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PubMed2002

Structure and inactivation of triosephosphate isomerase from Entamoeba histolytica.

Triosephosphate isomerase (TIM) has been proposed as a target for drug design. TIMs from several parasites have a cysteine residue at the dimer interface, whose derivatization with thiol-specific reagents induces enzyme inactivation and aggregation. TIMs lacking this residue, such as human TIM, are less affected. TIM from Entamoeba histolytica (EhTIM) has the interface cysteine residue and presents more than ten insertions when compared with the enzyme from other pathogens. To gain further insight into the role that interface residues play in the stability and reactivity of these enzymes, we determined the high-resolution structure and characterized the effect of methylmethane thiosulfonate (MMTS) on the activity and conformational properties of EhTIM. The structure of this enzyme was determined at 1.5A resolution using molecular replacement, observing that the dimer is not symmetric. EhTIM is completely inactivated by MMTS, and dissociated into stable monomers that possess considerable secondary structure. Structural and spectroscopic analysis of EhTIM and comparison with TIMs from other pathogens reveal that conformational rearrangements of the interface after dissociation, as well as intramonomeric contacts formed by the inserted residues, may contribute to the unusual stability of the derivatized EhTIM monomer.

Animals↗
PubMed1999

Different antiamebic antibody isotype patterns in the large and small intestine after local and systemic immunization of mice with glutaraldehyde fixed Entamoeba histolytica trophozoites.

We have determined the major immunoglobulin isotypes (IgG, IgA, IgM) of antiamebic antibodies induced in the serum and in the large and small intestine after local (oral and rectal) or systemic (intraperitoneal and intramuscular) immunization of mice with glutaraldehyde-fixed Entamoeba histolytica trophozoites (GFT). IgA predominated in the small intestine after immunization through all routes, whereas in the large intestine similar antibody levels of the major isotypes were induced by rectal, intraperitoneal and intramuscular immunization. The intramuscular route elicited intestinal responses lower than those induced by the rectal and intraperitoneal routes, but higher than the slight IgA antibody increase observed after oral immunization. The differences in antiamebic antibody response patterns at the large and small intestine suggest that there are different mucosal effector compartments. They also indicate that isotype analysis of mucosal antibodies from the sites where an infectious agent resides is needed to evaluate whether a vaccine candidate induces responses of higher protective value in the appropriate site, and that the study of antibody responses must not be limited to sampling the serum or mucosal sites distant to the relevant one.

Animals↗
PubMed1985

Entamoeba polecki infection in Southeast Asian refugees: multiple cases of a rarely reported parasite.

Recently, Entamoeba polecki was identified for the first time in our parasitology laboratory in stool specimens from eight Southeast Asian refugees. This ameba has been reported infrequently in the Western world; most reported cases have been from the New Guinea region. In most previously described patients and in our patients, no definite gastrointestinal symptoms could be directly attributed to E. polecki infection. Morphologically, E. polecki may mimic the pathogen E. histolytica and also nonpathogens such as E. coli. These species are most readily distinguished by studying encysted forms. In contrast to E. histolytica and E. coli, E. polecki characteristically has uninucleate cysts. Both pigs and monkeys naturally harbor E. polecki, but four of the patients in this series had no apparent contact with these animals. Other modes of infection may be human-to-human transmission or acquisition from other domestic animals. Six of our eight patients were treated successfully with one course of metronidazole in a regimen similar to that used for E. histolytica infection. In the two other patients, repeated courses of therapy eradicated the infection. Because of the recent increase in number of Southeast Asian immigrants to the United States, E. polecki may be identified more frequently than in the past. Physicians and laboratory personnel should be familiar with this organism, because it may be confused with E. histolytica or may act as a pathogen.

Adolescent↗
PubMedDate not supplied

Use of the ProSpecT microplate enzyme immunoassay for the detection of pathogenic and non-pathogenic Entamoeba histolytica in faecal specimens.

A commercial microplate enzyme immunoassay (ProSpecT EIA; Alexon Inc., Sunnyvale, CA 94089, USA) was compared with conventional microscopy for the diagnosis of Entamoeba histolytica infection. Using specimens known to be infected, the sensitivity of the ProSpecT EIA was 78% and its specificity was 99%. No cross reaction with other intestinal parasites was observed. The ProSpecT EIA and conventional microscopy (using merthiolate-iodine-formaldehyde direct wet mounts and concentration techniques) were then used to detect E. histolytica infections in 431 patients in a mental hospital in Taiwan. Using single stool specimens, microscopy detected infection in 10.9% of the patients, compared with 16.9% detected by ProSpecT EIA. The latter method was simple and quick, but more expensive, and could be used to complement microscopy if a prompt diagnosis is desired clinically. However, ProSpecT EIA cannot differentiate between pathogenic E. histolytica and non-pathogenic strains (= E. dispar), which limits its usefulness.

