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PubMed2002

Nitric oxide inhibits cysteine proteinases and alcohol dehydrogenase 2 of Entamoeba histolytica.

Nitric oxide is involved in the neutrophil and macrophage killing of the protozoan parasite Entamoeba histolytica. In the present study, we found that cysteine proteinases, significant contributors to amebic virulence and alcohol dehydrogenase 2, an enzyme absolutely required for the survival of the parasite, are both significantly inhibited by S-nitroso-glutathione, a physiological nitric oxide donor, within the concentration range 0.5-2.0 m M.

Alcohol Dehydrogenase↗
PubMed2003

The monocyte locomotion inhibitory factor produced by Entamoeba histolytica inhibits induced nitric oxide production in human leukocytes.

The monocyte locomotion inhibitory factor, an anti-inflammatory pentapeptide produced by Entamoeba histolytica, inhibits the in vitro production of nitric oxide induced by cytokines (INF-gamma, TNF-alpha) or PMA in human leukocytes. This can be added to the other previously reported functional effects of this factor, such as the inhibition of monocyte locomotion and the synthesis of reactive oxygen intermediates in both monocytes and neutrophils. The decreased nitric oxide production may interfere with the killing of amebas by neutrophils in the early invasive stages of amebiasis, when oxidative mechanisms are used [reactive oxygen and nitrogen intermediates either individually or synergistically via peroxynitrite (ONOO(-))], and in the advanced stages, when both non-oxidative and oxidative (including nitric oxide) mechanisms are employed by macrophages. Diminished nitric oxide production by leukocytes may also contribute to the paucity of late inflammatory components in amebic abscess of the liver and other amebic lesions.

Animals↗
PubMed2003

Involvement of signaling through protein kinase C and phosphatidylinositol 3-kinase in the excystation and metacystic development of Entamoeba invadens.

Using an axenic excystation system in vitro, we examined the effect of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K), which are signaling molecules responsible for numerous cellular responses, on the excystation and metacystic development of Entamoeba invadens. Excystation, which was assessed by counting the number of metacystic amoebae after the induction of excystation, was inhibited by the PKC inhibitors staurosporine, chelerythrine chloride and calphostin C in a concentration-dependent manner during incubation, compared with the controls. As cyst viability was not affected by these inhibitors, reduced excystation was not due to their direct toxic effects on cysts. Metacystic development, when determined by the number of nuclei in the amoebae, was delayed by these PKC inhibitors, because the percentage of 1-nucleate amoebae was lower than in controls at day 3 of incubation. Wortmannin, a potent inhibitor of PI3K, also inhibited excystation and metacystic development of E. invadens in a concentration-dependent manner, compared with the controls. These results indicate that signaling through PKC and PI3K contributes to the excystation and metacystic development of E. invadens.

Androstadienes↗
PubMed2004

Different effects of cytochalasins on the growth and differentiation of Entamoeba invadens.

The effect of five different cytochalasins on the growth, encystation and excystation of Entamoeba invadens was examined. At 10 microM, cytochalasins B, D, E and dihydrocytochalasin B markedly inhibited growth. Encystation was inhibited by cytochalasin D at 1 microM but not by other cytochalasins at the same concentration, whereas it was inhibited by 10 microM of cytochalasins B, E and dihydrocytochalasin B as well as cytochalasin D. Excystation, which was assessed by counting the number of metacystic amoebae after inducing excystation, was markedly enhanced by cytochalasin D as previously demonstrated, whereas the enhancing effect of cytochalasins A, B and dihydrocytochalasin B was weak. In contrast, cytochalasin E at 10 microM inhibited excystation and metacystic development. These results indicate that there is a difference in the effect of different cytochalasins on the growth and differentiation of E. invadens, depending on differences in their chemical structure.

Animals↗
PubMed2005

Entamoeba histolytica: focal adhesion kinase and Erk phosphorylation are altered in the cytoskeleton mutant BG-3.

