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PubMedDate not supplied

Effect of zinc-treated Entamoeba histolytica on the human polymorphonuclear respiratory burst.

One of the mechanisms that Entamoeba histolytica uses to evade host immune response is inhibition of the polymorphonuclear (PMN) leukocyte respiratory burst. In studies previously conducted in a model used in our laboratory, we observed that when treating trophozoites with different zinc concentrations certain amebic functions are inhibited while significantly limiting development of hepatic abscess in golden hamsters (Mesocricetus aureatus). We carried out an in vitro study using a chemoluminescent method to assess the effect zinc-treated amebic trophozoites exercise on respiratory burst in human PMNs. We measured response of PMNs incubated with E. histolytica trophozoites from cultures with TYI-S33 medium alone and with zinc. Zinc concentrations between 0.1 and 1.0 mM did not affect amebic trophozoite viability, and PMNs in contact with these in a zinc-free medium had an oxidative response similar to that obtained with zymosan and significantly greater (p <0.05) than that generated by cells co-incubated with amebas cultured in TYI-S33 medium alone. These results suggest that zinc alters the amebic mechanism that inhibits the oxidative function of human polymorphonuclear leukocyte.

Animals↗
PubMed2006

Methods for the investigation of diversity in Entamoeba histolytica.

The ability to distinguish variants of a species has many potential applications. In Entamoeba histolytica the first method to detect variation was based on isoenzyme analysis. However, this approach has been superseded by DNA-based analysis. In this review I discuss the basis of the variation detected in E. histolytica by the various molecular methods that have been published to date. Information on diversity in other species is mentioned where such information exists.

Animals↗
PubMed2006

Epigenetic silencing of gene expression in Entamoeba histolytica.

Transcriptional silencing of an amebapore (ap-a) gene occurred in Entamoeba histolytica following the transfection of plasmids containing a DNA segment (473 bp) homologous to the 5' upstream region of the gene. This segment contains the promoter region of the ap-a gene, a T-rich stretch, followed by a truncated SINE1 (short interspersed element) that is transcribed from the opposite strand. The downstream silencing of the ap-a gene did not occur with plasmids containing the entire SINE1 sequence or lacking the entire SINE1 sequences including the T-rich stretch. Such plasmids promoted the overexpression of the ap-a gene. The transcription of the SINE element required both the T-rich stretch as well as sequences from the 5' end of SINE. RNA extracts from gene-silenced cultures showed small amounts of short (approximately 140 nt), single-stranded molecules with homology to SINE1 transcripts but no siRNA. Chromatin immunoprecipitation (ChIP) analysis of silenced G3 trophozoites with an antibody against methylated K4 of histone H3 revealed a demethylation of K4 at the domain of the ap-a gene indicating transcriptional inactivation. These results suggest the involvement of the SINE1 element in triggering the gene silencing and the role of histone modification in its epigenetic maintenance. The avirulent phenotype of the silenced trophozoites was demonstrated in various assays and the results suggest they may have a potential use for vaccination.

Animals↗
PubMed2006

The cytoskeleton of Entamoeba histolytica: structure, function, and regulation by signaling pathways.

Pathogenesis in the parasite Entamoeba histolytica has been related to motility of the trophozoites. Motility is an important feature in amebas as they perform multiple motile functions during invasion of host tissues. As motility depends on the organization and regulation of the cytoskeleton elements, in particular of the actin cytoskeleton, the study of the molecular components of the machinery responsible for movement has been a key aspect to study in this parasite. Although many of the components have high homology in amino acid sequence and function to those characterized in higher eukaryotic cells, there are important differences to suggest that parasitic organisms may have developed adaptative differences that could be useful as targets to stop invasion. The purpose of this review is to evaluate current knowledge about the cytoskeleton of E. histolytica and the ways in which the parasite controls motility.

Animals↗
PubMed2006

Calcium-binding proteins of Entamoeba histolytica.

Calcium plays an essential role in many fundamental processes in almost all eukaryotic cells including protozoan parasite Entamoeba histolytica. Many of the calcium-mediated processes are carried out through the help of calcium-binding proteins (CaBPs). A few of these E. histolytica CaBPs have been described before. These proteins are unique to this organism and are thought to be essential. Availability of genome sequence has opened up the possibility of studying CaBPs at the whole genome level. In this preliminary report, we describe the complement of CaBPs present in E. histolytica. A large fraction of these genes are expressed in the trophozoites and are likely to be functional. The results suggest a number of pathways that are involved in calcium signaling and may be unique for this organism.

Animals↗
PubMed2006

New insights into molecular mechanisms of phagocytosis in Entamoeba histolytica by proteomic analysis.

