Electrophoretic Isoenzyme Patterns of Entamoeba histolytica and Entamoeba chattoni in a Primate Survey1

Stocks of Entamoeba histolytica grown in a monoxenic culture system from the feces of nonhuman primates are compared with the eleven zymodemes of E. histolytica so far demonstrated from man. In a similar fashion, Entamoeba chattoni has also been grown and identified. Both E. histolytica and E. chattoni have been demonstrated in keepers of the primate collections. Comparisons have been made using the electrophoretic patterns of three enzymes: glucosephosphate isomerase [(GPI) E.C.5.3.1.9], phosphoglucomutase [(PGM) E.C.2.7.5.1], and L-malate—NADP⁺ oxidoreductase (oxaloacetate-decarboxylating) [(ME) E.C. 1.1.1.40]. Enzyme patterns of E. histolytica from the apes were found to be identical with three of those already demonstrated from man. The enzyme pattern of E. chattoni was distinctly different from that of any of the E. histolytica zymodemes. Other protozoa found in the single fecal sample examined from each subject are also listed.     

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.     

Entamoeba histolytica: effect of growth conditions and bacterial associates on isoenzyme patterns and virulence

In xenic culture, isolates of Entamoeba histolytica from asymptomatic carriers are characterized, with rare exception, by possession of a nonpathogenic zymodeme. During the process of axenizing such an isolate, strain CDC:0784:4, a change in the pattern of the isoenzymes from nonpathogenic zymodeme I to pathogenic zymodeme II was observed 40 days after the amebae had been transferred from a medium for xenic cultivation to one used for axenic cultivation, but before axenization of the amebae had actually occurred. Axenization was accomplished by feeding the amebae lethally irradiated bacteria while suppressing and finally eradicating with antibiotics the bacterial flora accompanying the amebae in the original xenic culture. The change in zymodeme was accompanied by a change in virulence as evidenced by the ability of the amebae to produce hepatic abscesses in hamsters and to destroy monolayers of tissue culture cells. Two explanations are offered for the observed changes in zymodeme and virulence: a zymodeme is not a stable inherent property of the ameba. Alternatively, the original isolate consisted of two zymodeme populations and the conditions of growth selected for one or the other of the populations. In either case, our results suggest that the finding of a particular zymodeme in a culture of E. histolytica isolated from an asymptomatic carrier of the parasite cannot be used to predict a clinical condition or serve as a basis for the recommendation of therapy.     

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.     

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.     

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     

Glucose transport in Entamoeba histolytica

That the uptake of glucose by the parasitic amoeba Entamoeba histolytica occurs by an equilibrative transport system is supported by the following observations. 1. The rate of glucose uptake is several orders of magnitude greater than the uptake by pinocytosis. 2. The uptake of glucose exhibits saturation kinetics, with K(m)=1.6mm and V(max.) ranging from 2 to 5mumol/min per ml of cells at 37 degrees C. 3. The glucose analogues 2-deoxyglucose, 3-O-methylglucose and d-xylose are transported by the glucose system although with much less affinity. Competitive inhibition was observed between pairs of substrates, with K(i) values for any sugar closely coincident with the corresponding K(m). 4. d-Xylose, a sugar not metabolized by the cells, equilibrated with 80% of the amoebal cell water. 5. Cells equilibrated with xylose exhibited countertransport of this sugar against its concentration gradient when another substrate was added to the medium. 6. Blocking of glycolysis by iodoacetate or F(-) has no immediate effect on transport. The presence of a glucose-transport system in E. histolytica contrasts with the situation found in the non-parasitic amoeba, where pinocytosis seems to be the only mechanism of solute uptake.     

Multidrug resistance in Entamoeba histolytica

Drug resistance in protozoan parasites has been emerging in the past decade as an obstacle to their control. Amoebiasis, caused by Entamoeba histolytica, is a worldwide disease that provokes high rates of morbidity and mortality. Reports of failed drug treatment and differences in drug susceptibilities among E. histolytica strains probably herald the development of drug resistance in this parasite. In this review, Esther Orozco and co-workers summarize recent progress on the elucidation of physiological and molecular evidence of multidrug resistance in this parasite.     

Entamoeba histolytica and Entamoeba dispar: prevalence infection in a rural Mexican community

The frequency of Entamoeba histolytica and Entamoeba dispar infection was analyzed in a rural community in the state of Morelos, Mexico, through PCR technique by using specie specific primer. The E. histolytica specie was detected in 33 of 290 analyzed stool samples (11.4%), E. dispar specie was observed in 21 samples (7.2%) and both species of Entamoeba were detected in seven samples (2.4%). So a higher E. histolytica than E. dispar frequency infection was detected (13.8 versus 9.6%). Even though in our design we did not considered the follow-up of included individuals, the absence of invasive amebiasis cases in the studied population during our stay in town was unexpected.     

Thioredoxin-linked metabolism in Entamoeba histolytica

Entamoeba histolytica, an intestinal protozoan that is the causative agent of amoebiasis, is exposed to elevated amounts of highly toxic reactive oxygen species during tissue invasion. In this work, we report the molecular cloning, from E. histolytica genomic DNA, of the genes ehtrxr and ehtrx41, respectively coding for thioredoxin reductase (EhTRXR) and thioredoxin (EhTRX41). The genes were expressed in Escherichia coli cells, and the corresponding recombinant proteins were purified and characterized. EhTRXR catalyzed the NADPH (Km=4.5 microM)-dependent reduction of 5,5'-dithiobis-(2-nitrobenzoic) acid (Km=1.7 mM), EhTRX41 (Km=3.6 microM), and E. coli TRX (Km=4.6 microM). EhTRXR and EhTRX41 could be assayed as a functional redox pair that, together with peroxiredoxin, mediate the NADPH-dependent reduction of hydrogen peroxide and tert-butyl hydroperoxide. It is proposed that this detoxifying system could be operative in vivo. Results add value to the genome project information and advise reconsideration of key metabolic pathways operating in E. histolytica.     

