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Entamoeba histolytica: generation and characterization of hybrid clones.

Transference of DNA to Entamoeba histolytica was carried out by polyethylene glycol fusion of two amebic clones with different phenotypes. Clone C9, strain HM1:IMSS, was the donor. It is emetine-resistant, highly phagocytic and virulent, and grows in soft agar. Clone L6, strain HM1:IMSS, the recipient, is emetine-sensitive, phagocytic, and virulence-deficient, and it does not grow in soft agar. Clones L6 and C9 have shown high stability in their virulence phenotypes since their isolation more than 5 years ago. Before fusion experiments, clone C9 was incubated in 20 micrograms/ml bromodeoxyuridine for 24 hr, and then, irradiated with 310 nm light to complete inactivation. Controls ensured that all irradiated trophozoites died after 24 hr of incubation at 37 degrees C. Irradiated C9 trophozoites were fused with L6 trophozoites, and hybrids were selected by their ability to grow in the presence of emetine. All hybrids, independently generated, grew poorly in soft agar and showed both an intermediate emetine-resistance and rate of phagocytosis. Some of them destroyed efficiently cell monolayers, but interestingly, they showed differences in their ability to produce hepatic abscesses in hamsters.

Animals↗

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.

Animals↗

Ubiquitin of Entamoeba histolytica deviates in six amino acid residues from the consensus of all other known ubiquitins.

The amino acid sequence of ubiquitin from Entamoeba histolytica, as deduced from a cDNA nucleotide sequence, deviated at six positions from the consensus of all other known ubiquitins (ranging from Trypanosoma cruzi to Homo sapiens). The corresponding residues were scattered over the primary sequence, but came close together on the surface of the folded protein structure. We conclude that (i) E. histolytica branched off very early from the main eukaryotic line, and (ii) this organism may yield clues as to the evolutionary development of the ubiquitin system.

Amino Acid Sequence↗

Pore-forming peptide of Entamoeba histolytica. Significance of positively charged amino acid residues for its mode of action.

Amoebapore is a 77-residue pore-forming peptide from Entamoeba histolytica with antibacterial and cytolytic properties. It contains eight lysine residues and one histidine residue. Chemical modifications of amoebapore with various reagents affecting either both types of cationic residues or lysine and histidine residues separately resulted in virtually complete loss of pore-forming activity. The activity was restored by reversal of modifications. Whereas amoebapore was no longer capable of binding to phospholipid vesicles when its lysine residues were modified, the modification of the single histidine primarily affected oligomerization of the peptide upon membrane association.

Amino Acid Sequence↗

Inositol(1,3,4,5) tetrakisphosphate plays an important role in calcium mobilization from Entamoeba histolytica.

Calcium release from internal stores of Entamoeba histolytica, a parasitic protozoan, was observed by measuring fluorescence of Fura-2. Emptying of inositol(1,4,5)trisphosphate (Ins(1,4,5)P3)-sensitive calcium pools in permeabilized E. histolytica did not significantly affect subsequent calcium release by inositol(1,3,4,5)tetrakis-phosphate (Ins(1,3,4,5)P4). Similarly, prior depletion of Ins(1,3,4,5)P4-sensitive stores did not have any influence on subsequent calcium release by Ins(1,4,5)P3. The EC50 for calcium release was 0.15 microM with Ins(1,4,5)P3 and 0.68 microM with Ins(1,3,4,5)P4. In conclusion, the Ins(1,3,4,5)P4-sensitive calcium store in E. histolytica is separate and independent from the Ins(1,4,5)P3-sensitive pool.

Animals↗

Relative importance of inositol (1,4,5)trisphosphate and inositol (1,3,4,5)tetrakisphosphate in Entamoeba histolytica.

[3H]Inositol tetrakisphosphate (Ins(1,3,4,5)P4) binding sites which were poorly displaced by unlabelled inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3) were detected in membrane fractions of Entamoeba histolytica. Similarly, unlabelled Ins(1,3,4,5)P4 was 30-fold less efficient in displacing [3H]Ins(1,4,5)P3 binding. pH sensitivities of binding of the two isomers were markedly different. Scatchard analysis of the data revealed single binding sites and similar receptor densities for each of the two isomers. Formation of both Ins(1,4,5)P3 and Ins(1,3,4,5)P4 in E. histolytica was also demonstrated. Calcium release studies showed that after treatment with a saturating dose of either Ins(1,4,5)P3 or Ins(1,3,4,5)P4 the other inositol polyphosphate could partially revive the response to a subsequent addition of the first inducer. Our data clearly demonstrate that Ins(1,4,5)P3 and Ins(1,3,4,5)P4 are two equally important but independent second messengers in E. histolytica.

