other proteins of E. histolytica involved in phagocytosis, which present putative SUMOylation sites and the ΨKXE/D binding motif.

The splicing factor U2AF84 elicits intron retention impacting the virulence of the protozoan parasite Entamoeba histolytica

Entamoeba histolytica is the causative agent of amoebic dysentery and liver abscesses. The parasite infects approximately 1% of the human population, resulting in circa 100,000 deaths annually. The determining factors resulting in amebiasis are not well understood. The proteome of in vivo assembled Early splicing complexes identified a huge set of E. histolytica proteins involved in RNA processing, among them U2AF84, which is the ortholog of the human U2AF65. The Carboxyl-terminus domain of this factor is 240 amino acids longer than U2AF65, of which the last 136 residues correspond to the KH-QUA2 acidic domain, involved in the regulation of gene expression through Intron Retention. Such domains are able to dimerize members of the STAR family of proteins, such as Splicing Factor 1 (SF1), a factor that binds to the intronic Branch point Sequence (BS) in the Early splicing complex though its own KH-QUA2. Recent transcriptomic data showed upregulated expression of U2AF84 in amoebas recovered from liver abscesses. As a hypothesis, we propose that the interaction between the U2AF84- and SF1-KH-QUA2 domains reduces splicing via intronic retention and the virulence of E. histolytica. Our objective was analyze the impact of the interaction between the U2AF84- and SF1-KH-QUA2 domains on splicing and on pathogen virulence. KH-QUA2 deleted U2AF84 amoeba transformants displayed increased splicing of target transcripts in vivo. EMSA, UV and formaldehyde crosslinking, and overlay assays showed that recombinant SF1- and U2AF84-KH-QUA2 domains interact with each other, even in the absence of a BS radioactive probe. Also, the U2AF84-KH-QUA2 domain blocks partial replacement of the SF1-KH-QUA2 by the U2 snRNA from the BS, suggesting that splicing inhibition occurs by blockage of splicing complex E to A transition. All amoeba transformants were tested for their virulent phenotypes. Changes in the U2AF84-KH-QUA2 domain resulted in more aggressive amoebas, suggesting that an increase in splicing (therefore decreased intron retention) of target transcripts is required for appropriate amebiasis outcome. To test this, we constructed a chimeric U2AF84 protein combining the amino terminus of E. histolytica U2AF84 with the longer KH-QUA2 of the non-virulent human commensal Entamoeba dispar. The transfected chimeras were less aggressive than the U2AF84-KH-QUA2 deleted or mutated transformants, confirming, at least in part, our hypothesis. We conclude that a more extensive transcriptome resulting from intron retention, and an additional layer of gene expression regulation are necessary for appropriate amebiasis establishment. Our work links for the first-time splicing downregulation and pathogen virulence.

Network-Pharmacology and DFT Based Approach Towards Identification of Leads from Homalomena aromatica for Multi-Target In-Silico Screening on Entamoeba histolytica Proteins

Background: Entamoeba histolytica is the primary protozoan that causes amoebic dysentery and is prioritized as the third most prevalent protozoan causing parasitosis. Drug of choice in amoebic dysentery is metronidazole but it has unpleasant side effects with reports of development of resistance in certain cases. Homalomena aromatica Schott. is a plant which is used in different ethnomedicinal practices of South-east Asia to treat stomach ailments against intestinal parasites. Objective: the present study, a docking weighted network pharmacology-based approach was employed to understand the effects of a library of 71 natural molecules reported from Homalomena aromatica with reference to four proteins of Entamoeba histolytica namely thioredoxin reductase, cysteine synthase, glyceraldehyde-3-phosphate dehydrogenase, and ornithine decarboxylase. Method: Molecular docking of the phytoconstituents of H. aromatica was performed in Biovia Discovery Studio 2017 R2 software suite on the selected proteins of E. histolytica. A connection was established between the proteins and molecules through network pharmacology weighted docking studies with the help of Cytoscape V3.4.0 software to select three molecules namely HM 7, HM 23 and HM 24 on the basis of the generated network between the molecules and targets. Quantum mechanics based Density Functional Theory (DFT) analysis was performed on the filtered molecules to ascertain their viability with respect to LUMO-HOMO orbital energies of the filtered molecules. Results: On the basis of the docking studies of the natural molecules on the selected protein targets, a network of molecules was built. DFT based minimum energy gap was analysed to further ascertain the most potential inhbitors. Three molecules from H. aromatica; 3,7-dimethylocta-1,6-dien-3- yl acetate, α -methyl-α-(4-methyl-3-pentenyl)-oriranemethanol, and 7-octadiene-2,6-diol-2,6- dimethyl were predicted to be potential lead molecules against amoebiasis. Conclusion: The present study provides important evidence for the development of new drug molecules to treat amoebiasis.