Animals↗
PubMed2002

Structure and function of the Entamoeba histolytica Gal/GalNAc lectin.

Gal/GalNAc lectin is a novel multifunctional virulence factor of the human parasite Entamoeba histolytica. The native protein is a 260-kDa heterodimer consisting of a type 1 membrane protein disulfide bonded to a lipid-anchored protein. Each subunit has several isoforms that may form functionally different heterodimers, analogous to the integrin family of proteins. Recently a second 150-kDa Gal/GalNAc lectin has been identified in E. histolytica that associates with the 260-kDa lectin. The functions of the 260-kDa lectin have been characterized using specific monoclonal antibodies. This lectin plays roles in many of the critical aspects of this parasite's pathogenicity including adherence, cytolysis, invasion, resistance to lysis by complement, and also perhaps encystment. Current knowledge regarding both the structure and function of this unique multifunctional virulence factor are discussed.

Acetylgalactosamine↗
PubMed1982

Biochemical homogeneity of Entamoeba histolytica isolates, especially those from liver abscess.

The electrophoretic mobility patterns of four enzymes from trophozoites of Entamoeba histolytica were examined to determine the zymodeme of the amoebae populations involved. E. histolytica populations from aspirated liver pus belonged exclusively to types II and XI of eighteen zymodemes. E. histolytica from cases of amoebic dysentery--i.e., those patients presenting with intestinal ulceration and/or with haematophagous trophozoites in the faeces--also belonged to zymodemes II and XI. However, symptom-free cyst passers harboured E. histolytica belonging to one of several zymodemes none of which corresponded wth the zymodemes of the patients with liver abscesses or amoebic dysentery.

Dysentery, Amebic↗
PubMed1984

Entamoeba histolytica cyst passers: clinical features and outcome in untreated subjects.

To examine whether Entamoeba histolytica is the cause of a variety of intestinal disorders, as often implicated, a two-part study was conducted. In part 1, 184 patients attending a gastroenterology clinic and needing a sigmoidoscopy had a rectal biopsy specimen and a stool sample cultured for E histolytica. Part of the biopsy specimen was examined histologically. In addition, serum was tested for antibody to E histolytica. 34 (18.7%) patients were culture positive. There was no correlation between the culture results and patients' symptoms except for diarrhoea, which was more common in the culture-negative group, or with histological or serological findings. In the second part of the study, 15 culture-positive patients were followed up untreated for a mean (+/- SD) of 8.6 (+/- 4.9) months, range 1-18. In all 15 patients spontaneous eradication of the parasite occurred, and in none did symptoms of invasive amoebiasis develop. Thus E histolytica seems not to be responsible for many of the bowel complaints commonly attributed to it. In these cyst passers the cysts are probably incidental to an irritable bowel syndrome or some other organic disease. There seems to be little justification for treating cyst passers since eradication of the parasite occurs spontaneously and the danger of invasive amoebiasis is negligible. Such a practice would result in an enormous saving on the cost of drugs.

Amebiasis↗
PubMed1985

Seroepidemiological study of antibody responses to the zymodemes of Entamoeba histolytica.

In 303 apparently healthy subjects and 54 patients with confirmed invasive amoebiasis amoebae were isolated where possible, and Entamoeba histolytica was classified into its various zymodems. 20% of the healthy subjects were antibody-positive; 17 healthy subjects proved to be symptomless carriers of pathogenic zymodemes and, like patients with invasive disease, all these were seropositive. Furthermore, of subjects infected with pathogenic zymodemes 94-100% were strongly seropositive, compared with 2-4% of subjects with non-pathogenic zymodemes. Pathogenic zymodemes are therefore believed to be in constant contact with the host's tissues, even in symptom-free individuals; the presence of these potentially invasive organisms is thought to be directly related to the seropositivity in a given population, and seroepidemiological surveys would therefore provide information only on the distribution of this form of the parasite. Quantitative serological data constitute valuable information on the endemicity of an area, and the results will gain reliability from use of two complementary tests instead of one.

Antibody Formation↗
PubMed1985

Frequency distribution of Entamoeba histolytica zymodemes in a rural South African population.