The Entamoeba histolytica mutant BG-3 has several altered cytoskeletal properties, including the distribution of actin and certain surface characteristics such as osmolarity and electrophoretic mobility. By Western blot analysis and assays for cell adhesion to collagen, we demonstrate that mutant BG-3 shows an increase in the phosphorylation levels of protein kinases that participate in proliferation, adhesion and migration, such as focal adhesion kinase and MAP kinase (Erk2), and that it has also altered its capacity of binding to collagen type I. These results indicate that E. histolytica cytoskeleton integrity plays an important role in adhesion and thus invasion of the host.

Animals↗
PubMed2005

Entamoeba histolytica sequences and their relationship with experimental liver abscesses in hamsters.

The purpose of the present paper was to analyse the association between sequences of Entamoeba histolytica and their relationship with the development of hepatic abscesses in hamsters, using a complementary DNA library for E. histolytica. From the sequences obtained, we designed oligonucleotides for amplification by PCR. Trophozoites were isolated from faeces of 11 patients in whom cysts from E. histolytica were identified, and these trophozoites were then subjected to monoaxenic culture. Then 1 x 10(5) trophozoites were inoculated into hamster liver, with three hamsters used for every culture. Sequences were obtained for ubiquitin, lectin surface precursor, replication factor MCM3 and surface antigen. The associations between sequences and hepatic abscesses were: 11/11 for ubiquitin, 9/11 for lectin precursor, 4/11 for replication factor and 1/11 for surface antigen. These results suggest that ubiquitin could be an important protein involved in the mechanism of E. histolytica invasion.

Animals↗
PubMed2005

Could the homologous sequence of anti-inflammatory pentapeptide (MLIF) produced by Entamoeba histolytica in the N protein of rabies virus affect the inflammatory process?

Amebiasis and rabies are public health problems, and they have in common a poor inflammatory effect in the target organs that they affect. In the GenBank, it was found that the anti-inflammatory peptide monocyte locomotion inhibitory factor (MLIF) produced by Entamoeba histolytica homologates 80%, with a fragment of the N protein of the rabies virus. We speculated if the N protein could contribute to the scant inflammatory reaction produced by rabies virus in central nervous system. The N protein was obtained and studied in vitro and in vivo. The N protein, as MLIF, inhibited the respiratory burst in human mononuclear phagocytes (43%, p<0.05), but in contrast to MLIF, it increased chemotaxis and it did not significantly inhibit delayed hypersensitivity skin reaction to 1-chloro-2-4-dinitrobenzene in guinea pigs. Therefore, the full peptide sequence has to be present or it has to be cleaved-free from the large recombinant N protein molecule (55 kDa) to become active.

Animals↗
PubMed2005

Cytokine expression in CD4(+) cells exposed to the monocyte locomotion inhibitory factor produced by Entamoeba histolytica.

Entamoeba histolytica produces monocyte locomotion inhibitory factor (MLIF), a pentapeptide with in vitro and in vivo anti-inflammatory properties. MLIF may interfere with leukocyte migration, disturbing the balance of pro- and anti-inflammatory cytokines secreted by CD4(+) T lymphocytes. We evaluated the effect of MLIF on expression of pro- and anti-inflammatory cytokines in human CD4(+) T lymphocytes. Regulatory cytokines [interleukin-1 beta (IL-1beta), IL-2, interferon gamma (IFN-gamma), IL-5, IL-6, and IL-10] were studied by enzyme-linked immunosorbent assay method in CD4(+)-cell supernatant fluids. Proinflammatory cytokines were produced per se by MLIF (IL-1beta, IL-2, and IFN-gamma) and also anti-inflammatory cytokines (IL-5, IL-6, and IL-10) with 1-phorbol-12 myristate-13 acetate + MLIF; the IL-1beta, IFN-gamma, IL-5 and IL-6 production was inhibited but not that of IL-10 which disclosed increase in its expression. MLIF disturbs the pro- and anti-inflammatory balance, and it induces inhibition of IL-1beta (principal proinflammatory cytokine) and increases IL-10 (prototype of an anti-inflammatory cytokine).

