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PubMed1984

Relationship of cytotoxins of axenically cultivated Entamoeba histolytica to virulence.

Clarified homogenates prepared from different strains of axenically reared Entamoeba histolytica were assayed for acid and neutral proteinase activity, cytotoxicity, and protein content. The homogenates of the more virulent strains contained more acid or neutral proteinase and more toxin units per 10(6) trophozoites. Toxicity and neutral proteinase activities had a similar pattern of distribution. The cytotoxicity of the gel-filtered proteins of the studied strains followed the same rank order as their reported virulence. The more virulent strains (HM-1 and Rahman) had significantly more neutral proteinase activity than less virulent strains (HK-9, Laredo and Huff). Purified extracts of HM-1 and Rahman strains contained more toxin per milligram of protein and exhibited a lower cytotoxicity end point than those of the less virulent strains. The most cytotoxic fraction of each strain had a molecular weight of approximately 24,000, and all cytotoxins were antigenically similar. A more virulent strain (HM-1) secreted more cytotoxic activity when incubated under tissue culture conditions than did a less virulent strain (HK-9). Cytotoxic material secreted by strain HM-1 during axenic growth had the same molecular weight as that isolated from trophozoites. The Entamoeba histolytica toxin studied may be a neutral proteinase that is present in greater quantities in more virulent strains, thus accounting for their greater cytotoxicity in vitro and invasiveness in vivo.

Animals↗
PubMed1995

[Detection of pathogenic Entamoeba histolytica DNA in liver abscess aspirates with polymerase chain reaction].

Polymerase chain reaction (PCR) was used to diagnose amebic liver abscess by detecting pathogenic Entamoeba histolytica DNA in liver aspirates from patients with liver abscess. Oligonucleotide primers specific for gene encoding of the 30,000 molecule of pathogenic Entamoeba histolytica were used in the test. Liver aspirates from 23 cases with amebic liver abscess as evidenced by typical clinical findings or very high titres of anti-E. Histolytica antibodies with ELISA were found to be positive with PCR. Fourteen control samples (3 cases of bacterial liver abscess, 1 of liver cancer and 10 of abscess in other sites) were all negative to PCR. The results suggest that PCR is a sensitive and useful method for diagnosing amebic liver abscess.

Animals↗
PubMed1995

Entamoeba histolytica stimulates the unstable transcription of c-fos and tumour necrosis factor-alpha mRNA by protein kinase C signal transduction in macrophages.

Macrophages play an important role in the control of and resistance to Entamoeba histolytica (E. histolytica). However, E. histolytica infections are characterized by suppression of cell-mediated immunity. To elucidate the molecular mechanisms whereby amoebae modulate host defences, we investigated whether the parasite elicits the 'immediate early' gene c-fos and cytokine tumour necrosis factor-alpha (TNF)-alpha mRNA and determined the signal transduction pathways involved in naive bone marrow-derived macrophages (BMM). E. histolytica stimulated a rapid and transient expression of c-fos and low levels of TNF-alpha mRNA, whereas the non-pathogenic Entamoeba moshkovshii (E. moshkovskii) did not. Inhibition of the protein kinase C (PKC) pathway with the pharmacological inhibitor H7 and by PKC depletion with phorbol myristate acetate showed that E. histolytica modulates TNF-alpha and c-fos gene expression through a PKC-dependent stimulus-response coupling event. E. histolytica activated and translocated PKC to the membrane fraction in BMM demonstrating a rapid and direct effect on PKC enzyme activity. Unlike lipopolysaccharide (LPS), BMM stimulated with E. histolytica had reduced stability of both c-fos and TNF-alpha mRNA transcripts (> 50%) and failed to secrete TNF-alpha protein. BMM treated with amoebic proteins and stimulated with LPS, or interferon-gamma (IFN-gamma)+LPS, resulted in a 33% and 50% reduction in TNF-alpha mRNA levels, respectively. These data argue that although E. histolytica stimulates c-fos and TNF-alpha gene expression through PKC signal transduction, the rapid degradation of the mRNAs, the lack of secreted TNF-alpha protein and the observed decreased responsiveness to a stimulatory signal may be a novel mechanism whereby the parasite modulates host defence mechanisms.

Animals↗
PubMed1996

Physiology and molecular biology of multidrug resistance in Entamoeba histolytica.