Purine salvage in Entamoeba histolytica

Pulse-labeling of the nucleotide pool in Entamoeba histolytica with radioactive precursors, and subsequent high performance liquid chromatographic (HPLC) analysis of the radiolabeled nucleotides, indicate that E. histolytica is incapable of de novo synthesis of purine nucleotides. Hypoxanthine, inosine and xanthine could not be converted to nucleotides in E. histolytica, which suggests the absence of interconversion between adenine nucleotides and guanine nucleotides through formation of IMP. Adenosine was actively incorporated into nucleotides at an initial rate of 130 pmoles per minute per 10(6) trophozoites. Adenine, guanosine and guanine were also incorporated at much lower rates. The rate of adenine incorporation was enhanced by the presence of guanosine; the rate of guanine incorporation was significantly increased by adenosine. These stimulatory effects suggest that the ribose moiety of adenosine or guanosine can be transferred to another purine base to form a new nucleoside, and that the purine nucleosides are the immediate precursors of E. histolytica nucleotides. HPLC results showed that the radiolabel in adenine was exclusively incorporated into adenine nucleotides and that guanine was found only among guanine nucleotides, whereas the radioactivity associated with the ribose moiety of adenosine or guanosine was distributed among both adenine and guanine nucleotides.     

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.     

Nucleic Acids of Entamoeba histolytica

This review will concentrate on certain aspects of the nucleic acids of Entamoeba histolytica. Utilization and synthesis of purines and pyrimidines will initially be briefly discussed, e.g. salvage vs. de novo pathways, uptake studies and recognition of at least 4 transport loci. Data will be presented which show that the distribution and synthesis of RNA (to a lesser extent DNA) in the nucleus is basically the opposite one finds in other eukaryotes, viz. most RNA (ribosomal?) is synthesized (or accumulates) in the peripheral chromatin (functional equivalent of nucleolus?). The DNA is distributed and synthesized primarily throughout the nucleus. It is usually so dispersed that it will not stain with e.g. the standard Feulgen technique, unless the DNA condenses around the endosome (not a nucleolar equivalent) prior to nuclear division. Isolation of rRNA was difficult due, in part, to potent and difficult to inhibit RNase(s), some of which are apparently intimately bound to ribosomal subunits. The 25S (1.3 kDa), 178 (0.8 kDa) and 58 rRNA were recovered after isolation with a high salt SDS-DEP technique. This is the only procedure which enables us to obtain high yields of 258 rRNA: guanidine or guanidinium which permits isolation of intact functional mRNA results in isolation of small amounts of 28 RNA relative to 178 RNA. The 258 RNA is “nicked” (apparently during nuclear processing) and dissociates readily into 1 78 (0.7 kDa) and 168 (0.6 kDa) species, and a more rigidly bound 5.88 species. A small amount of “unnicked” 258 RNA was recovered with guanidine. Two DNA-dcpendent RNA polymerases (I and II) with a pronounced preference for denatured DNA as template were eluted from DEAE-Sephadex in reverse order of what occurs in other eukaryotes, except Physarum polycephalum. This conclusion was based on salt optima and alpha-amanitin sensitivity studies. Initial characterization of DNA isolated with a procedure capable of isolating > 100-kbp Leishmania DNA showed that undigested DNA migrates as a broad band between markers 6 and 24 kbp. The persistent recovery of such a “band” by us and Perez-Mutul et al. no larger than ca. 24 kbp (with the exception of >48 kbp DNA isolated by Hernandez et al. using an in situ lysis technique which did not include a proteinase) may be due to nicks introduced during isolation; or, perhaps much of the amebal DNA exists in vivo as gene sized fragments. However, preliminary data generated using orthogonal pulse-field agarose gel electrophoresis do suggest that amebal DNA may be in small chromosomes.     

Nucleic acids of Entamoeba histolytica

This review will concentrate on certain aspects of the nucleic acids of Entamoeba histolytica. Utilization and synthesis of purines and pyrimidines will initially be briefly discussed, e.g. salvage vs. de novo pathways, uptake studies and recognition of at least 4 transport loci. Data will be presented which show that the distribution and synthesis of RNA (to a lesser extent DNA) in the nucleus is basically the opposite one finds in other eukaryotes, viz. most RNA (ribosomal?) is synthesized (or accumulates) in the peripheral chromatin (functional equivalent of nucleolus?). The DNA is distributed and synthesized primarily throughout the nucleus. It is usually so dispersed that it will not stain with e.g. the standard Feulgen technique, unless the DNA condenses around the endosome (not a nucleolar equivalent) prior to nuclear division. Isolation of rRNA was difficult due, in part, to potent and difficult to inhibit RNase(s), some of which are apparently intimately bound to ribosomal subunits. The 25S (1.3 kDa), 17S (0.8 kDa) and 5S rRNA were recovered after isolation with a high salt SDS-DEP technique. This is the only procedure which enables us to obtain high yields of 25S rRNA; guanidine or guanidinium which permits isolation of intact functional mRNA results in isolation of small amounts of 25S RNA relative to 17S RNA. The 25S RNA is "nicked" (apparently during nuclear processing) and dissociates readily into 17S (0.7 kDa) and 16S (0.6 kDa) species, and a more rigidly bound 5.8S species. A small amount of "unnicked" 25S RNA was recovered with guanidine.(ABSTRACT TRUNCATED AT 250 WORDS)