Loop-mediated isothermal amplification (LAMP) reaction as viable PCR substitute for diagnostic applications: a comparative analysis study of LAMP, conventional PCR, nested PCR (nPCR) and real-time PCR (qPCR) based on Entamoeba histolytica DNA derived from faecal sample

BackgroundThis study reports the analytical sensitivity and specificity of a Loop-mediated isothermal amplification (LAMP) and compares its amplification performance with conventional PCR, nested PCR (nPCR) and real-time PCR (qPCR). All the assays demonstrated in this study were developed based on Serine-rich Entamoeba histolytica protein (SREHP) gene as study model.ResultsA set of SREHP gene specific LAMP primers were designed for the specific detection of Entamoeba histolytica. This set of primers recorded 100% specificity when it was evaluated against 3 medically important Entamoeba species and 75 other pathogenic microorganisms. These primers were later modified for conventional PCR, nPCR and qPCR applications. Besides, 3 different post-LAMP analyses including agarose gel electrophoresis, nucleic acid lateral flow immunoassay and calcein-manganese dye techniques were used to compare their limit of detection (LoD). One E. histolytica trophozoite was recorded as the LoD for all the 3 post-LAMP analysis methods when tested with E. histolytica DNA extracted from spiked stool samples. In contrast, none of the PCR method outperformed LAMP as both qPCR and nPCR recorded LoD of 100 trophozoites while the LoD of conventional PCR was 1000 trophozoites.ConclusionsThe analytical sensitivity comparison among the conventional PCR, nPCR, qPCR and LAMP reveals that the LAMP outperformed the others in terms of LoD and amplification time. Hence, LAMP is a relevant alternative DNA-based amplification platform for sensitive and specific detection of pathogens.

Escherichia coli mediated resistance of Entamoeba histolytica to oxidative stress is triggered by oxaloacetate.

Amebiasis, a global intestinal parasitic disease, is due to Entamoeba histolytica. This parasite, which feeds on bacteria in the large intestine of its human host, can trigger a strong inflammatory response upon invasion of the colonic mucosa. Whereas information about the mechanisms which are used by the parasite to cope with oxidative and nitrosative stresses during infection is available, knowledge about the contribution of bacteria to these mechanisms is lacking. In a recent study, we demonstrated that enteropathogenic Escherichia coli O55 protects E. histolytica against oxidative stress. Resin-assisted capture (RAC) of oxidized (OX) proteins coupled to mass spectrometry (OX-RAC) was used to investigate the oxidation status of cysteine residues in proteins present in E. histolytica trophozoites incubated with live or heat-killed E. coli O55 and then exposed to H2O2-mediated oxidative stress. We found that the redox proteome of E. histolytica exposed to heat-killed E. coli O55 is enriched with proteins involved in redox homeostasis, lipid metabolism, small molecule metabolism, carbohydrate derivative metabolism, and organonitrogen compound biosynthesis. In contrast, we found that proteins associated with redox homeostasis were the only OX-proteins that were enriched in E. histolytica trophozoites which were incubated with live E. coli O55. These data indicate that E. coli has a profound impact on the redox proteome of E. histolytica. Unexpectedly, some E. coli proteins were also co-identified with E. histolytica proteins by OX-RAC. We demonstrated that one of these proteins, E. coli malate dehydrogenase (EcMDH) and its product, oxaloacetate, are key elements of E. coli-mediated resistance of E. histolytica to oxidative stress and that oxaloacetate helps the parasite survive in the large intestine. We also provide evidence that the protective effect of oxaloacetate against oxidative stress extends to Caenorhabditis elegans.

