A fast electrophoretic isoenzyme technique for the identification of invasive and non-invasive Entamoeba histolytica and "E. histolytica-like" organisms

Cellulose acetate electrophoresis (CAE) was used to separate glucosephosphate isomerase, hexokinase, malic enzyme, and phosphoglucomutase extracted from invasive and non-invasive Entamoeba histolytica and "E. histolytica-like" organisms. Each of these morphologically similar organisms possessed a unique CAE isoenzyme profile that can be used as an aid in their identification. The CAE technique used to obtain these isoenzyme profiles is rapid, simple, and economical, and it requires neither specialized training nor elaborate equipment.     

Determination of taxonomic status of pathogenic and nonpathogenic Entamoeba histolytica zymodemes using isoenzyme analysis.

Entamoeba histolytica infection results in either asymptomatic colonization or invasion of host tissues leading generally to clinical symptoms. Zymodemes studies have demonstrated a correlation between isoenzyme profiles and clinical presentation. Thus, strains have been attributed to pathogenic or nonpathogenic groups according to their zymodeme. To determine the taxonomic relationship of these two groups, the isoenzyme profiles of 14 loci of 38 E. histolytica strains (pathogenic and nonpathogenic) and seven strains of other species of the same genus were analyzed. Genetic distance analysis clearly demonstrates the existence of two separate groups within the species E. histolytica.     

Determination of Taxonomic Status of Pathogenic and Nonpathogenic Entamoeba histolytica Zymodemes Using Isoenzyme Analysis

ABSTRACT Entamoeba histolytica infection results in either asymptomatic colonization or invasion of host tissues leading generally to clinical symptoms. Zymodeme studies have demonstrated a correlation between isoenzyme profiles and clinical presentation. Thus, strains have been attributed to pathogenic or nonpathogenic groups according to their zymodeme. To determine the taxonomic relationship of these two groups, the isoenzyme profiles of 14 loci of 38 E. histolytica strains (pathogenic and nonpathogenic) and seven strains of other species of the same genus were analyzed. Genetic distance analysis clearly demonstrates the existence of two separate groups within the species E. histolytica .     

Entamoeba histolytica: is conversion of "nonpathogenic" amebae to the "pathogenic" form a real phenomenon?

Entamoeba histolytica isolates have been shown to fall into two groups based on isoenzyme analysis. These groupings ("pathogenic" and "nonpathogenic") correlate well with the clinical course of the infection. A controversy exists over whether isoenzyme patterns are stable or whether under certain circumstances an isolate can convert from one form to the other. Resolution of this uncertainty is of importance since the nonpathogenic pattern has never been observed in amebae isolated from cases of active disease. This implies that, if the patterns are stable, carriers of amebae with this nonpathogenic pattern may never develop invasive disease. Although we set out to study isoenzyme conversion, we have been unable to replicate the two published accounts of this phenomenon. We have examined all of the variables proposed to be involved in the triggering of conversion, both individually and in combination. In none of the experiments was an alteration in the isoenzyme pattern observed. We now believe that isoenzyme patterns are stable and that all available evidence, other than the reported conversions, points to pathogenic and nonpathogenic E. histolytica being distinct species.     

Reducing agents and Entamoeba histolytica

Entamoeba histolytica remains an important but enigmatic parasite. It displays both non-pathogenic and invasive pathogenic types, which can be distinguished clinically and by isoenzyme markers. Yet as debated in Parasitology Today last year(1), the relationship between these two forms remains unclear. Bacterial associates and reducing agents are known to play on important role in the culture of E. histolytica, and possibly in its differentiation and invasive mechanisms. This article briefly reviews available information on the role o f reducing agents, and explores the possibility that bacteria may play a role in reduction o f toxic oxygen product - thereby promoting the virulence of E. histolytica. The review is not definitive, but should help to stimulate further research in this neglected area.     

Elucidation of cellular population and nature of anti-amoebic antibodies in cytotoxicity to Entamoeba histolytica (NIH:200)

Interactions between trophozoites of Entamoeba histolytica and cells of the immune system were studied in vitro by mixing effector cells obtained from normal and immunized guinea pigs with trophozoites in a ratio of 10:1. Crude amoebic extract (CAE) and its highest molecular weight (MW 650,000) fraction (F-I) were used for priming the effector cells in vivo. The effector cells were collected from the spleens of normal and immunized animals. Lymphocytes were prepared by allowing splenic mononuclear cells to adhere to plastic, followed by passage through nylon wool column. There was no significant difference between the killing capacity of mononuclear cells, monocyte depleted mononuclear cells or nylon wool fractionated lymphocytes, indicating that probably monocytes and B-cells were not involved in the cytotoxicity against E. histolytica. The data suggest that effector cells probably belonged to the T-cytotoxic and K-cell category. Both CAE and F-I sensitization could induce cytotoxicity of lymphocytes against trophozoites. Similarly, anti-CAE and anti-F-I sera were found to enhance the killing capacity of effector cells in vitro. The ability of anti-amoebic serum to induce cytotoxicity was found to be independent of complement involvement and resided in IgG but not in IgM.     