Animals↗
PubMed2006

The second cysteine protease inhibitor, EhICP2, has a different localization in trophozoites of Entamoeba histolytica than EhICP1.

The genome of Entamoeba histolytica contains two genes encoding inhibitors of cysteine proteases of the chagasin family. In contrast to that of EhICP1, the derived primary structure of the second inhibitor, EhICP2, possesses a typical N-terminal signal sequence. Processed EhICP2 is as weakly related to amoebiasin-1 (27% identity) as to chagasin (identity 30%), indicating a different evolutionary origin of both amebic genes. By Northern blots, we confirmed the expression of the ehicp2 gene, and in Western blots, the presence of the 11.5-kDa protein in trophozoite extracts was demonstrated. The inhibitor localized to large intracellular structures clearly differs from those containing EhICP1 as shown by indirect immunofluorescence. Recombinant EhICP2 significantly inhibited the cysteine protease activity of the amebic cell extract but with a lower extent than EhICP1. An overlay assay using a crude trophozoite extract demonstrated binding affinity of the amebic cysteine protease EhCP1 to EhICP2.

Amino Acid Sequence↗
PubMed2000

The endocytic pathway in Entamoeba histolytica.

We studied the endocytic pathway of Entamoeba histolytica using (a) confocal laser scanning microscopy to observe living cells labeled with fluorescent probes and (b) transmission electron microscopy of cells incubated in the presence of gold-labeled proteins or in a cytochemical medium designed for the localization of acid phosphatase activity. Images of acridine-orange-labeled cells showed that most of the intracellular vacuoles were acidic and were also labeled with Lucifer yellow, a fluorescent dye widely used for labeling of compartments of the endocytic pathway. A similar labeling pattern was observed when the cells were incubated in the present of fluorescein-labeled bovine albumin. However, no labeling was observed when fluorescein-labeled transferrin was used. Gold-labeled proteins (albumin, transferrin, horseradish peroxidase, and lactoferrin) were used for further characterization of the endocytic pathway. With the exception of transferrin, all the proteins bound to the protozoan surface and were subsequently internalized, appearing in small peripheral vesicles and some tubular structures. The ingested molecules accumulated in large vesicles located in the more central portion of the cell, which also presented acid phosphatase activity.

Animals↗
PubMed2001

Entamoeba histolytica: production of nitric oxide and in situ activity of NADPH diaphorase in amebic liver abscess of hamsters.

Entamoeba histolytica trophozoites were inoculated into the liver of hamsters and serum nitrate/nitrite levels [expressed as nitric oxide (NO) production] were determined at different times during amebic liver abscess (ALA) development. We also tested the effects of NO synthase (NOS) inhibitors such as N(G)-nitro-L-arginine methyl ester (L-NAME), aminoguanidine, and dexamethasone during ALA production. Since NOS activity has been correlated with expression of reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) in tissues, we performed histochemistry studies to determine the activity of the latter in livers infected with E. histolytica trophozoites. Production of NO in serum was directly proportional to the size of ALAs, and NOS inhibitors caused low levels of NO and smaller ALAs. Our data suggest that NO does not have any lytic effect on E. histolytica trophozoites and is therefore incapable of providing protection against the amebic liver infection. In addition, NADPHd activity was detected histochemically in hepatocytes and inflammatory cells associated with focal necrosis containing trophozoites. The positive reactivity observed in these parasites may be attributable to a close biochemical similarity of NADPHd to the NADPH:flavin oxidoreductase described in E. histolytica by other investigators.

Animals↗
PubMed2001

Protection of hamsters from amebic liver abscess formation by immunization with the 150- and 170-kDa surface antigens of Entamoeba histolytica.

