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PubMed2006

Increased expression of the major cysteine proteinases by stable episomal transfection underlines the important role of EhCP5 for the pathogenicity of Entamoeba histolytica.

The protozoan Entamoeba histolytica causes intestinal inflammation and liver abscess. Cysteine proteinases (CPs) have been proposed as important virulence factors for amoebiasis. To test the role of the various CPs for amoeba induced pathology, the three major enzymes of the parasite, namely EhCP1, EhCP2 and EhCP5 accounting for about 90% of total proteinase activity, were overexpressed by stable episomal transfection. Total CP activity of recombinant amoebae increased by three- to six-fold depending on the gene transfected. Interestingly, overexpression of the genes for EhCP1 or EhCP2 increased the activity of the corresponding enzyme only, whereas overexpression of the gene for EhCP5 increased the activity of all three enzymes, which is consistent with enzyme-converting activity of EhCP5. Cytopathic activity, measured by in vitro monolayer disruption, was dramatically increased in ehcp5-transfectants (five-fold) but showed only a modest increase in ehcp1- or ehcp2-transfectants (1.5-2-fold). In addition, overexpression of ehcp5 but not of ehcp1 or ehcp2 significantly increased amoebic liver abscess formation in laboratory animals. Moreover, transfection and overexpression of ehcp5 was able to compensate the reduction of in vivo pathogenicity in parasites, which have been silenced for the gene encoding the pore-forming protein amoebapore A. In summary, these results further support the important role of EhCP5 in E. histolytica pathogenicity.

Animals↗
PubMed2006

EhGEF2, a Dbl-RhoGEF from Entamoeba histolytica has atypical biochemical properties and participates in essential cellular processes.

Dbl proteins are a family of factors that exchange the guanine nucleotide which promote the activation of Rho small GTPases. This paper reports the molecular, structural, biochemical and functional characterization of EhGEF2, a new member of the Dbl family. EhGEF2 is the second GEF studied in parasites and in the protozoan Entamoeba histolytica, and it is also the first member of the Dbl family that was found to have Arm repeats. The catalytic domain (DH) of EhGEF2 has the conserved residues T421, N590 and E591, which are important for the activation of the GTPases. Biochemical studies on EhGEF2 showed that it could activate in vitro the amoebic GTPases EhRacA, EhRacB, EhRacC, EhRacD, EhRacG, EhRacH and EhCdc42, being EhRacG its main target. It was found that the DH domain binds specifically phosphatidic acid (PA); docking and lipid dot blot studies indicated that this binding does not interfere with the contact surface of EhRacG. Functional studies showed that both the Arm repeats and the catalytic domain of EhGEF2 participate in its localization at the amoebic membrane. Expression of a negative dominant version of EhGEF2 protein in E. histolytica provoked a 30% decrease in its ability to phagocyte human erythrocytes as well as severe effects on both the proliferation and the cellular chemotaxis which suggest that EhGEF2 participates in these cellular processes.

Amino Acid Motifs↗
PubMed2006

SINE elements of Entamoeba dispar.

Entamoeba histolytica and E. dispar are closely related protozoan parasites; the former causes clinical amoebiasis in humans while the latter appears to be non-pathogenic. The molecular biology of E. histolytica shows a number of unusual features, one of which is the abundance of polyadenylated but apparently untranslatable mRNAs produced; many of these are the product of at least three families of SINEs (EhSINE1-3). Here we show that the genome of E. dispar contains numerous copies of a SINE element (EdSINE1) whose 5'- and 3'-ends are very similar to those of EhSINE1 but with a much less similar middle portion. Twelve out of 18 copies examined had target site duplications. In none out of six cases examined was there a SINE element in the homologous region of the E. histolytica genome but a single copy of EdSINE1 is present in E. histolytica where it is identified as EhSINE3.

Amino Acid Sequence↗
PubMed2004

In vitro and in vivo interaction of Entamoeba histolytica Gal/GalNAc lectin with various target cells: an immunocytochemical analysis.

