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BACKGROUND: Entamoeba histolytica is a pathogenic protozoan accountable for amoebiasis, while Entamoeba moshkovskii is considered non-invasive. Despite morphological similarity, the molecular mechanisms underlying their different pathogenicity remain largely undefined. METHODS: Trophozoite proteins from axenic cultures of E. histolytica and E. moshkovskii were separated and identified using GeLC-MS/MS, and classified using Gene Ontology. Selected and differentially expressed proteins were validated by peptide-specific antibody production, ELISA, and immunofluorescence to determine cellular localization. RESULTS AND DISCUSSION: A total of 1,077 and 1,201 proteins were identified from E. histolytica and E. moshkovskii, respectively. The 801 of Entamoeba common proteins included kinases, GTPase-activating proteins, and heat shock proteins, reflecting conserved cellular processes. E. histolytica-unique proteins involved in nitrogen compound metabolism, vesicle-mediated transport, and catalytic activities, whereas E. moshkovskii proteins were related to lipid metabolism and environmental resilience. Subcellular localization revealed species-specific distribution of MmpL and AIG1-family proteins, suggesting potential roles in pathogenicity and host-immune response. A large proportion of hypothetical proteins was identified, highlighting gaps and opportunities for future study. CONCLUSIONS: Our study highlights conserved and divergent functions and cellular locations of Entamoeba species-specific proteins as insights for distinct pathogenicity and adaptation. MmpL and AIG1 proteins were proposed as potential targets for further diagnostic and therapeutic development.
This is the first description of a method for growing axenized Entamoeba histolytica as colonies from single cells in agar suspension. Factors affecting the efficiency of colony formation included unknown characteristics of the amebal strain, age of cells used, and the type and concentration of agar employed. Colony formation was dependent upon filling deep vessels (culture tubes or tissue culture flasks) with agar, probably to maintain reduced oxygen tension, and upon solidifying the agar rapidly at 0 C. It was demonstrated in a study with the drug metronidazole that the agar colony method was useful for quantifying cell viability in drug testing. Eleven of 12 E. histolytica strains tested, as well as one Entamoeba terrapinae, one Entamoeba barreti, and 3 Entamoeba invadens strains formed colonies in agar suspension. E. histolytica-like amebae (Laredo type) and E. moshkovskii formed only tiny colonies in agar.
Rhabdoviruses have been described in plants, arthropods and vertebrates including man. Members of the group are of agricultural, veterinary and medical importance. The presence of a rhabdovirus in Entamoeba histolytica and Entamoeba invadens is the first record of their existence within protozoa. The morphology of this virus is described and its significance discussed, in relation to a possible lysogenic state and pathogenecity of Entamoeba species.
Cultures of 14 stocks of Entamoeba histolytica and one only of Entamoeba coli were compared by electrophoretic patterns of three enzymes: glucose-phosphate isomerase, phosphoglucomutase and L-malate: NADP+ oxidoreductase (oxaloacetate-decarboxylating). Easily distinguished patterns divided E. histolytica into three groups, whilst a distinctly different pattern for E. coli was also seen.
One hundred and fifteen persons with asymptomatic Entamoeba histolytica or E. hartmanni infection, or both, were given metronidazole (750 mg three times daily for 5 days), tinidazole (1 g twice daily on 2 consecutive days), or a starch placebo. Three post-treatment stools were examined in the 2 weeks following initiation of treatment. Cysts of E. histolytica reappeared in the stools of 37% of 30 given metronidazole, 62% of 34 given tinidazole, and 70% of 31 given placebo. Cysts of E. hartmanni reappeared in the stools of 46% of 24 given metronidazole, 69% of 16 given tinidazole, and 90% of 10 given placebo. Rapid absorption and short duration of treatment make both drugs ineffective for the treatment of ameba carriers.