In this paper, we present the most relevant facts on multidrug resistance (MDR) in the protozoan parasite Entamoeba histolytica. MDR in E. histolytica presents characteristics similar to transformed mammalian cells. E. histolytica drug resistant mutants show cross-resistance to several drugs, and as in mammalian cells the resistance is reverted by verapamil. Six P-glycoprotein-like genes (EhPgp) have been cloned and characterized. Apparently, four of these genes are transcribed in drug-resistant mutants (EhPgp1, EhPgp2, EhPgp5 and EhPgp6), although only EhPgp1, EhPgp5 and EhPgp6 transcripts were clearly detected. The open reading frame (ORF) of the four completely full length genes is about 1300 amino acids long. EhPgp1, EhPgp2 and EhPgp5 have between 64 and 67% of positional identity among them, while EhPgp6 shows 38 to 46% positional identity to the other ameba genes. Interestingly, the phylogenetic tree suggested that Entamoeba P-glycoproteins are more related to the human and mouse P-glycoproteins than to the Plasmodium and Leishmania P-glycoproteins. Differential gene expression in drug-resistant mutants was detected when specific probes for each Ehpgp gene were used. To understand the differential expression of EhPgp genes we initiated the characterization of the upstream flanking regions of EhPgp1 and EhPgp5 genes. Upstream sequences showed between 53 and 66% of positional identity to Dictyostelium discoideum promoters.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Molecular characterization of myosin IB from the lower eukaryote Entamoeba histolytica, a human parasite.

The complete amino acid sequence of the Entamoeba histolytica unconventional myosin IB (Eh-myosin IB) is reported. Sequencing of overlapping cDNA fragments reveals a single open reading frame which predicts a 130 kDa protein of 1049 aa. Eh-myosin IB presents the three characteristics domains of myosins I subclass 1. This protein presents homology with myosins IB from other amoebae, but striking homologies with vertebrate unconventional myosins were also observed. The predicted actin and ATP-binding sites are located in the head domain. The heavy chain phosphorylation region is homologous to metazoan myosins I with an acidic residue present at the phosphorylation site. In the neck domain, an IQ motif indicates potential binding of calmodulin to the myosin I heavy chain. In the tail of Eh-myosin IB the three characteristic regions of myosin I are found. A putative membrane binding domain a very short domain rich in alanine and proline we demonstrate to be functional for actin binding, and the src-homology 3 domain. The Entamoeba histolytica myosin IB is the first unconventional myosin so far described in a lower eukaryote.

Actins↗
PubMed1997

Does Entamoeba histolytica cause irritable bowel syndrome?

BACKGROUND: Symptoms of patients with irritable bowel syndrome (IBS) closely mimic those of patients with non-dysenteric amebic colitis. AIM: To examine the clinical relevance of presence and types of Entamoeba histolytica in stools of patients with IBS. METHODS: IBS was diagnosed by Manning's criteria. Stool examination was done 4-weekly for 48 weeks to detect E. histolytica cysts or trophozoites. Patients underwent initial sigmoidoscopy. Sera of 22 IBS patients, 23 asymptomatic cyst passers and 36 healthy volunteers whose stools were also examined were tested for presence of antiamebic antibodies. Stools were cultured for amebae; positive cultures were subjected to polyacrylamide-gel electrophoresis (PAGE) using hexokinase (HK) isoenzyme to distinguish between pathogenic (fast-moving band) E. histolytica infection and nonpathogenic (slow band) species of Entamoeba dispar. RESULTS: E. histolytica cultured from stool samples of four IBS patients had slow-moving band of HK on PAGE. All patients spontaneously eradicated the infection during the next eight to 24 weeks; all had negative serology for antiamebic antibodies, and normal rectal mucosa on sigmoidoscopy. No change in symptom score occurred on follow up in IBS patients, although all of them cleared the infection. Three additional E. histolytica isolates from IBS patients obtained from another laboratory also showed nonpathogenic isoenzyme pattern. CONCLUSION: Bowel symptoms in IBS patients were not related to E. histolytica infection. The term non-dysenteric amebic colitis thus appears to be inappropriate, since it may be used erroneously for patients with IBS with nonpathogenic ameba, leading to injudicious treatment with antiamebic drugs.

Abdominal Pain↗
PubMed1998

Crithidia fasciculata induces encystation of Entamoeba invadens in a galactose-dependent manner.