The Gal/GalNAc lectin of Entamoeba histolytica trophozoites plays an important role in adhesion. The distribution and final destiny of the lectin during the interaction with host cells are poorly understood. Using monoclonal and polyclonal antibodies against the lectin we studied by immunocytochemistry the in vitro and in vivo interaction of E. histolytica trophozoites with human and hamster hepatocytes. We also analyzed the presence and distribution of the lectin in a mouse model of intestinal amoebiasis. In all cases, trophozoites were highly labeled by anti-lectin antibodies. Cultured human and hamster hepatocytes in contact with, or localized at the vicinity of parasites were also labeled by anti-lectin antibodies. Most of the labeled hepatocytes showed variable degrees of cell damage. Hepatocytes distantly localized from the parasites were also stained with the anti-lectin antibodies. Immunolabeling of tissue sections from different stages of the development of experimental amoebic liver abscess in hamsters showed inflammatory foci containing lectin-labeled trophozoites, hepatocytes, and sinusoidal and inflammatory cells. Lectin-containing hepatocytes had vacuolated cytoplasm with some nuclei with a condensed appearance. Damaged intestinal epithelium also was labeled with anti-lectin antibodies in a mouse model of intestinal amoebiasis. Electron microscopy of axenically cultured trophozoites using gold-labeled monoclonal and polyclonal anti-lectin antibody showed that plasma membrane, vacuole membranes and areas of cell cytosol were labeled. Higher deposits of gold particles in plasma membrane suggestive of cell secretion were observed. Our results demonstrated that Gal/GalNAc lectin was bound and captured by different target cells, and that host cells containing the lectin showed signs of cell damage. The contribution of lectin transfer to host cells in adherence and cell injury remains to be determined.

Animals↗
PubMed2005

Isolation and functional analysis of a chk2 homologue from Entamoeba histolytica.

Mammalian Chk2 is a Ser/Thr kinase required for cell-division arrest induced by DNA damage. We found six new kinase genes of Entamoeba histolytica by analysis in silico. One of the kinase genes was a homologue of human chk2 gene. The chk2 homologue gene (Eh chk2) was expected to encode 398 amino acids and showed nearly 50% homology to human Chk2 in amino acid sequence. Eh chk2 had a catalytic domain of Ser/Thr kinase and a fork head-associated (FHA) domain that is highly conserved among Chk2 homologues in vertebrates. To examine the biological functions of Eh chk2, we synthesized Eh chk2 mRNA in vitro and injected it into immature frog eggs (Xenopus laevis oocytes) as a model system of cell division. Eh chk2 markedly delayed the cell division of frog eggs by disrupting transition of G2 phase to M phase. Eh chk2 also inhibited the activation of p42 MAPK and Cdc2 kinase which are representative events induced by cell division. These results suggest that Eh chk2 gene should be a cell-division regulator in E. histolytica.

Amino Acid Sequence↗
PubMed2006

Isolation of pure compound R/J/3 from Pluchea indica (L.) Less. and its anti-amoebic activities against Entamoeba histolytica.

The plant Pluchea indica is known for its anti-inflammatory, anti-ulcer, anti-pyretic, hypoglycemic, diuretic and anti-microbial activities besides many other pharmacological activities. We have isolated and purified seven compounds from the methanolic root extract of this plant by column chromatography. The compounds were identified by spectroscopic analyses. The anti-amoebic activities of the pure compound R/J/3 was investigated against the HM1 strain of Entamoeba histolytica. The compound, R/J/3 showed the most pronounced anti-proliferative activity at a dose of 50 microg/ml. It also showed a marked activity on cell lysis of trophozoites, 4h after administration. The cell lytic activity was compared with metronidazole (5 microg/ml) as positive control.

Animals↗
PubMed2005

Risk factors for infection by the Entamoeba histolytica/E. dispar complex: an epidemiological study conducted in outpatient clinics in the city of Manaus, Amazon Region, Brazil.

An epidemiological study was conducted on a population attending outpatient clinics in Manaus, Amazon, Brazil to determine the prevalences of infection by the Entamoeba histolytica/E. dispar complex and by E. histolytica alone, as well as to identify the risk factors involved in transmission. The study was conducted in two phases: survey and case-control. Face-to-face interviews were carried out and faecal samples collected from 1578 individuals. Faeces were examined by optical microscopy and tested for the pathogenic E. histolytica specific antigen. Positivity to E. histolytica/E. dispar was 21.5% (340 cases). Cases were compared with 340 control samples, negative for the E. histolytica/E. dispar complex based on examination by optical microscopy. The analysis was conducted by logistic regression. The risk factors identified were: place of residence, age, ingestion of raw vegetables, quality of water consumed, number of rooms and bedrooms per house, and having other protozoan infections. Specific antigen detection tests identified 22 participants infected by E. histolytica (6.8%) among those positive for E. histolytica/E. dispar. There was a higher proportion of males among participants infected by E. histolytica than among those with E. dispar infections. The study population was asymptomatic or presented non-specific symptoms that could be attributed to amoebiasis.

Adult↗
PubMed2004

Prevention of intestinal amebiasis by vaccination with the Entamoeba histolytica Gal/GalNac lectin.

