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Cysts of Entamoeba invadens obtained under axenic conditions were stained with wheat germ agglutinin labelled with colloidal gold. Microscopic observation of encysting cultures revealed that upon staining, cysts acquired a red coloration, while trophozoites remained unstained. E. histolytica and E. coli cysts obtained from asymptomatic patients were also stained red by this technique. Electronmicroscopic examination of stained cells showed a layer of colloidal gold granules attached to the surface of cysts, while trophozoites remained free of gold granules. The results of the staining procedure developed, agreed with the known facts that wheat germ agglutinin is a lectin that binds specifically to chitin and its oligomers, and that chitin is present in the cyst wall of E. invadens. The results suggested that chitin is also present in the wall of cysts of E. histolytica and E. coli.
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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.
Restriction enzyme digestion patterns of total genomic DNA revealed the presence of highly repeated DNA in Entamoeba histolytica and E. histolytica-like (Laredo type) amoebae of humans, E. invadens and E. invadens and E. terrapinae of reptiles, and E. moshkovskii isolated from sewage polluted waters. Homology with Plasmodium berghei ribosomal DNA was striking. Northern blot analysis of E. histolytica RNA provided further evidence that the highly repeated DNA codes for ribosomal RNA. The distribution of restriction enzyme sites on repeated DNA of the Entamoeba was highly polymorphic. Thus it was possible, based on EcoRI digestion patterns, to distinguish between strains of E. histolytica and the other species of Entamoeba. Additionally, these investigations at the molecular level corroborate previously reported physiological and biochemical evidence indicating the E. histolytica-like amoebae and E. histolytica are not conspecific. EcoRI fragments from repeated DNA of E. histolytica (strain HM-1: IMSS) were cloned in the plasmid vector pTZ18R. A subfragment from one of these clones proved to be a useful hybridization probe in distinguishing E. histolytica from the other Entamoeba.
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.
The dynamics of cytoplasmic streaming, retrograde translocation of externally bound particles and locomotion by Entamoeba invadens were compared. Locomoting amoebae were monopodial, exhibited fountain flow cytoplasmic streaming and translocated externally bound erythrocytes to the rear of cells. The rates of rearward flow of peripheral cytoplasmic vacuoles and of the externally bound particles were equal to the rate of cell forward locomotion. Rhodamine-phalloidin staining revealed a distinct cortical polymerized actin cytoskelton. This was least evident about the periphery of the advancing pseudopod, increased in density toward the rear of the cell and was most concentrated in the uroid. A monoclonal anti-eucaryotic actin antibody, which recognized monomeric Entamoeba actin on immunoblots, stained trophozoites by indirect immunofluorescence throughout the cytoplasm, but not in the cortical regions stained by rhodamine-phalloidin. This and other evidence implied that the antibody recognized only unpolymerized actin in Entamoeba. We propose that locomotion, cytoplasmic streaming and translocation of externally bound particles are driven by a common actin-based mechanism in Entamoeba, possibly involving retrograde cortical actin flow and recycling.
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.
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.
We report the size-fractionation of Entamoeba histolytica and E. invadens deoxyribonucleic acid (DNA) by pulsed field gradient electrophoresis. Using 3 different electrophoretic conditions, we were able to resolve 6 to 9 bands between 300 and 2000 kilobases (kb), distributed in 16-22 large and up to 31 small chromosomes for E. histolytica DNA. For E. invadens, 4 to 5 bands between 300 and over 2000 kb were resolved and discriminated in 4 large and 6 small chromosomes. A ribosomal probe from Trypanosoma brucei hybridized with a 1100 kb band in E. histolytica strain HM1:IMSS and with a 1000 kb band in clone A of strain HM1:IMSS. Both cell lines also showed hybridization at the origin. The ribosomal probe hybridized only at the origin of the E. invadens DNA lane. A triosephosphate isomerase DNA probe from T. brucei hybridized only with a 2000 kb band in E. invadens, indicating that banding patterns are chromosomal bands and ruling out the possibility of DNA degradation.
A small number of Entamoeba isolates from humans, the best known of which is the 'Laredo' strain, have the ability to grow at room temperature. This peculiarity, along with other characteristics, distinguishes the strains from the human pathogen E. histolytica despite their being morphologically inseparable. In contrast, these 'E. histolytica-like' strains share several features with E. moshkovskii, which is most frequently isolated from polluted water. To examine the taxonomic relationships among these morphologically similar organisms, we have used polymerase chain reaction amplification of the small subunit ribosomal RNA gene combined with restriction fragment length polymorphism analysis, 'riboprinting'. The results clearly show that the 'E. histolytica-like' amoebae are indeed strains of E. moshkovskii, and not closely related to E. histolytica.
We describe the first serum-free, partly defined medium (PDM-805) for cultivating the human enteric pathogen, Entamoeba histolytica, and the reptilian amebae E. barreti, E. invadens, and E. terrapinae. PDM-805 was developed by the stepwise replacement of yeast extract, bovine serum, and a casein peptone digest in TYI-S-33, a medium widely used for the axenic cultivation of these parasites. The defined components include amino acids, carbohydrates, B vitamins, ascorbic acid, tocopherol, thioctic acid, nucleic acid precursors, trace metals, and phosphate buffers. The undefined components include a highly purified bovine serum albumin, a lipoprotein-cholesterol solution from bovine serum, and a dialyzable, autoclavable, water-soluble growth factor(s) having a molecular weight of less than 3,500 prepared from casein peptone. To date, studies on the growth requirements of E. histolytica, strain 200:NIH, show the following are essential for sustained multiplication of this ameba: iron, glucose, biotin, folic acid, niacinamide, pantothenate, pyridoxal, riboflavin, thiamine, cysteine, an ammonium moiety (in addition to that present in cysteine), bovine serum albumin, lipoprotein-cholesterol, and casein peptone dialysate.
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.
The effect of Entamoeba histolytica extracts (E.h.e.) on proliferation in vitro of human peripheral blood lymphocytes, cord blood lymphocytes, and lymphocytes enriched for T cells or non-T cells was investigated. As tested by 3H-thymidine uptake and by morphologic criteria, E.h.e. stimulate T lymphocyte proliferation. Non-T lymphocytes do not respond to E.h.e. The response of lymphocytes to E.h.e. differed from their response to Con A in several respects, indicating either that E.h.e. and Con A act on different subsets of T cells or that E.h.e. act by different mechanism on the Con A reactive cells.
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