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PubMed1998

Expression and characterization of recombinant pyruvate phosphate dikinase from Entamoeba histolytica.

The parasite Entamoeba histolytica is an organism whose main energetic source comes from glycolysis. It has the singularity that several of its glycolytic enzymes use pyrophosphate as an alternative phosphate donor. Thus, pyruvate phosphate dikinase (PPDK), an inorganic pyrophosphate (PPi)-dependent enzyme, substitutes pyruvate kinase present in humans. We previously cloned and sequenced the gene that codifies for PPDK in E. histolytica. We now report its expression in a bacterial system and its purification to 98% homogeneity. We determined its K(m) for phosphoenolpyruvate, AMP and PPi (21, < 5 and 100 microM, respectively). Unlike PPDK from maize and bacteria and pyruvate kinase from other cells, EhPPDk is dependent on divalent cations but does not require monovalent cations for activity. The enzyme has an optimum pH of 6.0, it is labile to low temperatures and has a tetrameric structure. Since EhPPDK is a PPi-dependent enzyme, we also tested the effect of some pyrophosphate analogs as inhibitors of activity. Studies on the function and structure of this enzyme may be important for therapeutic research in several parasitic diseases, since it has no counterpart in humans.

Animals↗
PubMed1998

Cloning and bacterial expression of adenosine-5'-triphosphate sulfurylase from the enteric protozoan parasite Entamoeba histolytica.

A gene encoding adenosine-5'-triphosphate sulfurylase (AS) was cloned from the enteric protozoan parasite Entamoeba histolytica by polymerase chain reaction using degenerate oligonucleotide primers corresponding to conserved regions of the protein from a variety of organisms. The deduced amino acid sequence of E. histolytica AS revealed a calculated molecular mass of 47925 Da and an unusual basic pI of 9.38. The amebic protein sequence showed 23-48% identities with AS from bacteria, yeasts, fungi, plants, and animals with the highest identities being to Synechocystis sp. and Bacillus subtilis (48 and 44%, respectively). Four conserved blocks including putative sulfate-binding and phosphate-binding regions were highly conserved in the E. histolytica AS. The upstream region of the AS gene contained three conserved elements reported for other E. histolytica genes. A recombinant E. histolytica AS revealed enzymatic activity, measured in both the forward and reverse directions. Expression of the E. histolytica AS complemented cysteine auxotrophy of the AS-deficient Escherichia coli strains. Genomic hybridization revealed that the AS gene exists as a single copy gene. In the literature, this is the first description of an AS gene in Protozoa.

Amino Acid Sequence↗
PubMed2000

Extracellular matrix-induced signaling in Entamoeba histolytica: its role in invasiveness.

Entamoeba histolytica trophozoites can invade the mucosa of the host large intestine to cause disease. Here, Isaura Meza describes recent work indicating that the trophozoites respond to the binding of extracellular matrix proteins by activating receptor-mediated signal transduction pathways. Activation of phosphokinase C or adenylyl cyclase induces protein phosphorylation, actin gene expression and reorganization of the actin cytoskeleton to form adhesive structures that promote interaction with the substrate and the release of proteases. The release of proteases at the sites of contact, with the subsequent degradation of the substrate and generation of chemotactic peptides, facilitates locomotion and dissemination of the invading trophozoites.

Adenylyl Cyclases↗
PubMed1987

Sensitivity of protein and RNA synthesis to emetine in axenic Entamoeba histolytica.

The action of emetine on the incorporation of (14C)1-DL-valine and (14C)-8-adenine in TCA insoluble fraction of axenic Entamoeba histolytica was investigated. These compounds were almost exclusively incorporated in the 'protein' and 'RNA' fractions, respectively. Incorporation of the purine showed a significantly higher sensitivity to emetine. While the percentage inhibition of valine incorporation by emetine decreased with increasing pH, the susceptibility of adenine incorporation was greater in the alkaline range.

Adenine↗
PubMedDate not supplied

Congo red effect on cyst viability and cell wall structure of encysting. Entamoeba invadens.