A monoclonal antibody, EH3015, prevents in vitro adherence of Entamoeba histolytica trophozoites to mammalian cells and inhibits amebic liver abscess formation in hamsters. By immunoaffinity chromatography with the monoclonal antibody, purified E. histolytica antigens with molecular masses of 150 and 170 kDa under non-reduced conditions were obtained. Hamsters were immunized with these antigens (group I) or with fractions further purified by polyacrylamide gel electrophoresis (group II). Pooled immune sera from the two groups inhibited in vitro amebic adherence to Chinese hamster ovary cells by 98% at 1:10 dilutions. The immunized hamsters were challenged by the intrahepatic injection of E. histolytica trophozoites. Complete protection from abscess formation was observed in 38% of hamsters in group I and 67% in group II, whereas all control hamsters inoculated only with adjuvant developed amebic liver abscesses. In the immunized hamsters, the abscesses in the two groups were significantly smaller than in the controls. These results demonstrate that the E. histolytica antigens are possible vaccine candidates for amebiasis.

Animals↗
PubMed2001

Inhibition of encystation of Entamoeba invadens by wortmannin.

Using an axenic encystation system in vitro, we examined the effect of wortmannin, a potent inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), which is a signaling molecule responsible for numerous cellular responses, on the encystation of Entamoeba invadens. Wortmannin inhibited both encystation and growth of E. invadens strain IP-1 in a dose-dependent manner, the former being more resistant to the drug than the latter. There was little decrease in the number of trophozoites after 3 days of culture in encystation medium containing wortmannin; and the cells remained motile, suggesting that the inhibitory effect of the drug on encystation was not due to its toxic effect on trophozoites. The addition of wortmannin after the induction of encystation was also inhibitory for encystation. Trophozoites incubated for 1 day in encystation medium with wortmannin did not encyst after removal of the drug, suggesting that the drug effect was not reversible in encystation medium. In contrast, trophozoites cultured in growth medium with wortmannin did encyst after their transfer to encystation medium without the drug. Encystation with wortmannin was more strongly inhibited among trophozoites grown in the presence of the drug than among those grown in the absence of the drug. The process of cyst maturation was slightly affected by wortmannin. These results suggest a possible role for PI 3-kinase in the signaling involved in the encystation of E. invadens.

Androstadienes↗
PubMed2002

DNA polymorphism in north indian isolates of Entamoeba histolytica detected by PCR fingerprinting.

Entamoeba histolytica infection in humans is still one of the leading causes of parasitic diseases among the developing countries, including India. It therefore becomes extremely important to characterize this parasite with the aim of preventing and controlling the amoebiasis which it causes. The present study describes for the first time the ability of random amplified polymorphic DNA (RAPD)-PCR fingerprinting strategies to subtype amoebae and to detect considerable genetic variability present among the various North Indian isolates of E. histolytica studied. The number of rapdemes generated by the application of arbitrary primers in RAPD-PCR showed a significant variation in these isolates, both in the size and number of bands, thus revealing the presence of considerable genetic polymorphism in them. Hence, this rapid and easy method could suitably be employed in carrying out significant molecular epidemiological studies and also in the large-scale epidemiological typing of this parasite.

Animals↗
PubMed2000

Appearance of a stage-specific immunodominant glycoprotein in encysting Entamoeba invadens.

The appearance of cyst-specific proteins in encysting Entamoeba invadens and their immunogenicity were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting using an axenic encystation system in vitro. A rabbit antiserum against trophozoites of E. invadens reacted with a number of proteins of cysts after 1-4 days of incubation. Thus, a number of cyst proteins remained antigenically unchanged as common antigens of the two forms after transformation from trophozoites to cysts. A rabbit antiserum against cysts also reacted with the trophozoite proteins as well as the cyst proteins. The most interesting result was that the rabbit anticyst serum reacted predominantly with an 88-kDa protein of cysts after 1 day of incubation. The 88-kDa protein reacted with the anticyst serum absorbed with trophozoite proteins and was thus cyst-specific. The reactivity of the 88-kDa protein of cysts with the absorbed anticyst serum decreased as encystation proceeded. When soluble and particulate fractions prepared from cysts after 1 day of incubation were examined by electrophoresis and immunoblotting, the 88-kDa protein that had reacted with the absorbed anticyst serum was found to be present in the particulate fraction, which was rich in cell-wall fragments, and stained with periodic acid-Schiff's reagent, indicating that it is a glycoprotein. The results indicate that encystation is accompanied by appearance of the cyst-specific 88-kDa glycoprotein, which is immunodominant and most abundantly expressed in cysts after 1 day of incubation and appears to be associated with the cyst wall.