Pathophysiology of amoebiasis

Few organisms are more aptly named than Entamoeba histolytica, an intestinal protozoan parasite that can lyse and destroy human tissue. Within the past four years, new models of E. histolytica infection have begun to illuminate how amoebic trophozoites cause intestinal disease and liver abscess, and have expanded our understanding of the remarkable killing ability of this parasite. Here, I summarize recent work on the interactions between E. histolytica and human intestine, and between E. histolytica and hepatocytes, and discuss what these studies tell us about the role of inflammation and programmed cell death in the pathogenesis of amoebiasis.     

Detection of Entamoeba histolytica/Entamoeba dispar in stool specimens by using enzyme-linked immunosorbent assay

Entamoeba histolytica actually comprises two genetically distinct but morphologically indistinguishable species. E. histolytica can cause invasive intestinal and extra intestinal disease, while E. dispar cannot. Identification and differentiation of E. dispar and E. histolytica in stool sample by microscopy is imprecise. Several weeks of culture and isoenzyme analysis are required to differentiate E. histolytica from E. dispar. In this study, we have used an enzyme-linked immunosorbent assay (ELISA) for detection of E. histolytica/E.dispar and compared it with microscopy. Eighty-eight samples were evaluated, trichrome staining was positive in 20.4% (18) and ELISA was positive in 29.5% (26). Both tests were positive in 14 (15.9%) samples, 4 (4.5%) only with direct microscopy, and 12 (13.6%) only with ELISA. Both tests were negative in 58 (65.9%) samples. Microscopy has low sensitivity and high specificity, low negative predictive value and high positive predictive value in comparison with ELISA. E. histolytica/E. dispar antigen detection ELISA tests are inexpensive compared to the specific tests, yield objective results and do not require experienced microscopists and can therefore be recommended for screening of stools worldwide and the results can be taken for treatment that are fitting with its clinic.     

Detection of Entamoeba histolytica antigen in stool samples in Mersin, Turkey

Entameoba histolytica, 1 of the 2 Entamoeba species with similar morphology that infect humans, causes invasive intestinal and extraintestinal diseases, whereas Entamoeba dispar is found commensally and is noninvasive. Because of their morphologic similarity, E. histolytica and E. dispar cannot be differentiated microscopically. The antigens of E. histolytica and E. dispar, however, may be detected by the ELISA method. Previous studies have found that the detection of antigens in the stool is as sensitive and specific as cultures and isoenzyme analyses. Stool samples from 272 patients with diarrhea in the province of Mersin, Turkey, were examined for the presence of Entamoeba species microscopically and for Entamoeba (E. histolytica/E. dispar) antigens using the ELISA method. An E. histolytica-specific ELISA test was used to examine 29 E. histolytica/E. disparpositive samples. Twenty-four (8.82%) of the samples tested positive for E. histolytica/E. dispar by trichrome staining, and 29 (10.6%) of the samples tested positive for E. histolytica/E. dispar by the Entamoeba screening test. Entamoeba histolytica was positive in 21 (7.72%) and E. dispar positive in 8 (2.94%) samples. The detection of true E. histolytica infection is possible with the use of E. histolytica-specific antigen ELISA tests. Thus, real cases of amoebiasis can be detected and treated, and overtreatment of the patients with E. dispar, which is the nonpathogenic species, will be prevented.     

Variations in cytotoxicity and isoenzyme patterns of uncloned and cloned cultures of axenic Entamoeba histolytica

Five clones of axenic Entamoeba histolytica (HMI) grown as discrete colonies in semisolid agar medium were adapted in liquid medium and labelled as HMI-C121, HMI-C131, HMI-C143, HMI-C144 and HMI-C145. The clone HMI-C121 was more cytotoxic to the cultured Baby Hamster Kidney (BHK) cells while all other clones were significantly (P less than 0.001) less cytotoxic as compared to the cloned HMI-C121 and uncloned E. histolytica (HMI). The uncloned Indian axenic E. histolytica (KCG:0986:11) as well as E. histolytica (NIH:200) cultures were significantly (P less than 0.001) less cytotoxic to cultured BHK cells. No difference in the electromobility of maleate NADP oxidoreductase (ME) or glucophosphate isomerase (GPI) isoenzyme in the lysates of all the cloned and uncloned cultures of E. histolytica was observed. The clones HMI-C121, HMI-C131, HMI-G143 and HMI-C144 had three bands of hexokinase (HK) while all uncloned cultures and one of clones, HMI-C145 had only two bands. Though cloned and uncloned cultures had a single PGM band, the relative electromobility (rf) of phosphoglucomutase (PGM) for clone HMI-C131, HMI-C143 HMI-C144 was relatively less (rf 0.075) and these were also significantly (P less than 0.001) less cytotoxic to BHK cells as compared to clone HMI-C121. It is felt that axenic E. histolytica culture consists of several populations (clones) and expression of isoenzymes pattern or cytotoxic potentials would depend upon the population which predominantly multiples and outgrows other populations in the culture system.     