Genetic characterization of Entamoeba histolytica reveals a significant association of parasite genotype with disease outcome

Amoebiasis caused by the enteric parasite Entamoeba histolytica has variable disease outcomes. The association between parasite genotypes and outcome of amoebic infection still remains obscure and requires to be explored. Genetic characterization of clinical isolates is certainly a useful method to explore this relation. E. histolytica isolates from different disease outcomes has been genetically characterized based on the widely used repetitive marker, Serine rich E. histolytica protein gene (Srehp). Genetic organizations of our study isolates are unique in compare to previous reports. Most of the identified repeat patterns are outcome specific. Significant association between parasite genotypes and outcome of amoebic infection has also been established.

Entamoeba histolytica: protein arginine transferase 1a methylates arginine residues and potentially modify the H4 histone.

BackgroundIn eukaryotes, histone arginine methylation associates with both active and repressed chromatin states depending on the residues involved and the status of methylation. Even when the amino-terminus of Entamoeba histolytica histones diverge from metazoan sequences, these regions contain arginine residues that are potential targets for methylation. However, histone arginine methylation as well as the activity of arginine methyltransferases (PRMTs) has not been studied in this parasite. The aim of this work was to examine the dimethylation of arginine 3 of H4 histone (H4R3me2) and to identify the parasite PRMT that could be responsible for this modification (EhPRMT1).MethodsTo examine the presence of H4R3me2 in E histolytica, we performed Western blot and immunofluorescence assays on trophozoites using an antibody against this epigenetic mark. To recognize the PRMT1 enzyme of this parasite that possibly perform that modification, we first performed a phylogenetic analysis of E. histolytica and human PRMTs. RT-PCR assays were carried out to analyze the expression of the putative PRMT1 genes. One of these genes was cloned and expressed in Escherichia coli. The recombinant protein was tested by its recognition by an antibody against human PRMT1 and in its ability to form homodimers and to methylate commercial histones.ResultsThe arginine 3 of human H4, which is subjected to post translational methylation, was aligned with the arginine 8 of E. histolytica H4, suggesting that this residue could be methylated. The recognition of an 18 kDa nuclear protein of E. histolytica by an antibody against H4R3me2 confirmed this assumption. We found that this parasite expresses three phylogenetic and structural proteins related to PRMT1. Antibodies against the human PRMT1 detected E. histolytica proteins in cytoplasm and nuclei and recognized a recombinant PRMT1 of this parasite. The recombinant protein was able to form homodimers and homotetramers and displayed methyltransferase activity on arginine 3 of chicken H4.ConclusionAll these results suggest that E. histolytica contains as a minimum one structural and functional protein ortholog to PRMT1, enzyme that potentially dimethylates H4R8. This modification may play an important role in the gene expression regulation of this microorganism.

A genomewide overexpression screen identifies genes involved in the phosphatidylinositol 3-kinase pathway in the human protozoan parasite Entamoeba histolytica.