Animals↗

Mucin and nonmucin secretagogue activity of Entamoeba histolytica and cholera toxin in rat colon.

Depletion of colonic mucus occurs before invasion of the colonic mucosa by Entamoeba histolytica trophozoites. It is hypothesized that E. histolytica releases a mucus secretagogue; this was studied in a rat colonic loop model. In colonic loops exposed to live amebae, mucus secretion was quantitated by release of acid-precipitable [3H]glucosamine-labeled luminal glycoprotein and by specific immunoassay. Mucus secretion increased in dose-dependent fashion in response to greater than or equal to 1 X 10(5) trophozoites; cholera toxin (20 micrograms per loop), a known mucus secretagogue, elicited a similar response. Thin-section histological analysis of amebae and cholera toxin-exposed loops showed increased mucus release and streaming from mucosal goblet cells with cellular cavitation compared with control loops. Sepharose-4B chromatography of amebae and cholera toxin-stimulated glycoproteins demonstrated secretion of mucins and an 80%-90% increase in low-molecular-weight proteins. E. histolytica trophozoites and cholera toxin enhanced the secretion of preformed and newly synthesized mucin glycoproteins and stimulated colonic glycoprotein synthesis. The level of mucus secretion elicited by axenic E. histolytica strains correlated with their virulence in vivo and in vitro. The amebic secretagogue was released into the culture medium and was heat stable. Mucus secretagogue activity of E. histolytica may contribute to depletion or alteration of the protective mucus blanket, facilitating pathogenesis of invasive amebiasis.

Animals↗

Clinical survey of Entamoeba gingivalis by multiple sampling in patients with advanced periodontal disease.

A clinical survey of Entamoeba gingivalis was conducted in patients with advanced periodontal disease. A total of 100 specimens were taken from 10 patients (four females and six males) who were each sampled at 10 disto-facial random sites. The oral hygiene of the persons scored from good to fair to poor and very poor. The age of the test persons ranged from 20 to 68 years. All persons examined harbored E. gingivalis: the minimum prevalence was four sites positive out of 10 and the maximum prevalence was 10 sites positive out of 10 for the protozoan. It is recommended that for reliable testing of the presence of E. gingivalis at least five-10 different sites should be examined within the periodontium of each patient. Data reported earlier in the literature on the incidence of E. gingivalis--obtained after a single sampling per individual--are believed to be on average too low and, therefore, should be interpreted with caution.

Adult↗

Growth studies on xenic cultures of Entamoeba gingivalis using established media.

Wantland's egg medium, modified Shaffer-Frye (MSF) medium and Tryptose-Trypticase-Yeast Extract-Serum-Blood (TTY-SB) medium were compared with variations of the latter two media for their ability to support xenic growth of Entamoeba gingivalis. Wantland's egg medium was unsuitable for growth of E. gingivalis. Accompanying bacteria became resistant to penicillin and streptomycin, overwhelming the amoeba culture. MSF medium was also unsuitable for the cultivation of E. gingivalis. Bacterial growth was heavy and protozoan growth sparse. MSF medium without mercaptosuccinic acid, but with rice starch, dextran or levan substituted for glucose and with Yersinia enterocolitica added, supported limited growth of the amoeba. Unmodified TTY-SB medium did not sustain growth of E. gingivalis. However, when rice starch suspension was substituted for glucose, L-cysteine HCl was deleted, and a Crithidia sp. was added to the E. gingivalis culture grown xenically, enhanced growth of the oral amoeba resulted in this modified TTY-SB medium. E. gingivalis is very sensitive to changes in incubation temperature. Optimum growth was found to be in the narrow range from 34.5 to 35 degrees C for all media tested.

Animals↗

Entamoeba histolytica: a simplified method to quantify its cytotoxicity.