Prevention of intestinal infection by Entamoeba histolytica would block both invasive disease and parasite transmission. The amebic Gal/GalNAc lectin mediates parasite adherence to the colonic surface and fecal anti-lectin IgA is associated with protection from intestinal reinfection in children. We tested if vaccination with the E. histolytica Gal/GalNAc lectin could prevent cecal infection in a C3H mouse model of amebic colitis. Two trials using native lectin purified from the parasite and two trials using a 64 kDa recombinant fragment ("LecA") were performed with a combined intranasal and intraperitoneal immunization regimen using cholera toxin and Freund's adjuvants, respectively. Two weeks after immunization mice were challenged intracecally with trophozoites, and 4-12 weeks after challenge mice were sacrificed for histopathologic evaluation of infection. Vaccination prevented intestinal infection with efficacies of 84 and 100% in the two native lectin trials and 91 and 34% in the two LecA trials. Mice with detectable pre-challenge fecal anti-lectin IgA responses were significantly more resistant to infection than mice without fecal anti-lectin IgA responses. These results show for the first time that immunization with the Gal/GalNAc lectin can prevent intestinal amebiasis in mice and suggest a protective role for fecal anti-lectin IgA in vivo.

Administration, Intranasal↗
PubMed2003

Cockroaches (Periplaneta americana and Blattella germanica) as potential mechanical disseminators of Entamoeba histolytica.

Since cockroaches feed on feces, they may disseminate infective agents with the fecal-oral route. A field survey and experiments were conducted to determine the role of cockroaches in the dissemination of Entamoeba histolytica. Periplaneta americana (n=299) and Blattella germanica (n=29) were collected from 11 primary schools in an urban area of South Taiwan. E. histolytica/E. dispar cysts were found on the cuticle and/or in the digestive tract of P. americana (25.4%) whereas cysts were only isolated from the digestive tract of B. germanica (10.3%). E. histolytica cysts were found on the cuticle and/or in the digestive tract of both species after exposure to 1000 or 100 cysts/g feces for 24 h. These findings indicate that cockroaches may harbor the E. histolytica cysts and play a role as potential mechanical disseminators.

Animals↗
PubMed2000

RAPD in the analysis of isolates of Entamoeba histolytica.

Genetic variability in Entamoeba histolytica was analyzed by random amplified polymorphic DNA (RAPD) using ten arbitrary primers. Due to intrinsic characteristics of the RAPD technique only axenic samples were analyzed. since the presence of any microorganism in the cultures interfered in the DNA profile by generating RAPDs not pertaining to E. histolytica. The RAPD profiles of E. histolytica samples isolated from patients with different clinical manifestations from different regions of the Americas shared about 70% of the bands produced. These profiles were compared to those obtained for E. moshkorskii, and E. invadens. The combined data for the ten primers were used in the phenetic analysis of all the isolates studied by using the Dice similarity coefficient as the genetic distance measure between the samples. Three distinct groups could be separated by phenon line: one including E. moshkovskii samples, which shared > 90% of the RAPDs produced by the different primers; one consisting solely of E. invadens; and a third comprising samples of E. histolytica, which showed considerable intraspecific variability.

Animals↗
PubMed2001

Novel putative saposin-like proteins of Entamoeba histolytica different from amoebapores.

Amoebapores, the pore-forming proteins of Entamoeba histolytica, have been shown to play a pivotal role in the pathogenicity of the protozoan parasite. They belong to the functionally diverse family of saposin-like proteins (SAPLIPs) characterized by a conserved pattern of cysteine residues and the ability to interact with lipids. Here, we report the identification of genomic sequences encoding presumably novel SAPLIPs in E. histolytica and classify them in the structural and functional context provided by known family members. The genes of altogether 15 SAPLIPs are transcribed in the axenically cultured trophozoites as evidenced by reverse transcriptase-polymerase chain reaction. Interestingly, a remarkable sequence variety with a strong resemblance to that of known, functionally diverse SAPLIPs is present in this archaic, unicellular organism.

Amino Acid Sequence↗
PubMed2002

Entamoeba histolytica: immunohistochemical study of hepatic amoebiasis in mouse. Neutrophils and nitric oxide as possible factors of resistance.

Studies in mice have not rendered conclusive data on cell and humoral factors to support the resistance of this rodent to Entamoeba histolytica infection. In Balb/c and C3H/HeJ mice inoculated with live or fixed trophozoites, we studied the evolution of the hepatic lesion, the kinetics of inflammatory cells, and the participation of some humoral factors in the development of the hepatic amoebic lesion. From the first hour, amoebae were surrounded by neutrophils containing inducible nitric oxide synthase (iNOS); macrophages also expressing iNOS appeared lately, whereas NK cells were not part of the inflammatory infiltrates. On the fourth day, neutrophils, macrophages, T and B lymphocytes, plasma cells, and some NK cells limited the lesions and anti-amoeba antibodies appeared when most parasites had been eliminated. Therefore, the resistance of the mice to E. histolytica probably lies in non-specific immune responses, among which the activation of neutrophils and the production of nitric oxide (NO) may be important amoebicide factors.