The protozoan parasite Entamoeba histolytica ingests microorganisms and mammalian cells. Phagocytosis is essential for cell growth and is implicated in pathogenesis of E. histolytica. Phagocytosis consists of a number of steps including recognition of and binding to ligands on the target cells via a galactose/N-acetylgalactosamine-specific lectin, activation of a signaling pathway leading to cytoskeletal reorganization, and vesicle trafficking, all of which play distinct but coordinated roles in phagocytosis. Recent studies of proteomic analysis of purified phagosomes or affinity-purified Gal/GalNAc-binding proteins using reversed phase capillary liquid chromatography and ion trap tandem mass spectrometry enabled high throughput identification of proteins involved in phagosome biogenesis. These studies provided a list of proteins involved in the pathway and also shed light on the dynamic process of phagosome maturation. These approaches should provide significant insights into molecular mechanisms of phagosome biogenesis and help to elucidate the pathogenesis of this important parasite.

Animals↗
PubMed2004

Sequence divergence of Entamoeba histolytica tubulin is responsible for its altered tertiary structure.

Atypical microtubular structures of the protozoan parasite Entamoeba histolytica (Eh) have been attributed to amino acid sequence divergence of Eh tubulin. To investigate if this sequence divergence leads to significant differences in the tertiary structure of the Eh alphabeta-tubulin heterodimer, we have modeled alphabeta-tubulin heterodimer of Eh based on the crystal structure of mammalian tubulin. The predicted 3D homology model exhibits an overall resemblance with the known crystal structure of mammalian tubulin except for the 16 residue long carboxy terminal region of Eh beta-tubulin. We propose that this C-terminal region may provide steric hindrance in the polymerization of Eh alphabeta-tubulin for microtubule formation. Using docking studies, we have identified the binding sites for different microtubule specific drugs on Eh beta-tubulin. Our model provides a rational framework, both for understanding the contribution of Eh beta-tubulin C-terminal region to alphabeta-tubulin polymerization and design of new anti-protozoan drugs in order to control amoebiasis.

Amino Acid Sequence↗
PubMed2004

Inhibition of gene expression with double strand RNA interference in Entamoeba histolytica.

In order to inhibit gene expression in Entamoeba histolytica, we have developed a method based on expressing double strand RNA interference constructs in stable transformants. The 5' end of Eh Dia was cloned head to head with an intervening non-specific stuffer fragment in the E. histolytica expression vector pJST4. This construct was transformed in E. histolytica HM1:IMSS trophozoites and stable transformants were selected with 20microg/ml G418. Our results show that expression of Eh Dia was completely inhibited in these transformants. These stable transformants could be maintained indefinitely without expression of Eh Dia. This method therefore provides an effective tool to study the phenotypic changes, which occur due to inhibition of gene expression in the absence of mutants and other microbiological manipulations in this protozoan parasite.

Animals↗
PubMed2005

The ABP-120 C-end region from Entamoeba histolytica interacts with sulfatide, a new lipid target.

EhABP-120 is the first filamin identified in the parasitic protozoan Entamoeba histolytica. Filamins are a family of cross-linking actin-binding proteins that promote a dynamic orthogonal web. They have been reported to interact directly with more than 30 cellular proteins and some phosphoinositides. The biochemical consequences of these interactions may have either positive or negative effects on the cross-linking function and also form a link between the cytoskeleton and plasma membrane. In this study, the EhABP-120 carboxy-terminal domain (END) was biochemically characterized. This domain was able to associate to 3-sulfate galactosyl ceramide, a new lipid target for a member of the filamin family. Also, the END domain was able to dimerize "in vitro." Molecular modeling analysis showed that the dimeric region is stabilized by a disulfide bond. Electrostatic and docking studies suggest that an electropositive concave pocket at the dimeric END domain interacts simultaneously with several sulfogalactose moieties of the sulfatide.

Amino Acid Sequence↗
PubMed2006

Cloning and partial characterization of Entamoeba histolytica PTPases.

Reversible protein tyrosine phosphorylation is an essential signal transduction mechanism that regulates cell growth, differentiation, mobility, metabolism, and survival. Two genes coding for protein tyrosine phophatases, designed EhPTPA and EhPTPB, were cloned from Entamoeba histolytica. EhPTPA and EhPTPB proteins showed amino acid sequence identity of 37%, both EhPTPases showed similarity with Dictyostelium discoideum and vertebrate trasmembranal PTPases. mRNA levels of EhPTPA gene are up-regulated in trophozoites recovered after 96h of liver abscess development in the hamster model. EhPTPA protein expressed as a glutathione S-transferase fusion protein (GST::EhPTPA) showed enzymatic activity with p-nitrophenylphosphate as a substrate and was inhibited by PTPase inhibitors vanadate and molybdate. GST::EhPTPA protein selectively dephosphorylates a 130kDa phosphotyrosine-containing protein in trophozoite cell lysates. EhPTPA gene codifies for a 43kDa native protein. Up-regulation of EhPTPA expression suggests that EhPTPA may play an important role in the adaptive response of trophozoites during amoebic liver abscess development.