A simplified and reliable method to quantify Entamoeba histolytica cytotoxicity was standardized. Mice spleen leucocytes were utilized as target cells. Interaction time was reduced to 1 h by pelleting interacting cells. To assess target-cell killing by amoebae, a nigrosine exclusion test was employed. Fixation with glutaraldehyde stabilized the percentage of stained target cells. Similar results were obtained when cytotoxicity of the E. histolytica HM1 strain was tested by the traditional and proposed methods. The new method allowed quantification of the contribution of cytolysis and cytophagocytosis to amoebic cytotoxicity. It was also demonstrated that uncloned E. histolytica HM1 strain is a heterogeneous population with respect to cytotoxicity expression.

Animals↗

An antibiotic-free medium for the xenic cultivation of Entamoeba gingivalis.

Diamond's TYI-S-33 (Trypticase-Yeast Extract-Iron-Serum) medium was used as the basis for a new antibiotic-free medium for xenic growth of Entamoeba gingivalis. Nutritional requirements of the oral protozoan were determined in an effort to optimize growth. TYI-S-33 medium did not support E. gingivalis growth prior to modification. The changes included: (a) deletion of L-cysteine.HCl and thioctic acid, (b) substitution of glucose for dextran I (mol. wt 185,000) or rice starch, (c) reduction of concentrations of tryptone (2.5 g l-1), yeast extract (1.25 g l-1) and dextran I (1 g l-1), (d) increased concentration of ferric ammonium citrate (0.2 g l-1), and (e) addition of gastric mucin (2.4 g l-1). Dextran I was chosen as the major carbon source; its use in the medium limited growth of accompanying bacteria. This new antibiotic-free medium significantly increased E. gingivalis growth (16-20 E. gingivalis trophozoites observed per field) as compared to growth in Diamond's TYSGM-9 (Trypticase-Yeast Extract-Serum-Gastric Mucin) medium (six to 10 E. gingivalis trophozoites observed per field).

Animals↗

Detection of pathogenic Entamoeba histolytica DNA in liver abscess fluid by polymerase chain reaction.

A sensitive method for detection of pathogenic Entamoeba histolytica DNA in drained fluids from liver abscess patients, using the polymerase chain reaction (PCR), has been developed. The PCR employs oligonucleotide primers specific for the gene encoding the 30 kDa molecule of pathogenic E. histolytica. Liver abscess fluids (19 samples), from 14 patients with a presumptive amebic liver abscess, were examined microscopically and by the PCR method. Only two of the 19 samples were positive microscopically, whereas all 19 samples tested positive by PCR. This technique can be used to confirm the diagnosis of amebic liver abscess.

Animals↗

Serum-independent and serum-dependent cytoadherence in the interaction of Entamoeba histolytica with mammalian target cells.

Entamoeba histolytica kills target cells only on direct contact, suggesting that trophozoite-mediated cytolysis is initiated by the contact between trophozoites and target cells. We have shown that adherence between E. histolytica and target cells (polymorphonuclear granulocytes, erythrocytes, Chinese hamster ovary cells, human colon carcinoma cells) was inhibited by specific carbohydrates, and adherence between E. histolytica and polymorphonuclear granulocytes (PMN) was enhanced by preincubation of the trophozoites with serum. Inhibition of adherence clearly paralleled inhibition of cytolysis and phagocytosis of target cells. Cytolysis of PMN, however, was not increased by preincubation of the trophozoites with serum. These results suggest that the effector functions of trophozoites are only dependent on carbohydrate-specific adherence mechanisms mediated by the amoebic Gal/GalNAc-binding lectin. E. histolytica trophozoites themselves can be killed by PMN, depending on the virulence of the trophozoites. PMN could not kill E. histolytica trophozoites more effectively when the adherence was enhanced by preincubation of the trophozoites with serum or when adherence was only mediated by serum-dependent mechanisms.

Adenocarcinoma↗

Influence of bacteria on electrophoretic proteinase patterns of Entamoeba histolytica isolates.