Entamoeba histolytica is a protozoan parasite that causes amoebic dysentery and liver abscess. E. histolytica relies on motility, phagocytosis, host cell adhesion, and proteolysis of extracellular matrix for virulence. In eukaryotic cells, these processes are mediated in part by phosphatidylinositol 3-kinase (PI3K) signaling. Thus, PI3K may be critical for virulence. We utilized a functional genomics approach to identify genes whose products may operate in the PI3K pathway in E. histolytica. We treated a population of trophozoites that were overexpressing genes from a cDNA library with a near-lethal dose of the PI3K inhibitor wortmannin. This screen was based on the rationale that survivors would be overexpressing gene products that directly or indirectly function in the PI3K pathway. We sequenced the overexpressed genes in survivors and identified a cDNA encoding a Rap GTPase, a protein previously shown to participate in the PI3K pathway. This supports the validity of our approach. Genes encoding a coactosin-like protein, EhCoactosin, and a serine-rich E. histolytica protein (SREHP) were also identified. Cells overexpressing EhCoactosin or SREHP were also less sensitive to a second PI3K inhibitor, LY294002. This corroborates the link between these proteins and PI3K. Finally, a mutant cell line with an increased level of phosphatidylinositol (3,4,5)-triphosphate, the product of PI3K activity, exhibited increased expression of SREHP and EhCoactosin. This further supports the functional connection between these proteins and PI3K in E. histolytica. To our knowledge, this is the first forward-genetics screen adapted to reveal genes participating in a signal transduction pathway in this pathogen.

The Cell Surface Proteome of Entamoeba histolytica

Surface molecules are of major importance for host-parasite interactions. During Entamoeba histolytica infections, these interactions are predicted to be of prime importance for tissue invasion, induction of colitis and liver abscess formation. To date, however, little is known about the molecules involved in these processes, with only about 20 proteins or protein families found exposed on the E. histolytica surface. We have therefore analyzed the complete surface proteome of E. histolytica. Using cell surface biotinylation and mass spectrometry, 693 putative surface-associated proteins were identified. In silico analysis predicted that ∼26% of these proteins are membrane-associated, as they contain transmembrane domains and/or signal sequences, as well as sites of palmitoylation, myristoylation, or prenylation. An additional 25% of the identified proteins likely represent nonclassical secreted proteins. Surprisingly, no membrane-association sites could be predicted for the remaining 49% of the identified proteins. To verify surface localization, 23 proteins were randomly selected and analyzed by immunofluorescence microscopy. Of these 23 proteins, 20 (87%) showed definite surface localization. These findings indicate that a far greater number of E. histolytica proteins than previously supposed are surface-associated, a phenomenon that may be based on the high membrane turnover of E. histolytica.

Effect of the sesquiterpene lactone incomptine A in the energy metabolism of Entamoeba histolytica

Entamoeba histolytica is the causative agent of human amoebiasis, which mainly affects developing countries. Although several drugs are effective against E. histolytica trophozoites, the control of amoebiasis requires the development of new and better alternative therapies. Medicinal plants have been the source of new molecules with remarkable antiprotozoal activity. Incomptine A isolated from Decachaeta incompta leaves, is a sesquiterpene lactone of the heliangolide type which has the major in vitro activity against E. histolytica trophozoites. However the molecular mechanisms involved in its antiprotozoal activity are still unknown. Using a proteomic approach based on two-dimensional gel electrophoresis and mass spectrometry (ESI-MS/MS) analysis, we evidenced that 21 E. histolytica proteins were differentially expressed in response to incomptine A treatment. Notably, three glycolytic enzymes, namely enolase, pyruvate:ferredoxin oxidoreductase and fructose-1,6-biphosphate aldolase, were down-regulated. Moreover, ultrastructural analysis of trophozoites through electronic microscopy showed an increased number of glycogen granules. Taken together, our data suggested that incomptine A could affect E. histolytica growth through alteration of its energy metabolism.