Sulfate activation in mitosomes plays an important role in the proliferation of Entamoeba histolytica

Mitochondrion-related organelles, mitosomes and hydrogenosomes, are found in a phylogenetically broad range of organisms. Their components and functions are highly diverse. We have previously shown that mitosomes of the anaerobic/microaerophilic intestinal protozoan parasite Entamoeba histolytica have uniquely evolved and compartmentalized a sulfate activation pathway. Although this confined metabolic pathway is the major function in E. histolytica mitosomes, their physiological role remains unknown. In this study, we examined the phenotypes of the parasites in which genes involved in the mitosome functions were suppressed by gene silencing, and showed that sulfate activation in mitosomes is important for sulfolipid synthesis and cell proliferation. We also demonstrated that both Cpn60 and unusual mitochondrial ADP/ATP transporter (mitochondria carrier family, MCF) are important for the mitosome functions. Immunoelectron microscopy demonstrated that the enzymes involved in sulfate activation, Cpn60, and mitochondrial carrier family were differentially distributed within the electron dense, double membrane-bounded organelles. The importance and topology of the components in E. histolytica mitosomes reinforce the notion that they are not "rudimentary" or "residual" mitochondria, but represent a uniquely evolved crucial organelle in E. histolytica.     

Differentiation of Entamoeba histolytica and Entamoeba dispar infections

In this article, Terry jackson and Jonathan Ravdin briefly review the latest information on monoclonal antibody-based ELISAs that use antigen capture as a tool in the differential detection of human infection with Entamoeba histolytica and E. dispar. Current technology of culture and isoenzyme analysis is not widely available, is cumbersome and too time-consuming. A further potential benefit of antigen detection tests is that they can be used to monitor the efficacy of therapy; this is a shortcoming of serological tests owing to the persistence of the antibody response after successful treatment.     

Analysis of the protein kinome of Entamoeba histolytica

Protein kinases play important roles in almost all major signaling and regulatory pathways of eukaryotic organisms. Members in the family of protein kinases make up a substantial fraction of eukaryotic proteome. Analysis of the protein kinase repertoire (kinome) would help in the better understanding of the regulatory processes. In this article, we report the identification and analysis of the repertoire of protein kinases in the intracellular parasite Entamoeba histolytica. Using a combination of various sensitive sequence search methods and manual analysis, we have identified a set of 307 protein kinases in E. histolytica genome. We have classified these protein kinases into different subfamilies originally defined by Hanks and Hunter and studied these kinases further in the context of noncatalytic domains that are tethered to catalytic kinase domain. Compared to other eukaryotic organisms, protein kinases from E. histolytica vary in terms of their domain organization and displays features that may have a bearing in the unusual biology of this organism. Some of the parasitic kinases show high sequence similarity in the catalytic domain region with calmodulin/calcium dependent protein kinase subfamily. However, they are unlikely to act like typical calcium/calmodulin dependent kinases as they lack noncatalytic domains characteristic of such kinases in other organisms. Such kinases form the largest subfamily of kinases in E. histolytica. Interestingly, a PKA/PKG-like subfamily member is tethered to pleckstrin homology domain. Although potential cyclins and cyclin-dependent kinases could be identified in the genome the likely absence of other cell cycle proteins suggests unusual nature of cell cycle in E. histolytica. Some of the unusual features recognized in our analysis include the absence of MEK as a part of the Mitogen Activated Kinase signaling pathway and identification of transmembrane region containing Src kinase-like kinases. Sequences which could not be classified into known subfamilies of protein kinases have unusual domain architectures. Many such unclassified protein kinases are tethered to domains which are Cysteine-rich and to domains known to be involved in protein-protein interactions. Our kinome analysis of E. histolytica suggests that the organism possesses a complex protein phosphorylation network that involves many unusual kinases.     

Circular DNA of Entamoeba histolytica encodes ribosomal RNA

The presence of repeated DNA sequences encoding RNA in Entamoeba histolytica has been reported. In the present study we demonstrate by agarose gel electrophoresis. DNase digestion and electron microscopic analysis that these genes are located on extrachromosomal circular DNA molecules with an approximate size of 26 kb. Detection of replication intermediates suggests the episomal nature of these molecules. Amplified, extrachromosomal rRNA genes appear to be a common feature among the lower eukaryotes, occurring more commonly as linear molecules and less commonly as circles. Entamoeba histolytica is 1 of the few organisms studied in which rRNA genes are located predominantly on extrachromosomal circles.     