Animals↗
PubMed2000

Galactose-specific adhesin and cytotoxicity of Entamoeba histolytica.

We examined the molecular mechanisms of the cytotoxicity of Entamoeba histolytica, using the loss of transepithelial electrical resistance (TER) of monolayers of Madin-Darby canine-kidney (MDCK) cells on their incubation with axenic trophozoites of the HM1-IMSS strain. Such loss of TER occurs very early (in 2-5 min) and is caused by the opening of tight junctions and the detachment of cells. We used specific inhibitors for three of the four molecules currently accepted as being responsible for cytotoxicity: galactose-specific adhesin(s), phospholipase A, and cysteine proteinases. We also used inhibitors of calcium channels. Axenic trophozoites of E. histolytica strain HM1-IMSS were preincubated with the different inhibitors for 1 h prior to their coincubation with MDCK-cell monolayers. The only inhibitor that effectively blocked the loss of TER caused by the parasite was galactose. We suggest that in this experimental model, galactose-specific adhesin(s) are essential for amebic cytotoxicity.

Animals↗
PubMed2000

Cloning and characterization of Entamoeba histolytica antigens recognized by human secretory IgA antibodies.

To identify the Entamoeba histolytica antigens capable of inducing secretory IgA (sIgA) responses in humans, a cDNA library from the strain HM1:IMSS was immuno-screened with saliva from patients with intestinal amebiasis or amebic liver abscess. Clones isolated with sIgA antibodies from patients with intestinal amebiasis corresponded to the known serine-rich protein isoform, a 29 kDa cysteine-rich protein and 1-alpha elongation factor. Clones corresponding to enolase, cyclophilin, ribosomal protein L23a, and an Hsp70 family protein were isolated with sIgA from a patient with amebic liver abscess. A glutamic acid-rich peptide (EhGARP) positive with sIgA from a patient with amebic liver abscess was also isolated; for EhGARP, no homologs were found in the protein databases. The antigens isolated are potentially useful in the development of an oral vaccine or new diagnostic tools for amebiasis.

Amino Acid Sequence↗
PubMed1996

Detection and characterization of DNA polymerase activity in Entamoeba histolytica.

DNA polymerase activity was detected and characterized in nuclear extracts from trophozoites of Entamoeba histolytica. The activity was high at pH 2 to pH 6, but at pH 8 and 10 the activity was very low. The presence of K+ was inhibitory for the activity and a higher concentration of K+ markedly inhibited the activity. Magnesium ions (Mg2+) were absolutely required for activity and its optimal concentration was 6 to 8 mM. The activity was markedly inhibited by aphidicolin which is an inhibitor of mammalian DNA polymerases alpha, delta, and epsilon and also by N-ethylmaleimide which is an inhibitor of DNA polymerases, alpha, gamma, delta and epsilon. However, inhibition of the activity by 2', 3'-dideoxythymidine-5'-triphosphate which is an inhibitor of DNA polymerases beta and gamma was relatively weak. Thus sensitivity of the E. histolytica enzyme to these inhibitors was similar to that of mammalian DNA polymerases (alpha, delta and epsilon) of the alpha family. Monoclonal antibodies against human DNA polymerase alpha did not bind to DNA polymerase of E. histolytica.

Animals↗