Entamoeba histolytica antigenic protein detected in pus aspirates from patients with amoebic liver abscess

Entamoeba histolytica is a causative agent of amoebic liver abscess (ALA) and is endemic in many underdeveloped countries. We investigated antigenic E. histolytica proteins in liver abscess aspirates using proteomics approach. Pus samples were first tested by real-time PCR to confirm the presence of E. histolytica DNA and the corresponding serum samples tested for E. histolytica-specific IgG by a commercial ELISA. Proteins were extracted from three and one pool(s) of pus samples from ALA and PLA (pyogenic liver abscess) patients respectively, followed by analysis using isoelectric focussing, SDS–PAGE and Western blot. Unpurified pooled serum samples from infected hamsters and pooled human amoebic-specific IgG were used as primary antibodies. The antigenic protein band was excised from the gel, digested and analysed by MALDI-TOF/TOF and LC-MS/MS. The results using both primary antibodies showed an antigenic protein band of ∼14kDa. Based on the mass spectrum analysis, putative tyrosine kinase is the most probable identification of the antigenic band.

Proteomic Analysis of the Cyst Stage of Entamoeba histolytica

BackgroundThe category B agent of bioterrorism, Entamoeba histolytica has a two-stage life cycle: an infective cyst stage, and an invasive trophozoite stage. Due to our inability to effectively induce encystation in vitro, our knowledge about the cyst form remains limited. This also hampers our ability to develop cyst-specific diagnostic tools.AimsThree main aims were (i) to identify E. histolytica proteins in cyst samples, (ii) to enrich our knowledge about the cyst stage, and (iii) to identify candidate proteins to develop cyst-specific diagnostic tools.MethodsCysts were purified from the stool of infected individuals using Percoll (gradient) purification. A highly sensitive LC-MS/MS mass spectrometer (Orbitrap) was used to identify cyst proteins.ResultsA total of 417 non-redundant E. histolytica proteins were identified including 195 proteins that were never detected in trophozoite-derived proteomes or expressed sequence tag (EST) datasets, consistent with cyst specificity. Cyst-wall specific glycoproteins Jacob, Jessie and chitinase were positively identified. Antibodies produced against Jacob identified cysts in fecal specimens and have potential utility as a diagnostic reagent. Several protein kinases, small GTPase signaling molecules, DNA repair proteins, epigenetic regulators, and surface associated proteins were also identified. Proteins we identified are likely to be among the most abundant in excreted cysts, and therefore show promise as diagnostic targets.Major ConclusionsThe proteome data generated here are a first for naturally-occurring E. histolytica cysts, and they provide important insights into the infectious cyst form. Additionally, numerous unique candidate proteins were identified which will aid the development of new diagnostic tools for identification of E. histolytica cysts.

Unique short tandem repeat nucleotide sequences in Entamoeba histolytica isolates from China

A few PCR-based DNA typing methods using repetitive elements contained within both protein-coding genes and noncoding DNAs have been reported for Entamoeba histolytica over the years. The serine-rich E. histolytica protein and tRNA-linked short tandem repeats (STRs) are most commonly used to investigate the relationship between parasite genotype and E. histolytica infection outcome. Many E. histolytica infections in China have been reported; however, little genome information has been provided. In the current paper, five Chinese E. histolytica samples were reported: three amoebic liver abscess cases, one combined case and one asymptomatic case. Our study is the first to report on the DNA typing information of E. histolytica in China. We included two city, one imported, and two country cases. Sequence analysis of serine-rich protein genes confirmed the presence of seven sequence types in five isolates. The STRs amplified from the samples revealed five STR variations in the A-L, four in the N-K2, and R-R loci, three in D-A, S(TGA)-D and S-Q loci. Two country patients were found to have a different outcome of infection with the same genotypes of E. histolytica, whereas in a city case, one E. histolytica strain had led to different outcome of the infection in one patient. Analyses of the results suggest that more genome information of E. histolytica strains from China through accurate methods is needed to interpret how the parasite genome plays a role in determining the outcome of E. histolytica infections.