BACKGROUND: The cell wall of Entamoeba invadens cysts is composed of chitin microfibrils as the main structural component. It has been demonstrated in yeast that the chitin cell wall assembly is altered by dyes such as Congo red (CR) and Calcofluor. METHODS: The purpose of this work was to study the cell wall assembly under the effect of CR dye on encysting E. invadens by means of light and electron microscopy, after the amebas were subjected to the effect of 100-2,000 micrograms CR/mL. Experiments were performed either in BI-S-33 or in mLG media. RESULTS: Trophozoite growth was not inhibited by 100-1,000 micrograms/mL CR after 8 days of incubation in BI-S-33 medium. However, low levels of growth were observed with 2,000 micrograms/mL of dye. No significant differences in morphologically viable (hyaline) cyst production occurred after 24-48 h, when 100 micrograms CR/mL was used, while the highest concentration of CR (2,000 micrograms/mL) resulted in a significant decrease of hyaline cyst yield; dead cysts prevailed in cultures, particularly at 72 h of CR treatment. Differentiation of amebas incubated in the presence of 500-2,000 micrograms/mL CR produced abnormal chitin deposits, rendering irregularly thick or double cell walls, as shown by transmission and scanning electron microscopy. Cyst cultures obtained under 100 micrograms/mL CR produced as many trophozoites as did the control when they were incubated in BI-S-33, but only low numbers of trophozoites were found in culture cysts obtained under higher CR doses. CONCLUSION: Our results suggest that CR affects E. invadens encystment, alters the cell wall formation, and also affects the cyst viability.

Animals↗
PubMedDate not supplied

Mg, Mn, and Co ions enhance the formation of Entamoeba histolytica cyst-like structures resistant to sodium dodecyl sulfate.

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

Animals↗
PubMedDate not supplied

Signal transduction in Entamoeba histolytica induced by interaction with fibronectin: presence and activation of phosphokinase A and its possible relation to invasiveness.

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

Actins↗
PubMed2002

Construction and immunogenicity of a codon-optimized Entamoeba histolytica Gal-lectin-based DNA vaccine.

Invasive amebiasis caused by Entamoeba histolytica is the third leading parasitic cause of mortality, and there are no vaccines available to help control the disease. The galactose-adherence lectin (Gal-lectin) is the parasite's major molecule allowing it to adhere to colonic mucin for colonization and to target cells for tissue destruction. It is immunodominant and is regarded as the most promising candidate molecule to be included in a subunit vaccine against amebiasis. In this study, we are reporting the construction of a codon-optimized DNA vaccine encoding a portion of the Gal-lectin heavy subunit that includes the carbohydrate recognition domain (CRD), and its in vivo testing in mice. The vaccine stimulated a Th1-type Gal-lectin-specific cellular immune response as well as the development of serum antibodies that recognized a recombinant portion of the heavy subunit, and that inhibited the adherence of trophozoites to target cells in vitro.

Amino Acid Sequence↗
PubMedDate not supplied

Protection in a gerbil model of amebiasis by oral immunization with Salmonella expressing the galactose/N-acetyl D-galactosamine inhibitable lectin of Entamoeba histolytica.

Infection with the enteric parasite Entamoeba histolytica can result in colitis and dysentery as well as abscesses at extra-intestinal sites. An effective vaccine must be able to protect against both mucosal and systemic disease. In this study an attenuated Salmonella strain that expressed a portion of the GalNAc lectin of E, histolytica was used to orally immunize gerbils. Animals were challenged by intrahepatic injection of amebic trophozoites. A significant decrease in size of amebic liver abscesses was observed in orally immunized animals. Oral immunization with a Salmonella-based vaccine was as effective as systemic immunization for protection against systemic challenge.

Administration, Oral↗
PubMed1999

DNA vaccination with the serine rich Entamoeba histolytica protein (SREHP) prevents amebic liver abscess in rodent models of disease.

Amebiasis remains one of the leading parasitic causes of death worldwide. A vaccine that prevented amebic liver abscess would significantly reduce mortality from this disease. To test the feasibility of a DNA vaccine to prevent amebic liver abscess, we immunized both mice and gerbils with plasmid DNA encoding the serine rich Entamoeba histolytica protein (SREHP). Animals receiving the SREHP DNA vaccine developed both antibody and cell mediated immune responses that recognized amebic trophozoites. A single dose of the SREHP DNA vaccine protected 80% of vaccinated mice and 60% of vaccinated gerbils from developing amebic liver abscess after direct hepatic inoculation of amebic trophozoites. Our study indicates that DNA vaccination with SREHP can provide high levels of protection against amebic liver abscess in animal models of disease.

Animals↗
PubMed2003

Collagen-induced STAT family members activation in Entamoeba histolytica trophozoites.