We have examined the sodium dodecyl sulfate (SDS)-induced autoproteolysis of E. invadens (PZ and IP101) and E. moshkovskii (FIC and Laredo) trophozoite lysates. Heat-treated lysates containing parahydroxy-mercuribenzoate (pHMB) of all four strains had undegraded protein patterns. Unheated pHMB-lacking lysates of PZ had two (99 and 90 kDa) and IP101 lysates had three (45, 99, 90 kDa) major proteins, whereas FIC and Laredo lysates had only one (90 kDa). Heat-treatment changed the remaining proteins to smaller ones: 37 in PZ and IP101, 33 and 37 kDa in FIC and Laredo. Unheated lysates run on gelatin-containing "substrate" gels had gelatinases whose sizes were higher in lysates containing pHMB and allowed us to detect a 200 kDa gelatinase in E. invadens strains. Our results indicate that SDS induces immediate autoproteolysis by CPs in non-histolytica trophozoites, whose proteinases appear to be processed by self-digestion, and Entamoeba proteinases vary considerably under the various conditions used to obtain and characterize them.
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E. histolytica, pathogenic, and E. dispar, non-pathogenic, being morphologically identical are, when recovered as cysts in feces, diagnosed as E. histolytica. A survey on The Seychelles demonstrated that when culture and zymodeme characterization was used to compare against microscopy alone, the risk of overdiagnosis of E. histolytica infections was drastically reduced. From a total of 313 subjects tested 21 cultures grew amebas. By zymodeme analysis eight were E. histolytica, 40 were E. dispar and the remainder were various species of non-pathogenic intestinal amebas. Two further small comparative surveys confirmed these findings.
Large numbers of 2 species of Entamoeba, Entamoeba coli and Entamoeba bovis, were recovered from captive white-tailed deer from Georgia. The trophozoite of the first ameba was a large form with a well-defined vesicular nucleus bearing a prominent eccentric endosome, abundant peripheral chromatin, and numerous periendosomal granules. Cytoplasmic vacuoles containing bacteria and other substances were common. Mature cysts were octonucleate. Glycogen vacuoles and irregularly shaped chromatoids were frequently present. This ameba was identified as Entamoeba coli. The trophozoite of the second ameba was small, with a typical Entamoeba nucleus. Cysts were uninucleate, containing numerous chromatoids resembling bars, splinters of wood, and irregular masses. There was occasionally a glycogen vacuole. This ameba was identified as Entamoeba bovis.
The ribosomal RNA genes of the protozoan parasite Entamoeba histolytica are highly repeated and display restriction fragment length polymorphism. Using a set of four DNA probes spanning the coding region and part of the flanking region of the E. histolytica ribosomal RNA genes, an analysis of the DNA bands generated by EcoRI digestion of Entamoeba DNA is presented. This analysis included five strains of E. histolytica, four strains of E. moshkovskii, and one strain each of E. invadens and E. terrapinae. No common bands were observed between E. histolytica and the other Entamoeba. Within E. histolytica, two bands were conserved in all strains while the others were polymorphic. Detailed analysis of DNA from independently isolated clones of the strain HM-1:IMSS of E. histolytica showed two bands to be highly polymorphic. Of these, the 4.4-kb band of clone 6 was further analyzed. Polymorphism in this band could even be demonstrated in cells of the same clone. Restriction enzyme analysis of this DNA band from two clones of HM-1:IMSS showed that the polymorphism may be due to variable numbers of DraI repeat units present in this DNA stretch.
The assertion that ingestion of human erythrocytes is restricted to invasive strains of Entamoeba histolytica has not been evaluated previously by comparative studies. In this report we describe the in vitro ingestion of human erythrocytes by pathogenic and nonpathogenic Entamoeba. Microscopic evaluation of erythrophagocytosis by eight different Entamoeba grown in culture revealed that strains of E. histolytica isolated from cases of human dysentery show a much higher rate of erythrocyte ingestion than nonpathogenic strains. However, all strains are able to phagocytize erythrocytes. The extremely high rate of phagocytic activity shown by pathogenic E. histolytica could be one of the properties related to the pathogenicity of this parasitic protozoan.