Profiles of Entamoeba histolytica–specific immunoglobulins in human sera

ObjectiveTo determine the profiles of anti-Entamoeba histolytica (E. histolytica) IgA, IgG, and IgM in sera of diarrheic and non-diarrheic individuals and partially characterize target antigens. MethodsSerum samples from thirty diarrheic and thirty non-diarrheic individuals were subjected to IgA, IgG, and IgM profiling through enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunoblot. ResultsELISA titer results showed that both diarrheic and non-diarrheic individuals possess high levels of E. histolytica-specific IgG compared to IgA and IgM. Flow cytometry data showed that diarrheic serum samples had higher mean reaction percentages against E. histolytica cells compared to non-diarrheic samples. Immunoreactive E. histolytica proteins with molecular weights ranging between 7 kDa and 292 kDa were recognized by diarrheic serum IgG, and 170 kDa and 250 kDa by non-diarrheic serum IgG. ConclusionsOur findings suggest that serum anti-E. histolytica IgG, compared with serum anti-E. histolytica IgA and IgM responses, was generally high in both diarrheic and non-diarrheic sera, indicating a past exposure to the organism both in symptomatic patients as well as in asymptomatic carriers, respectively. In addition, serum IgG from diarrheic and non-diarrheic patients were able to detect immunogenic E. histolytica proteins.

Characterization of a novelEntamoeba histolyticastrain from Burkina Faso, Africa, possessing a unique hexokinase-2 gene

An Entamoeba histolytica strain (BF-841 cl1) that originated from Burkina Faso, Africa presented with novel, polymorphic genotypes of the serine-rich E. histolytica protein and the anodic hexokinase-2 (HXK-2) isoenzyme band, which showed less electrophoretic mobility than that of an E. histolytica reference strain [HM-1:IMSS cl6 (zymodeme (Z)-II)] by starch gel electrophoresis and isoelectric focusing (IEF). The HXK-2 gene of BF-841 cl1 had amino acid variations at four positions compared to the sequence of HM-1: IMSS cl6. These variations were absent from the sequences of four other E. histolytica strains with different zymodemes [KU27 (Z-II), SAW1627 (Z-IIα-), SAW755CR clB (Z-XIV), and KU2 (Z-XIX)]. The results of IEF showed no difference in the substrate specificity of HXK (HXK-1 and HXK-2) between BF-841 cl1 and the three reference E. histolytica strains (HM-1:IMSS cl6, SAW755 clB, and KU27). It was also confirmed that BF-841 cl1 was able to form liver abscesses in Syrian hamsters.

Molecular and functional characterization of an Entamoeba histolytica protein (EhMLCI) with features of a myosin essential light chain

Entamoeba histolytica, a protozoan parasite of humans, relays on its striking motility to survive and invade host tissues. Characterization of the molecular components involved in motile processes is crucial to understand its pathogenicity. Although protein components of myosin II hexamers have been predicted from E. histolytica genome data, only a heavy chain of myosin, EhmhcA, has been characterized so far. We have cloned an E. histolytica cDNA sequence that best matched Dictyostelium discoideum myosin essential light chain and found that the cloned sequence is transcribed as an mRNA of 0.445kb which could encode a protein of 16.88kDa, within the predicted range for a myosin light chain. In silico analyses revealed that the protein sequence, named EhMLCI, shows two consensus domains for binding MHC, but lacks the N-terminal sequence for actin binding, as in A2 type myosin essential light chains. A single EF-hand calcium-binding domain was identified in the C-terminus and several high score predictability sites for serine and tyrosine phosphorylation. Antibodies to recombinant EhMLCI identified two proteins of approximately 17 and 15kDa in trophozoite extracts, the latter phophorylated in tyrosines. Serine phosphorylation was not detected. Immunomicroscopy revealed EhMLCI cortical and cytoplasmic distribution in trophozoites and true colocalization with EhmhcA determined by PCC. Co-immunoprecipitation corroborated EhMLCI interaction with EhmhcA. EhMLCI was also localized in actomyosin-containing complexes. Differential partition of phospho-tyrosinated EhMLCI into cell fractions containing the soluble form of EhmhcA and its lack of serine phosphorylation suggest its possible participation in a novel down regulatory mechanism of myosin II activity in E. histolytica.