Amino Acid Sequence↗
PubMed2006

Molecular modeling of some 1H-benzimidazole derivatives with biological activity against Entamoeba histolytica: a comparative molecular field analysis study.

Comparative molecular field analysis (CoMFA) was performed on a set of 1H-benzimidazole derivatives. Molecular modeling and 3D-QSAR were employed to determine the tautomeric form that would probably fit a target receptor in Entamoeba histolytica. CoMFA results suggest that the antiamoebic activity is favored with steric bulk at position 5 of the benzimidazole ring and low electron density on the group at position 2. To the best of our knowledge this is the first 3D-QSAR study performed for benzimidazoles as antiamoebic agents. The CoMFA models derived will be very valuable to design new and more potent compounds against E. histolytica.

Algorithms↗
PubMed2004

Involvement of protein kinases on the process of erythrophagocytis by Entamoeba histolytica.

Erythrophagocytic capacity of trophozoites of Entamoeba histolytica is considered a factor in the virulence of this pathogenic protozoan. We present evidence showing that such activity resembles the ingestion of microorganisms by highly differentiated phagocytic cells, such as macrophages. Previous treatment of the trophozoites with genistein or tyrphostin, inhibitors of tyrosine protein kinases, with staurosporine, a protein kinase C inhibitor, and wortmannin, a fungal metabolite that inhibits phosphoinositide 3-OH kinase, significantly inhibited their erythrophagocytic capacity.

Androstadienes↗
PubMedDate not supplied

Entamoeba histolytica: kinetic and molecular evidence of a previously unidentified pyruvate kinase.

We report the kinetic characterization of a previously unidentified pyruvate kinase (PK) activity in extracts from Entamoeba histolytica trophozoites. This activity was about 74% of the activity of pyruvate phosphate dikinase. EhPK differed from most PKs in that its pH optimum was 5.5-6.5 and was inhibited by high PEP concentrations (1-5mM); these are concentrations at which PK is usually assayed. The optimal temperature was above 40 degrees C with negligible activity below 20 degrees C. EhPK exhibited hyperbolic kinetics with respect to both PEP (K(m) = 0.018 mM) and ADP (K(m) = 1.05 mM). However, it exhibited a sigmoidal behavior with respect to PEP at sub-saturating ADP concentrations. EhPK did not require monovalent cations for activity. Fructose-1,6 bisphosphate was a potent non-essential activator; it increased the affinity for ADP without modification of the V(max) or the affinity for PEP. Phosphate, citrate, malate, and alpha-ketoglutarate significantly inhibited EhPK activity. A putative EhPK gene fragment found in EhDNA was analyzed. The data indicate that E. histolytica trophozoites contain an active PK, which might contribute to the generation of glycolytic ATP for parasite survival.

Adenosine Diphosphate↗
PubMedDate not supplied

Entamoeba histolytica: induction of cyclooxygenase-2 expression during amoebic liver abscess formation in hamsters (Mesocricetus auratus).

Experimental amoebic liver abscess in hamsters curses with an increase in both, systemic levels of prostaglandin E2 (PGE(2)) and local cyclooxygenase activity in liver microsomes. The cellular source of PGE(2) and the isoform of cyclooxygenase responsible are not completely evidenced. Cyclooxygenase-2 (COX-2) protein and gene expression were demonstrated on macrophages and polymorphonuclear cells as a result of Entamoeba histolytica infection in hamsters at 2, 4, and 7 days postinfection by immunohistochemistry and RT-PCR. E. histolytica trophozoites located in the lesion showed a strong positive signal for COX-2, however the enzyme was not detected in cultured trophozoites by Western blot. Our results indicate that the increment in PGE(2) is the result of COX-2 activity from cells of the reticuloendothelial system and reinforce the possibility that PGE(2) production by enzyme induction in macrophages may be a mechanism by which E. histolytica modulates the host immune response in this parasitic infection.

Animals↗
PubMedDate not supplied

Entamoeba histolytica: cDNAs cloned as 30kDa collagen-binding proteins (CBP) belong to an antioxidant molecule family. Protection of hamsters from amoebic liver abscess by immunization with recombinant CBP.