To determine the prevalence of Entamoeba histolytica and its zymodeme distribution pattern, isoenzyme electrophoresis was performed on isolates of the parasite from 1381 individuals of the population of a semirural endemic area south of Durban. About 10% of the population was found to harbour E histolytica. The zymodeme distribution was similar to that described in other surveys but two new zymodemes were observed, a non-pathogenic zymodeme XIX and a pathogenic zymodeme XX. Non-pathogenic zymodemes were more frequent in females than in males. In apparently healthy individuals there was a 1% prevalence of pathogenic E histolytica; the distribution was the same in males and females even though invasive amoebiasis was far more common in males. Symptoms of amoebic colitis subsequently developed in only 2 of the 14 symptomless individuals from whom pathogenic zymodemes of E histolytica were isolated.

Adolescent↗
PubMed1986

Is Entamoeba histolytica in homosexual men a pathogen?

Entamoeba histolytica (EH) in homosexual men is generally considered to be pathogenic. To test this hypothesis, the generally accepted features of invasion (haematophagous trophozoites in faeces; high-titre serum antibody; moderate to severe acute inflammatory change; and presence of EH in the mucosa on rectal biopsy) and the zymodeme pattern of cultured trophozoites were assessed in twenty-three EH excretors and eleven control homosexual men. No trophozoites or antibody to EH were found in either group. When other pathogens were excluded, no patient in either group had severe, acute histopathological proctitis. Moderately severe change was seen in 38% of EH excretors and 18% of controls (not significant). All the zymodemes were non-pathogenic. Successful eradication of EH did not result in even a trend towards normalisation of the moderate inflammatory histopathology. There are, therefore, no data here to suggest that EH is a pathogen in homosexual men.

Adult↗
PubMed1988

Immunological differentiation of pathogenic and non-pathogenic isolates of Entamoeba histolytica.

Entamoeba histolytica can act as a harmless commensal organism in the lumen of the large intestine, or can cause invasive amoebiasis. Some workers have suggested that there are two distinct subspecies of this organism, and that only one of these is associated with invasive disease. Present isoenzyme tests to identify the subspecies take several days to analyse: we report a technique that uses immunofluorescence with monoclonal antibodies, takes two days to perform, and may, therefore, assist in the clinical management of patients infected with this organism.

Animals↗
PubMed2000

Cloning, characterization and functional expression of a cyclophilin of Entamoeba histolytica.

Full-length Entamoeba histolytica cyclophilin gene (EhCyp) was isolated, characterized and recombinantly expressed in bacterial cells. The deduced amino acid sequence of EhCyp shows 60-70% identity with cyclophilins from other organisms and has conserved the cyclophilin signature motifs and residues involved in cyclosporin A binding. Upstream of the 501 bp open reading frame of EhCyp, sequences resembling the putative consensus E. histolytica CE1, CE2 and CE3 regulatory elements were found. Northern blot assays revealed a single transcript of 0.63 kb. The transcription start was determined by primer extension at position -13 relative to the initial ATG codon. Cyclosporin A binding and peptidyl-proplyl cis-trans isomerase activities characteristic of cyclophilin were detected in soluble extracts of E. histolytica trophozoites and in the recombinant protein. In both cases, the isomerase activity was inhibited by nanomolar concentrations of cyclosporin A. Treatment of cultured trophozoites with cyclosporin A decreased their proliferation with a 50% inhibition value of 1 microg/ml and was lethal in doses over 50 microg/ml.

Amino Acid Sequence↗
PubMed2000

An Entamoeba histolytica rab-like encoding gene and protein: function and cellular location.

We identified here a 576 bp rab-like gene (EhrabB) in Entamoeba histolytica. EhrabB is located 332 bp upstream from the start codon of the Ehcp112 encoding gene, but is transcribed from the complementary strand. The EhrabB open reading frame predicts a 192 amino acid polypeptide (EhRabB) with 40-42% identity to Rab proteins, involved in vesicle docking regulation in endo and exocytic pathways of eukaryotic cells. Transcripts of 0.6 and 0.97 kb were detected by the EhrabB probe in northern blot assays. Using specific antibodies, EhRabB was located in small cytoplasmic vesicles by confocal microscopy. During phagocytosis, EhRabB was initially translocated to the plasma membrane and to the phagocytic mouths. The protein diminished after 10 min phagocytosis, suggesting that EhRabB could be participating in the regulation of the endocytosis process.

Amino Acid Motifs↗
PubMed2001

A novel anti-inflammatory oligopeptide produced by Entamoeba histolytica.