New Assembly, Reannotation and Analysis of the Entamoeba histolytica Genome Reveal New Genomic Features and Protein Content Information

BackgroundIn order to maintain genome information accurately and relevantly, original genome annotations need to be updated and evaluated regularly. Manual reannotation of genomes is important as it can significantly reduce the propagation of errors and consequently diminishes the time spent on mistaken research. For this reason, after five years from the initial submission of the Entamoeba histolytica draft genome publication, we have re-examined the original 23 Mb assembly and the annotation of the predicted genes.Principal FindingsThe evaluation of the genomic sequence led to the identification of more than one hundred artifactual tandem duplications that were eliminated by re-assembling the genome. The reannotation was done using a combination of manual and automated genome analysis. The new 20 Mb assembly contains 1,496 scaffolds and 8,201 predicted genes, of which 60% are identical to the initial annotation and the remaining 40% underwent structural changes. Functional classification of 60% of the genes was modified based on recent sequence comparisons and new experimental data. We have assigned putative function to 3,788 proteins (46% of the predicted proteome) based on the annotation of predicted gene families, and have identified 58 protein families of five or more members that share no homology with known proteins and thus could be entamoeba specific. Genome analysis also revealed new features such as the presence of segmental duplications of up to 16 kb flanked by inverted repeats, and the tight association of some gene families with transposable elements.SignificanceThis new genome annotation and analysis represents a more refined and accurate blueprint of the pathogen genome, and provides an upgraded tool as reference for the study of many important aspects of E. histolytica biology, such as genome evolution and pathogenesis.

Crystal structure of the aspartyl-tRNA synthetase from Entamoeba histolytica

The crystal structure of the aspartyl-tRNA synthetase from the eukaryotic parasite Entamoeba histolytica has been determined at 2.8Åresolution. Relative to homologous sequences, the E. histolytica protein contains a 43-residue insertion between the N-terminal anticodon binding domain and the C-terminal catalytic domain. The present structure reveals that this insertion extends an arm of the hinge region that has previously been shown to mediate interaction of aspartyl-tRNA synthetase with the cognate tRNA D-stem. Modeling indicates that this Entamoeba-specific insertion is likely to increase the interaction surface with the cognate tRNAAsp. In doing so it may substitute functionally for an RNA-binding motif located in N-terminal extensions found in AspRS sequences from lower eukaryotes but absent in Entamoeba. The E. histolytica AspRS structure shows a well-ordered N-terminus that contributes to the AspRS dimer interface.

DNA characterization of simian Entamoeba histolytica-like strains to differentiate them from Entamoeba histolytica

Two simian Entamoeba histolytica-like strains, EHMfas1 and P19-061405, have been suggested to represent a new species based on genetic characterization. Sequence analyses of the hexokinase, glucose phosphate isomerase, and phosphoglucomutase genes supported the previous findings of isoenzyme analyses demonstrating a new zymodeme pattern. Phylogenetic studies of 18S rDNA, 5.8S rDNA, the chaperonin 60 gene, and the pyridine nucleotide transhydrogenase gene showed original clusters of simian E. histolytica-like strains below or near E. histolytica, respectively. Comparative studies of the chitinase and the serine-rich E. histolytica protein genes and locus 1-2 region revealed that most mutated units were shared among the simian E. histolytica-like strains. The similarities of each of the repeating units within the simian E. histolytica-like strains or E. histolytica and the differences of those between the both might be generated by concerted evolution. Our results indicate that EHMfas1 and P19-061405 should be considered to be the same species, despite that they were isolated from different monkey species and different habitats. Simian E. histolytica-like amebas may be endemic to macaque monkeys, as a counterpart to E. histolytica in humans, and should be differentiated from E. histolytica by the revival name Entamoeba nuttalli, as proposed for P19-061405.