Animals↗
PubMedDate not supplied

A Rho-like small GTPase of Entamoeba histolytica contains an unusual amino acid residue in a conserved GDP-stabilization region and is not a substrate for C3 exoenzyme.

The Entamoeba histolytica small GTP-binding protein EhRho1 has an unusual amino acid residue at a conserved site found in all known Ras superfamily proteins. EhRho1 has an isoleucine at position 45, which corresponds to position 28 of human Ras and Rac and position 30 of human Rho and Cdc42. All other known small GTPases have an aromatic residue (typically phenylalanine) at this position, and mutation to a leucine renders other Ras proteins constitutively active by reason of diminished affinity for GDP. It was determined that the EhRho1 protein has a half-time of GDP dissociation similar to that of a human Rho protein, HsRhoA, and therefore an isoleucine at this site in EhRho1 is not likely to render EhRho1 constitutively active. It was also found that EhRho1 is not a substrate for the Rho-specific C3 exoenzyme. Thus EhRho1 appears to be an unusual member of the Ras family.

ADP Ribose Transferases↗
PubMed2002

Entamoeba histolytica: acute granulomatous intestinal lesions in normal and neutrophil-depleted mice.

To study the role of neutrophils in the innate resistance to Entamoeba histolytica intestinal infection in mice, animals were treated with anti-neutrophil monoclonal antibodies prior to intracecal parasite inoculation and the resulting lesions were compared with normal mice that had been equally infected. In contrast to our previous finding that neutrophils are critical in eliminating E. histolytica infection in the liver, we show here that neutrophils are not absolutely required to eliminate E. histolytica infection from the intestine. Although the neutrophils are not critical for resolution of the E. histolytica infection, neutrophils do appear to provide some measure of protection as the intestinal amoeba burden was higher at early timepoints after infection in the neutropenic animals. In addition, we found that while both the normal and the neutrophil-depleted mice developed ulcerative lesions in the colon, the neutropenic mice had an increased frequency of granulomas that formed around the amoeba. Thus, our findings appear to be the first evidence showing that granulomatous inflammation can occur after intestinal infection in mice using axenically cultured amoeba.

Animals↗
PubMedDate not supplied

Entamoeba histolytica: sequence conservation of the Gal/GalNAc lectin from clinical isolates.

The Gal/GalNAc lectin gene of Entamoeba histolytica is a major amebic virulence protein responsible for interaction with host tissues. We investigated sequence differences in the Gal/GalNAc lectin heavy subunit in three isolates from Bangladesh and one isolate from Georgia, each of which was determined to be genetically distinct by SREHP AluI digestion. Interestingly, we observed only slight genetic diversity in the lectin gene as compared with the HM1:IMSS laboratory strain, originally a clinical isolate from Mexico. Genetic conservation of the Gal/GalNAc lectin between isolates may reflect that the lectin is under strong functional selection or possibly, that E. histolytica is a clonal population. Sequence conservation of the lectin indicates that immune responses against it should be cross-protective.

Amino Acid Sequence↗
PubMed2002

Entamoeba histolytica: rapid identification and differentiation of Indian isolates by riboprinting.

Entamoeba histolytica infection still remains one of the major public health problem for developing countries like India. A rapid and accurate detection of this parasite is essential for prevention and control of amoebiasis. In this study, using the method of 'riboprinting' (PCR-RFLP of rRNA genes from amoeba) we have analysed 15 stool samples from symptomatic patients of amoebiasis. All 15 patients of clinical amoebiasis had E. histolytica in their stool and two of the samples also showed mixed infection of E. dispar. Apart from the known restriction enzyme sites within the amoeba SSU-rRNA genes, a new Sau3A site having a discriminatory value is identified in these E. histolytica isolates from India. Hence, it is possible to rapidly identify E. histolytica DNA and differentiating it from E. dispar using minute amounts of clinical stool samples, thus eliminating the laborious parasite culturing process. Thus, riboprinting is advantageous for clearcut identification of E. histolytica in order to decide an effective antiamoebic therapy.

Animals↗
PubMedDate not supplied

Entamoeba histolytica: intracellular distribution of the proteasome.

We have studied the intracellular distribution of proteasome subunits, corresponding to the catalytic (20S) core and the regulatory (19S) cap, in the extracellular protozoan parasite Entamoeba histolytica. Contrary to all cell types described to date, notably mammalian and yeast, in which the proteasome is found in the nucleus and actively imported into it, microscopic analysis and subcellular fractionation of E. histolytica trophozoites show that the proteasome is absent from the nucleus of these cells. We speculate that, given the relative abundance of mono- and multinucleated trophozoites in culture, a relationship may exist between this unusual distribution of the proteasome and the frequent lack of synchrony between karyo- and cytokinesis in this primitive eukaryote.

Animals↗