Two P-glycoprotein genes (EhPgp1 and EhPgp2) from the protozoan parasite Entamoeba histolytica were sequenced from a genomic library made with the DNA of an emetine-resistant ameba mutant, which overexpresses mRNAs homologous to segments of the human mdr1 (P-glycoprotein) gene. The open reading frames for EhPgp1 and EhPgp2 were 1302 and 1310 amino acids long, respectively, and showed a 67% positional identity with each other and 41% and 40% positional identities, respectively, with human mdr1 gene. Within each ameba P-glycoprotein were the ATP-binding sites found twice in eukaryotic P-glycoproteins and once in prokaryotic transport proteins. Hydropathy plots of the ameba P-glycoproteins were nearly superimposable on that of the human mdr 1, showing 2 homologous halves, each containing an ATP-binding site and 6 hydrophobic transmembrane domains that form the putative channel. A phylogenetic tree showed that the Entamoeba P-glycoproteins are more related to the human and mouse P-glycoproteins than to the Plasmodium and Leishmania P-glycoproteins. Also identified in the E. histolytica genomic library were 2 P-glycoprotein pseudogenes, each with a frame shift and stop codons in identical places within the amino ATP-binding site. In conclusion, the 2 E. histolytica P-glycoproteins encoded by the EhPgp1 and EhPgp2 genes are similar in structure to the mammalian P-glycoproteins and so may be involved in energy-dependent drug efflux by this human parasite.
Colchicine has a temperature-dependent cytotoxic effect on Entamoeba sp. (Laredo isolate) that is most apparent when the drug is applied during the initiation of cultures at a concentration of 7.5 mM or higher. Continued transfer of cultures in medium containing progressively increasing concentrations of colchicine has resulted in a variant that grows prolifically in the presence of colchicine (7.5 mM) with a generation time comparable to that of the parent stock, Comparison of a number of parameters of the 2 variants revealed that colchicine resistance was accompanied by a change in cell shape, a reduced membrane permeability, which could partially be overcome by the addition of dimethyl sulfoxide (DMSO), and a reduced tolerance to osmotic stress. However, the parent strain and resistant variant were equally susceptible to cycloheximide and puromycin suggesting that the acquired colchicine resistance may not be explained on the basis of an entirely unspecific generalized reduced ability for drug uptake. Colchicine resistance and altered structure were found to be stable over a long period of time. The possible interdependence of these 2 parameters and their relation to cell motility in Entamoeba sp. are discussed.
Auto-antibodies against normal human liver have been detected in the sera of humans with highly positive indirect hemagglutination (IHA) amebiasis titers and with clinically-proven amebic liver abscess. Sera of amebiasis patients and rabbits immunized with killed Entamoeba histolytica were tested for anti-amebic antibodies by the IHA test and for auto-antibodies by the complement fixation test, using the antigens prepared from extracts of human liver and rabbit liver. A direct correlation was found to exist between high anti-Entamoeba antibody titers and the presence of anti-liver antibody in the serum. It is proposed that, in addition to direct parasite damage to host tissue, immunological damage could result from the attachment of circulating antigen to the cell surfaces of host tissues such as the liver.
Mixed and monoxenic cultures of rectal exudates from three patients with amebiasis of colon, and of feces from three asymptomatic carriers of Entamoeba histolytica obtained after administration of a saline cathartic, and from a case of exudate of the perianal cutaneous lesion border produced by this parasite, were done. The culture media used were for: mixed culture, Boeck and Drbohlav modified culture and, for monoxenic culture, Diamond's monophasic TP-S-1 medium without vitamin mixture and using bovine serum. In order to inhibit the bacterial growth in the mixed culture, streptomycin sulphate and pencillin G were used. In order to eliminate the bacterial flora kanamicin sulphate, chloramphenicol, gentamycin and disodic carbenicillin were added. Mixed cultures of seven strains of Entamoeba histolytica were obtained: HM22:IMSS, HM23:IMSS, HM24:IMSS, HM26:IMSS, HM27:IMSS, HM28:IMSS y HM29:IMSS, and monoxenic culture with Bacteroids symbiosus of the strains HM22:IMSS and HM27:IMSS, with a previous adaptation in mixed culture. The adaptation of HM26:IMSS strain in monoxenic culture was obtained by means of direct seeding of the rectal exudate with Fusobacterium symbiosus.