A cDNA expression library of Entamoeba histolytica was screened with antiserum to native amoebic collagen binding proteins (CBPs), and two clones C13 and C7 which partially encode for the 30 kDa CBP were obtained. The sequenced clones were 90% homologous. C7 had a 69 bp deletion at the 5' end that is present in C13 and encodes for a Glu-Cys-Lys rich region and a four amino acids repeat (Glu-Lys-Glu-Cys). Purified fusion proteins from these cDNA clones were able to bind native type I collagen gels in a pH, calcium, ionic strength, and temperature dependent way. The binding of pgtC13 to collagen gel was time and temperature stable, while pgtC7 binding was not, suggesting that the deleted region in C7 is important for the binding. The clones reported here partially encode a 30 kDa CBP that also belong to an antioxidant molecule family. We demonstrated that the fusion protein pgtC13 is immunogenic and partially protective as a subunit vaccine in the hamster model of amoebic liver abscess.

Amino Acid Sequence↗
PubMedDate not supplied

Entamoeba histolytica: correlation of assessment methods to measure erythrocyte digestion, and effect of cysteine proteinases inhibitors in HM-1:IMSS and HK-9:NIH strains.

Entamoeba histolytica trophozoites are able to degrade human erythrocytes; the loss of erythrocyte cellular matrix and degradation of plasma membrane were observed, along with the decrease in the average size of digestive vacuoles. Ninety-six percent of hemoglobin ingested was hydrolyzed by trophozoites within 3h, as evidenced by electrophoresis. Accordingly, X-ray spectroscopy revealed the presence of iron inside vacuoles after erythrophagocytosis, the concentration of which decreased to control levels in a similar period. Quantification of erythrocyte digestion at the early and late periods was determined by a spectrophotometric procedure, with t(1/2)=1.67 h and 35-min for HM-1:IMSS and HK-9:NIH trophozoites, respectively. In the latter, activity was due to the combined action of intracellular enzymatic activity and exocytosis. E-64c and leupeptin totally inhibited erythrocyte digestion within a 3-h period, thereafter hydrolysis took place at lower rate. Our results suggest that erythrocyte digestion in E. histolytica proceeds in different ways in these two amebic strains, and can be blocked by proteinase inhibitors.

Animals↗
PubMed2004

Entamoeba invadens: cysteine protease inhibitors block excystation and metacystic development.

We examined the effects of six cysteine protease inhibitors on the excystation and metacystic development of Entamoeba invadens. Excystation, which was assessed by counting the number of metacystic amoebae after the induction of excystation, was inhibited by the cysteine protease inhibitors Z-Phe-Ala-DMK and E-64d in a concentration-dependent manner during incubation compared to the controls. Neither inhibitor had a significant effect on cyst viability; thus, their inhibitory effects were not due to the toxic effect on cysts. Metacystic development, when determined by the number of nuclei in amoeba, was also inhibited by these protease inhibitors, because the percentage of 4-nucleate amoebae was higher than in the controls on Day 3 of incubation. Although other cysteine protease inhibitors, Z-Phe-Phe-DMK, E-64, ALLM, and cathepsin inhibitor III, had a weak or little effect on the excystation, they inhibited cysteine protease activity in the lysates of E. invadens cysts. Broad bands with gelatinase activity of metacystic amoebae, as well as cysts and trophozoites, were detected in the gelatin substrate gel electrophores and were inhibited by Z-Phe-Ala-DMK. There was a difference in the protease composition between cysts and trophozoites, and the protease composition of metacystic amoebae changed from cyst-type to trophozoite-type during development. These results strongly suggest that cysteine proteases contribute to the excystation and metacystic development of E. invadens, which leads to successful infection.

Animals↗
PubMed2005

Entamoeba histolytica: intracellular distribution of the sec61alpha subunit of the secretory pathway and down-regulation by antisense peptide nucleic acids.

The Sec61alpha protein is defined as a highly conserved essential integral component of the endoplasmic reticulum in eukaryotic cells. We report a detailed immunolocalization of the Entamoeba histolytica homologue of the Sec61alpha subunit (EhSec61alpha), which shows an irregular pattern throughout the cell and is also found on the cell surface, its effective down-regulation by means of antisense peptide nucleic acids and its effects on cell proliferation, subcellular distribution of two virulence factors, and the ability of the trophozoites to cause liver abscess in hamsters. Although Sec61alpha levels are specifically decreased in antisense PNA-treated trophozoites, which proliferate more slowly than the controls, mobilization of the cysteine protease 5 and amoebapore to the cell surface is not significantly impeded and the capacity to induce liver abscess in hamsters is largely unaffected. The implications of these findings are discussed in the context of the peculiar cell biology of E. histolytica.

Animals↗