The interaction of Entamoeba histolytica trophozoites with collagen type I and calcium induces a membrane to nuclei signaling. The transduction pathways involved in such phenomena are still poorly understood. Using a combination of immunoprecipitation assays, Western immunoblot analysis, electrophoretic mobility shift assays and immunocytochemistry we demonstrate here the expression, tyrosine phosphorylation, nuclear translocation and DNA binding of two members of the signal transducers and activators of transcription family of inducible transcription factors in the protozoan parasite E. histolytica. These results support the notion that the interaction of the extracellular matrix components with the parasite turns on a genetic program that facilitates the invasion of the host.

Animals↗
PubMed2002

LINEs and SINE-like elements of the protist Entamoeba histolytica.

A survey of whole genome shotgun sequences of the protozoan parasite Entamoeba histolytica revealed three families of non-long terminal repeat (LTR) retrotransposons or long interspersed elements (LINEs) (called EhLINEs in this report). The 4.8 kb EhLINEs each had a single open reading frame with a putative nucleic acid binding motif (CCHC) and restriction enzyme-like endonuclease domain located downstream of the reverse transcriptase (RT) domain. Phylogenetic analysis of the RT domain placed the EhLINEs in the R4 clade of non-LTR elements, a mixed clade of non-LTR elements that includes members from nematodes, insects, and vertebrates. EhLINE1 (which was previously identified as HMc and EhRLE) shared a common 3' end with a highly transcribed 0.55 kb short interspersed element (SINE)-like element previously identified as IE or ehapt2 and called EhLSINE1 in this report. Similarly, EhLINE2 shared a common 3' end with a highly transcribed 0.65 kb SINE-like element called EhLSINE2 in this report. The shared 3' end sequences of the EhLINEs and EhLSINEs suggested that EhLINEs are involved in the retrotransposition of the EhLSINEs. EhLSINEs were flanked by target site duplications and contained conserved 5' sequences, which likely regulate their transcription. The EhLSINEs are the first protist SINE-like elements identified that share a common 3' sequence with LINEs, and the first SINE-like elements that have been associated with the R4 clade of non-LTR elements.

Amino Acid Sequence↗
PubMed1996

Identification and analysis of the u6 small nuclear RNA gene from Entamoeba histolytica.

Among the small nuclear RNAs (snRNAs) involved in the spliceosomal processing of pre-mRNA, U6 is the most conserved. As a first evidence for the presence of the splicing machinery in the amitochondrial protozoan Entamoeba histolytica (Eh), we have cloned the u6 snRNA gene. We find that in this organism u6 is a single copy gene that is transcribed as a poly(A)- RNA molecule of approximately 105 nucleotides. We have mapped the 5' end of the U6 snRNA transcript, and identified typical elements of a putative polymerase III promoter. This is the first snRNA gene reported in Eh. Sequence analysis indicates that this gene contains all the conserved nucleotides known to be important for U6 snRNA function. These results, in conjunction with the earlier finding of genes that contain pre-mRNA introns, suggest that Eh has a functional spliceosomal complex.

Animals↗
PubMed1996

Identification of novel genes from Entamoeba histolytica by expressed sequence tag analysis.

Shotgun sequencing of cDNA clones is now an established approach to gain insight into the expressed nucleotide (nt) sequences in a given cell. We analysed 100 randomly picked cDNA clones of the protozoan parasite, Entamoeba histolytica, by nt sequencing, with a view to obtain novel gene sequences not detected so far by biochemical and genetic analyses. About 56% of the analysed clones showed significant homology with other genes in the database, including a number of genes whose presence may not be suspected in E. histolytica owing to its unusual subcellular organization. The results suggest that this approach can provide important clues to understand unique biochemical mechanisms in this parasite.

Animals↗
PubMed1998

Cloning and heterologous expression of Entamoeba histolytica adenylate kinase and uridylate/cytidylate kinase.