Functional characterization of spliceosomal introns and identification of U2, U4, and U5 snRNAs in the deep-branching eukaryote Entamoeba histolytica

Pre-mRNA splicing is essential to ensure accurate expression of many genes in eukaryotic organisms. In Entamoeba histolytica, a deep-branching eukaryote, approximately 30% of the annotated genes are predicted to contain introns; however, the accuracy of these predictions has not been tested. In this study, we mined an expressed sequence tag (EST) library representing 7% of amoebic genes and found evidence supporting splicing of 60% of the testable intron predictions, the majority of which contain a GUUUGU 5' splice site and a UAG 3' splice site. Additionally, we identified several splice site misannotations, evidence for the existence of 30 novel introns in previously annotated genes, and identified novel genes through uncovering their spliced ESTs. Finally, we provided molecular evidence for the E. histolytica U2, U4, and U5 snRNAs. These data lay the foundation for further dissection of the role of RNA processing in E. histolytica gene expression.     

Circular DNA of Entamoeba histolytica Encodes Ribosomal RNA

. The presence of repeated DNA sequences encoding RNA in Entamoeba histolytica has been reported. In the present study we demonstrate by agarose gel electrophoresis, DNase digestion and electron microscopic analysis that these genes are located on extrachromosomal circular DNA molecules with an approximate size of 26 kb. Detection of replication intermediates suggests the episomal nature of these molecules.
Amplified, extrachromosomal rRNA genes appear to be a common feature among the lower eukaryotes, occurring more commonly as linear molecules and less commonly as circles. Entamoeba histolytica is 1 of the few organisms studied in which rRNA genes are located predominantly on extrachromosomal circles.     

Recent developments in amoebiasis:the Gal/GalNAc lectins of Entamoeba histolytica and Entamoeba dispar

Amoebiasis is responsible for 50000-100000 deaths annually. Invasive amoebic disease begins with the attachment of Entamoeba histolytica trophozoites to colonic mucin, a process mediated by the amoebic Gal/GalNAc lectin. The non-pathogenic counterpart, E. dispar, is morphologically identical but genetically distinct. Investigations comparing the Gal/GalNac lectin from these two organisms are under way.     

Introns of Entamoeba histolytica and Entamoeba dispar.

The genome of Entamoeba histolytica is considered to possess very few intervening sequences (introns), as only 5 intron-containing genes from this protozoan parasite have been reported so far. However, while sequencing a number of genomic contigs as well as three independent genes coding for ribosomal protein L27a, we have identified 9 additional intron-containing genes of E. histolytica and the closely related species Entamoeba dispar, indicating that introns are more common in these organisms than previously suggested. The various amoeba introns are relatively short comprising between 46 and 115 nucleotides only and have a higher AT-content compared to the corresponding exon sequences. In contrast to higher eukaryotes, amoeba introns do not contain a well-conserved branch point consensus, and have extended donor and acceptor splice sites of the sequences G     

Kinetic characterization of methionine gamma-lyases from the enteric protozoan parasite Entamoeba histolytica against physiological substrates and trifluoromethionine, a promising lead compound against amoebiasis

Methionine gamma-lyase (MGL) (EC 4.4.1.11), which is present in certain lineages of bacteria, plants, and protozoa but missing in mammals, catalyzes the single-step degradation of sulfur-containing amino acids (SAAs) to alpha-keto acids, ammonia, and thiol compounds. In contrast to other organisms possessing MGL, anaerobic parasitic protists, namely Entamoeba histolytica and Trichomonas vaginalis, harbor a pair of MGL isozymes. The enteric protozoon En. histolytica shows various unique aspects in its metabolism, particularly degradation of SAAs. Trifluoromethionine (TFM), a halogenated analog of Met, has been exploited as a therapeutic agent against cancer as well as against infections by protozoan organisms and periodontal bacteria. However, its mechanism of action remains poorly understood. In addition, the physiological significance of the presence of two MGL isozymes in these protists remains unclear. In this study, we compared kinetic parameters of the wild-type and mutants, engineered by site-directed mutagenesis, of the two MGL isotypes from En. histolytica (EhMGL1 and EhMGL2) for various potential substrates and TFM. Intracellular concentrations of l-Met and l-Cys suggested that these SAAs are predominantly metabolized by EhMGL1, not by EhMGL2. It is unlikely that O-acetyl-l-serine is decomposed by EhMGLs, given the kinetic parameters of cysteine synthase reported previously. Comparison of the wild-type and mutants revealed that the contributions of several amino acids implicated in catalysis differ between the two isozymes, and that the degradation of TFM is less sensitive to alterations of these residues than is the degradation of physiological substrates. These results support the use of TFM to target MGL.