Gelatin SDS- polyacrylamide gel electrophoresis was used to compare proteinase banding patterns under reducing conditions from whole cell lysates of four axenic and four xenic pathogenic strains of Entamoeba histolytica. All strains shared major bands in the 34 and 66-68 kDa regions, whereas only the axenic strains produced major bands at 26, 28, 30 and 45 kDa. One axenic strain, NIH 200, when reassociated with mixed bacterial flora, reverted to an electrophoretic banding pattern characteristic of other xenic strains. These results suggest that the 26-30 kDa and 45 kDa proteinases of E. histolytica are induced by bacterial starvation while others are constitutively expressed. It is also proposed that the axenic bands of 45 and 34 kDa represent respectively, the reduced forms of the 56 and 40 kDa bands reported elsewhere under non-reducing conditions.

Animals↗

Monoclonal antibody-based immunohistochemical demonstration of Entamoeba histolytica in liver tissues of experimentally infected hamster (Mesocricetus auratus).

Histopathological changes and the presence of Entamoeba histolytica trophozoites was sequentially followed after intrahepatic inoculation of the parasites in 42 hamsters, 35 of which received no treatment whereas the remaining seven were treated with metronidazole. The liver tissues were examined for amoebic trophozoites by a monoclonal antibody (mAb)-based immunofluorescence assay (IFA), a mAb-based immunoperoxidase (IPx) and H & E staining. The number of hamsters developing abscesses was increased with time and was highest on day 30. Cellular infiltration with inflammatory cells and glycogen depletion were observed as early as day 5, followed thereafter by more intense inflammation of portal canals, periportal fibrosis, bile duct proliferation and hepatocyte degeneration. In 7 metronidazole-treated hamsters, no obvious pathological damage was seen. In a group of seven hamsters each, both IPx and IFA were positive in 3, 3, 4, 5 and 4 hamsters and in 3, 4, 3, 3 and 5 hamsters on days 5, 10, 15, 20 and 30, respectively. In 18 control hamsters, IPx, IFA and H & E were all negative. If the result from H & E was used as a gold standard, agreement between H & E and IFA and H & E and IPx were 91.4%, and 88.6%, respectively. Sensitivity and specificity were 93.8% and 89.5%, respectively for IFA, and 93.8% and 84.2%, respectively for IPx.

Animals↗

Pathogenic action of Entamoeba invadens: intestinal epithelium invasion by trophozoites in vitro.

Interactions between live Entamoeba invadens trophozoites and guinea-pig caecal explants were studied. A high percentage of amoebae adhering to the apical surface of epithelial cells was observed 10-20 min after infection, but no histopathological changes were observed. After 30 min, mild oedema at the base of the interglandular epithelium and death of some epithelial cells were evident. The epithelial barrier was invaded by amoebae at desquamating zones and phagocytosis of epithelial cells or cellular debris was occasionally observed. Invasion of the mucosa and tissue necrosis became more severe with increased time of incubation. The continuity of epithelial lining was severely compromised after 2 h of infection and erosive lesions were prominent in the mucosa. These results demonstrate that E. invadens is able to invade the intestinal epithelium although it reportedly lacks the powerful cytotoxic and cytolytic elements described for E. histolytica.

Animals↗

Necrotic colitis associated with Entamoeba histolytica infection in a cat.

A 3-year-old Persian cat developed bloody diarrhoea. On histological examination, a marked necrotic colitis with a large number of invading protozoan parasites was observed. The protozoan was identified as Entamoeba histolytica by light and electron microscopy. This is the first report describing spontaneous amoebiasis in cats.

Animals↗

A dot enzyme-linked immunosorbent assay for detection of antibodies against Entamoeba histolytica.

A visual micro-dot enzyme-linked immunosorbent assay (ELISA) based on detection of antibodies against soluble antigens of axenically grown cultures of Entamoeba histolytica is described. The antigen was spotted on a nitrocellulose sheet, the unsaturated sites blocked with bovine serum albumin (BSA) and incubated with 3-fold dilutions of patient sera followed by incubation with protein A conjugated to peroxidase. Enzymic activity was evidenced using the substrate 4-chloro-1-naphthol. A positive reaction produced a blue spot. The sensitivity of the assay was better and comparable to the indirect haemagglutination assay (IHA) and plate ELISA. The entire assay could be completed within 3 h. Antigen-loaded and pre-blocked nitrocellulose strips could be stored up to 3 months at room temperature and 37 degrees C.

Amebiasis↗