We have isolated two cDNA clones encoding Entamoeba histolytica nucleotide kinases, EhAK and EhUK, expressed them in E. coli and performed functional studies of the recombinant enzymes. Nucleotide sequence analysis showed that EhAK and EhUK genes exhibited the features characteristic of E. histolytica genes, such as transcripts with relatively short 5' and 3' untranslated flanking regions containing the conserved E. histolytica transcription promoter elements located 5' to the initiation codon and a polyadenylation signal in the 3' UTR, a distinctive codon usage bias for A or T in the third position and an AT bias greater than 75% in the flanking regions of the transcripts. At the protein level, both enzymes belong to the short variant nucleoside monophosphate (NMP) kinases, which lack a 29amino acid LID region present in the long variant isoenzymes. EhAK was 30-38% identical to the members of the adenylate kinase (AK) family while EhUK was more similar (48-49% identity) to UMP/CMP kinases. Both enzymes used ATP as preferred phosphate-group donor but each one exhibited strict specificity for the acceptor NMP, EhAK for AMP and EhUK for the pyrimidine nucleoside monophosphates UMP and CMP. Biochemical characterization of the enzymes and phylogenetic reconstruction showed that EhUK is an authentic and well conserved member of the UMP/CMP kinase group while EhAK is the most divergent member known of the AK1 isoenzymes.

Adenylate Kinase↗
PubMed2000

Compositional pressure and translational selection determine codon usage in the extremely GC-poor unicellular eukaryote Entamoeba histolytica.

It is widely accepted that the compositional pressure is the only factor shaping codon usage in unicellular species displaying extremely biased genomic compositions. This seems to be the case in the prokaryotes Mycoplasma capricolum, Rickettsia prowasekii and Borrelia burgdorferi (GC-poor), and in Micrococcus luteus (GC-rich). However, in the GC-poor unicellular eukaryotes Dictyostelium discoideum and Plasmodium falciparum, there is evidence that selection, acting at the level of translation, influences codon choices. This is a twofold intriguing finding, since (1) the genomic GC levels of the above mentioned eukaryotes are lower than the GC% of any studied bacteria, and (2) bacteria usually have larger effective population sizes than eukaryotes, and hence natural selection is expected to overcome more efficiently the randomizing effects of genetic drift among prokaryotes than among eukaryotes. In order to gain a new insight about this problem, we analysed the patterns of codon preferences of the nuclear genes of Entamoeba histolytica, a unicellular eukaryote characterised by an extremely AT-rich genome (GC = 25%). The overall codon usage is strongly biased towards A and T in the third codon positions, and among the presumed highly expressed sequences, there is an increased relative usage of a subset of codons, many of which are C-ending. Since an increase in C in third codon positions is 'against' the compositional bias, we conclude that codon usage in E. histolytica, as happens in D. discoideum and P. falciparum, is the result of an equilibrium between compositional pressure and selection. These findings raise the question of why strongly compositionally biased eukaryotic cells may be more sensitive to the (presumed) slight differences among synonymous codons than compositionally biased bacteria.

Animals↗
PubMed2000

The evolution of Entamoeba, a cautionary tale.

In addition to its medical importance, the parasite Entamoeba histolytica has been studied by evolutionary biologists because of its unusual structural and biochemical features. Recent molecular data have revealed that many of our assumptions regarding the relationships of the organism and its relatives have been incorrect.

Animals↗
PubMed2003

Humoral immune response against proteophosphoglycan surface antigens of Entamoeba histolytica elicited by immunization with synthetic mimotope peptides.

The protozoan parasite Entamoeba histolytica, which is responsible for intestinal amebiasis and amebic liver abscess, is causing significant morbidity and mortality worldwide. Proteophosphoglycans (PPGs, also known as lipophosphoglycans, LPGs, or lipopeptidophosphoglycans, LPPGs) are major surface components of E. histolytica. Passive immunization with a monoclonal antibody (EH5) directed against the PPGs protected severe combined immune-deficient mice from amebic liver abscess. The structure of the PPGs is very complex and only known in part. To find peptide mimics of E. histolytica PPG antigens, we had screened phage-displayed random peptide libraries with the antibody EH5. We identified various peptide mimics of E. histolytica PPGs, all sharing a consensus sequence Gly-Thr-His-Pro-X-Leu. Several of the phage clones induced a significant, specific IgG response against membrane antigens of E. histolytica after immunization of mice with whole phage particles. In the present work, in order to avoid the use of phage particles for immunization, we coupled two selected chemically synthesized peptides to keyhole limpet hemocyanin (KLH). The two KLH-conjugated peptides were immunogenic in mice and induced the production of high titers of anti-peptide antibodies, and one of the two peptides was also able to induce significant titers of antibodies against E. histolytica PPGs. Our results demonstrate that the KLH-conjugated peptides are able to mimic the EH5 epitope without the M13 phage sequences flanking the peptide inserts and independent of the structural framework of the phage.

Adjuvants, Immunologic↗