The ability of the flagellate Crithidia fasciculata to induce encystation of the reptile pathogen, Entamoeba invadens, was studied in vitro. A specific ratio of flagellate to amoeba was required; both live and heat-killed C. fasciculata induced amoebic encystation. The interaction between the Crithidia and Entamoeba cells was found to be galactose-mediated because the addition of galactose to the culture medium, or the pretreatment of the flagellate with galactosidase, eliminated its ability to induce encystation. Galactose was also found to prevent the amoeba amoeba aggregation that normally occurs in axenic cultures of encystation-induced E. invadens. Both galactose and glcNAc completely inhibited cyst formation of these induced cultures, although the latter sugar did not prevent cell aggregation. These results indicate that a galactose-mediated interaction between E. invadens cells is an early step in the in vitro encystation pathway.

Animals↗
PubMed1998

System of 14C-glucose incorporation in axenic cultures of Entamoeba invadens: IP-1 strain.

BACKGROUND: It has been described that the walls of the amebic cysts from Entamoeba invadens are composed mainly of chitin, a polysaccharide of amino-sugars. It is also known that the synthesis of this polysaccharide is closely related to the degradation of the intracellular glycogen in this organism. Nevertheless, it is not known whether the intracellular glycogen is really the source of the glucose requirements for the synthesis of the cell wall. METHODS: To determine the relationship between the wall cyst synthesis and glycogen degradation, it was considered to develop an in vitro culture cell system to label this polysaccharide with radioactive glucose. In this study, a system of 14C-glucose incorporation in axenic cultures of E. invadens was developed. The experiments in the study were carried out to recognize if an increase occurred in the 14C-glucose incorporation into ameba when the amount of the radioactivity used was increased, or whether this incorporation is a dependent metabolic stage. RESULTS: The results showed that the amount of glucose incorporation reached similar values of 4.5 x 10(-12) mmol per cell in both cases. A different slope in the glucose kinetic incorporation between the cultures previously subjected to glucose depletion and the standard cultures was observed. CONCLUSIONS: This axenic method of radioactive glucose incorporation in Entamoeba invadens could facilitate the analysis on a greater scale of the metabolism of this nutrient.

Animals↗
PubMed1963

A SAMPLE SURVEY OF SELECTED AREAS IN AND NEAR LITTLE ROCK, ARKANSAS, TO ASSESS THE PREVALENCE OF ENTAMOEBA HISTOLYTICA.

Laboratory confirmed clinical amoebiasis was diagnosed among more than 50 persons by the University of Arkansas Medical Center from 1965 to 1961. The presence of this number of patients prompted an investigation into the prevalence of Entamoeba histolytica in four selected areas in and around Little Rock, Arkansas. The results of the study, using a sample survey technique, showed that it is possible to have a seemingly large number of clinically significant cases of amoebiasis in a population without a high prevalence of the parasite. Evidence was also obtained of the beneficial effects of improvements in sanitary facilities, as reflected in the lower amoebic prevalence rate in a 0-4-year age-group from one of the four areas surveyed in which a piped, indoor city-water supply and sewage facilities had been installed six years previously.

Amebiasis↗
PubMed2002

Entamoeba histolytica: Clinical Update and Vaccine Prospects.

Amebiasis is extraordinarily common in children of the developing world. This realization has come from application of diagnostic techniques that distinguish the nonpathogenic parasite Entamoeba dispar from Entamoeba histolytica. E. histolytica infection is found in children with dysentery, diarrhea, and in many cases in children with no gastrointestinal symptoms. Genetically distinct strains of E. histolytica exist but evidence is too preliminary to judge if some strains are more virulent than others. A provocative study from Tanzania has shown that pregnant women with HIV infection who are coinfected with E. histolytica are at greater risk of delivering a low birth weight infant. Perhaps the most exciting new data is the identification of acquired immunity to recurrent E. histolytica infection. Acquired immunity is linked to a mucosal immune response against a major virulence factor of the parasite, a Gal/GalNAc lectin responsible for adherence and killing of the host. Prospects for a vaccine are thus brightening at the same time that the true burden of disease is comprehended.

Journal Article↗
PubMed1999

Amebiasis: Clinical Implications of the Recognition of Entamoeba dispar.

Entamoeba histolytica was recently reclassified to recognize the existence of two genetically distinct but morphologically indistinguishable species: E. histolytica, the protozoan parasite that causes amebic dysentery and liver abscess, and Entamoeba dispar, a nonpathogenic intestinal parasite. Acceptance of this redefinition has dramatically changed both our understanding of the true epidemiology of E. histolytica and the optimal methods for diagnosing amebiasis. Molecular-based diagnostic tests using polymerase chain reaction (PCR) to amplify amebic DNA or enzyme-linked immunosorbent assay (ELISA) to identify amebic antigens in stool samples have been developed to distinguish infection with E. histolytica from infection with E. dispar. Because of its ability to differentiate strains of E. histolytica, PCR is a very useful research tool. Only the ELISA-based test is simple to perform, making it clinically useful in the developing world. To avoid unnecessary treatment of individuals infected with E. dispar, the World Health Organization has stressed the importance of making a specific diagnosis of E. histolytica infection (not E. histolytica/E. dispar) before treating for amebiasis.

Journal Article↗
PubMed1980

Demonstration Of Entamoeba Histolytica Cyst From Urban And Rural School Children.

School children between 6 to 12 years of age were studied for the presence of Entamoeba histolytica cysts in a period of 6 months, form March to August 1980. Fecal specimens collected form urban and rural children were examined first by the Lugol stained technique and then by the Faust's iron alum hematoxylin method. A total of 731 specimens were examined. Of these 26.9 per cent, 197 specimens, harbored the cysts. The demonstration rate for the cysts by age group varied from 19.6 to 43.5 per cent. The demonstration rate in boys was 24.4 per cent and in girls 29.8 per cent. Similarily, the rate in urban and rural area was 32.4 and 24.1 per cent, respectively. It was found in the present study that there were no significant differences in the age and sex rates of demonstration between urban and rural children but there was a predominance of the small race of Entamoeba histolytica over the large race.

Journal Article↗
PubMed1989

[Prevalence of Entamoeba histolytica and other intestinal protozoa among the inhabitants of Poznan province over a period of 30 years].

The aim of the study was to present the past and actual prevalence of Entamoeba histolytica and other intestinal protozoa in Poznań Province. Although in Poland there are only few accurate data concerning the prevalence of intestinal protozoa, the results of some studies were found reliable. In a survey conducted in 1958 in an orphanage in Poznań (Kasprzak and Karlewiczowa) the intestinal protozoa were found with high prevalence (Table 1). After six years this prevalence decreased considerably (Karlewiczowa and Kasprzak, 1964). Because at the same time such a decrease in intestinal protozoa prevalence, especially that in Entamoeba coli, was observed in the whole population of the Poznań Province, long-term surveys were undertaken. The diagnosing of intestinal protozoa in faeces of 4165 persons, performed at more points in time in the period of 1963 to 1986 in the Poznań Province, were conducted by the same highly skilled staff. Decreasing rates of infection were seen for each amoeba species at nearly each point in time, particularly in the first eight years of observation (Table 2). By using the "amoebic index" and "amoebic prevalence rate" in assessing epidemiological factors (acc. WHO, 1969), the considerable reduction in both indices should be accounted for the improvement of the sanitation and socio-economic status in our country. After the Second World War some Polish authors expressed the fear that after returning to the country, the Polish soldiers who had fought in the endemic regions of invasive amoebiasis, infected with E. histolytica, would constitute sources for spreading amoebiasis and that the disease would be a nucleus of a new, hitherto unknown, social calamity in Poland. Fortunately, the fear did not come true. Although in this country the infection with amoebae, also with E. histolytica, decreased considerably after several years, unfortunately, some opinions still prevail, being the source of an amoebo-mania in the medical staff and corresponding amoebophobia in the patients.

Amebiasis↗
PubMed1997

Encystation of entamoeba parasites.

Entamoeba histolytica is a protozoan parasite of humans, and the causitive agent of intestinal amebiasis. The disease-causing stage of the parasite is an osmotically sensitive ameboid form, which differentiates into a thick-walled cyst for transmission from person to person. The conditions within the human intestine that induce encystment of the amoeba are unknown, but studies using an amoebic parasite of reptiles are now yielding information about the molecules and host:parasite interactions involved in the process. An understanding of the amoeba's obligatory encystment pathway should provide an approach for interrupting the transmission of this parasite, for which there is currently no vaccine.

Amebicides↗
PubMed2000

Ectonucleotide diphosphohydrolase activities in Entamoeba histolytica.

In this work, we describe the ability of living cells of Entamoeba histolytica to hydrolyze extracellular ATP. In these intact parasites, whose viability was determined by motility and by the eosin method, ATP hydrolysis was low in the absence of any divalent metal (78 nmol P(i)/h/10(5) cells). Interestingly, in the presence of 5 mM MgCl(2) an ecto-ATPase activity of 300 nmol P(i)/h/10(5) cells was observed. The addition of MgCl(2) to the extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 1.23 mM MgCl(2). Both activities were linear with cell density and with time for at least 1 h. The ecto-ATPase activity was also stimulated by MnCl(2) and CaCl(2) but not by SrCl(2), ZnCl(2), or FeCl(3). In fact, FeCl(3) inhibited both Mg(2+)-dependent and Mg(2+)-independent ecto-ATPase activities. The Mg(2+)-independent ATPase activity was unaffected by pH in the range between 6.4 and 8. 4, in which the cells were viable. However, the Mg(2+)-dependent ATPase activity was enhanced concomitantly with the increase in pH. In order to discard the possibility that the ATP hydrolysis observed was due to phosphatase or 5'-nucleotidase activities, several inhibitors for these enzymes were tested. Sodium orthovanadate, sodium fluoride, levamizole, and ammonium molybdate had no effect on the ATPase activities. In the absence of Mg(2+) (basal activity), the apparent K(m) for ATP(4-) was 0.053 +/- 0.008 mM, whereas at saturating MgCl(2) concentrations, the corresponding apparent K(m) for Mg-ATP(2-) for Mg(2+)-dependent ecto-ATPase activity (difference between total and basal ecto-ATPase activity) was 0.503 mM +/- 0.062. Both ecto-ATPase activities were highly specific for ATP and were also able to hydrolyze ADP less efficiently. To identify the observed hydrolytic activities as those of an ecto-ATPase, we used suramin, a competitive antagonist of P(2) purinoreceptors and an inhibitor of some ecto-ATPases, as well as the impermeant agent 4'-4'-diisothiocyanostylbenzene-2'-2'-disulfonic acid. These two reagents inhibited the Mg(2+)-independent and the Mg(2+)-dependent ATPase activities to different extents, and the inhibition by both agents was prevented by ATP. A comparison among the ecto-ATPase activities of three amoeba species showed that the noninvasive E. histolytica and the free-living E. moshkovskii were less efficient than the pathogenic E. histolytica in hydrolyzing ATP. As E. histolytica is known to have a galactose-specific lectin on its surface, which is related to the pathogenesis of amebiasis, galactose was tested for an effect on ecto-ATPase activities. It stimulated the Mg(2+)-dependent ecto-ATPase but not the Mg(2+)-independent ATPase activity.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1993

Expression of sequences related to c-myc in Entamoeba.

The evolutionarily conserved proto-oncogene c-myc is involved in both proliferation and differentiation processes of higher eukaryotic cells. We report here the identification and characterization of sequences homologous to c-myc in different Entamoeba species using a fragment of the mammalian c-myc gene as a probe. This probe hybridized with fragments of 3.5 and 3.4 kilobases (kb) in E. histolytica HindIII of EcoRI digested DNA. In E. invadens it recognized fragments of 3.1 and 2.8 kb, and in Laredo strain (reported as E. moshkovskii by Clark and Diamond in 1991) the probe hybridized with fragments of 17 kb. The c-myc probe identified transcripts of 3.3 and 1.5 kb in E. histolytica, transcripts of 1.8 and 1.3 kb in Laredo strain, and transcripts of 3.7, 1.8, 1.5 and 1.1 kb in E. invadens. Antibodies against a highly conserved region of the c-myc protein recognized in E. histolytica polypeptides of 35, 40, and 60 kDa. The expression of the 60 kDa polypeptide was temperature-inducible in Laredo strain. In E. invadens a 110 kDa strong band was detected by the antibodies. Surprisingly, E. invadens myc-like sequences and proteins showed greater homology to mammalian c-myc gene and proteins. Expression of proteins antigenically related to c-myc varied according to the cell cycle phase of E. histolytica. These proteins peaked during D, G1, and S phases and declined during G2.

Animals↗
PubMed1984

Lectin-induced agglutination of trophozoites of different species and strains of Entamoeba.

In vitro agglutinability of trophozoites of three Entamoeba histolytica strains, cultivated under axenic conditions in the presence of concanavalin A (Con A), was shown to be related to the degree of their pathogenicity for experimental animals and of the concentration of Con A. Seven strains of E. invadens tested also agglutinated in the presence of Con A, and the degree of agglutination was proportional to the concentration of the lectin. Three strains of E. histolytica-like "Laredo-type" amebae, a strain of E. terrapinae, and a strain of E. moshkovskii agglutinated very slightly, only in the presence of the highest concentration of Con A tested.

Agglutination↗