The monocyte locomotion inhibitory factor (MLIF), a heat-stable oligopeptide found in the supernatant fluid of Entamoeba histolytica axenic cultures was isolated by ultra-filtration, gel-sieve chromatography and high powered liquid chromatography (HPLC), and its primary structure (Met-Gln-Cys-Asn-Ser) established by Edman sequencing and mass-spectrometry (MS). A synthetic peptide had the same selective anti-inflammatory features as the native material in comparable concentrations: in vitro inhibition of the locomotion in human peripheral blood monocytes, and of the respiratory burst in the same cells and in human neutrophil polymorphonuclear leucocytes; and in vivo depression of delayed hypersensitivity skin reactions to dinitrochlorobenzene in guinea pigs. This oligopeptide is apparently synthesized by the ameba as suggested by [(35)S]-Cys and Met incorporation, probably as part of a larger molecule, from which it is cleaved by proteolysis. The full sequence was not found in the 431 available E. histolytica protein sequences. The factor may contribute to the unexpected paucity of the late inflammatory reaction found in advanced invasive amebiasis and, perhaps in consequence, to the regeneration without scarring (restitutio ad integrum) of the affected organs that is observed following successful treatment of this disease

Adult↗
PubMed2002

Cysteine proteinases from distinct cellular compartments are recruited to phagocytic vesicles by Entamoeba histolytica.

Cysteine proteinases, which are encoded by at least seven genes, play a critical role in the pathogenesis of invasive amebiasis caused by Entamoeba histolytica. The study of these enzymes has been hampered by the inability to obtain significant quantities of the individual native proteinases. We have now expressed functionally active recombinant ACP1 (EhCP3) and ACP2 (EhCP2) proteinases in baculoviral expression vectors. The purified recombinant ACP1 and ACP2 proteinases exhibited similar activities for fluorogenic peptide substrates, especially in their preference for an arginine residue at the P2 position. Although ACP1 and ACP2 are structurally cathepsin L, homology modeling revealed that the aspartic acid in the S2 pocket would result in a substrate specificity for positively charged amino acids, like cathepsin B. The hydrolysis of peptide substrates was strongly inhibited by small peptidyl inhibitors specifically designed for parasitic cysteine proteinases. Confocal and immunoelectron microscopy localization of the proteinases with monoclonal and monospecific antibodies raised to the recombinant enzymes and peptides demonstrated that ACP2 was membrane-associated while ACP1 was cytoplasmic. Following phagocytosis of erythrocytes, ACP1, as well as the membrane-associated cysteine proteinase, ACP2, were incorporated into phagocytic vesicles. These studies suggest that E. histolytica has a redundancy of cysteine proteinases for intracellular digestion and that they may be recruited from different cellular compartments to the site of digestion of phagocytosed cells. The production of active proteinases in baculovirus and large scale recombinant enzymes in bacteria should further our understanding of the role of different cysteine proteinase gene products in virulence.

Animals↗
PubMed2002

Context-dependent roles of the Entamoeba histolytica core promoter element GAAC in transcriptional activation and protein complex assembly.

Transcriptional control of the hgl5 gene of Entamoeba histolytica is mediated through an unusual core promoter composed of TATA, GAAC and Initiator elements. In the hgl5 promoter the GAAC element (AATGAACT) determines the site and rate of transcription initiation. Here we tested the role of the GAAC element in transcription activation from upstream regulatory elements (UREs) in the hgl5 promoter. We also examined the function of the GAAC element in the ferredoxin (fdx) promoter and characterized the protein binding to the GAAC element. Electrophoretic mobility shift assays (EMSA) demonstrated that the GAAC region is necessary for higher-order nuclear protein complex assembly. The function of the GAAC element in transcription activation mediated by UREs revealed that mutation of the GAAC element did not affect transcription activation mediated by the hgl5 URE4 but abrogated activation by the hgl5 URE3. We compared the role of the GAAC elements in the hgl5 and fdx promoters. Competitive gel shift assays were consistent with the same nuclear protein binding to the GAAC elements in both genes. Mutation of the GAAC element in the fdx gene decreased reporter gene expression, however, in contrast to hgl5 gene, had no effect on the site of transcription initiation. These results support a role for the GAAC element in assembly of nuclear proteins at the core promoter and in transcription activation mediated by URE3. The differing effect on transcription initiation in the hgl5 and fdx genes upon mutation of the GAAC element suggests a context-dependence of the GAAC-binding protein in gene expression.

Animals↗
PubMed2002

Characterization of a Rab7-like GTPase, EhRab7: a marker for the early stages of endocytosis in Entamoeba histolytica.

We have identified a 667 base pair Rab7-like cDNA (EhRab7) from Entamoeba histolytica. The EhRab7 cDNA predicts a polypeptide of at least 206 amino acids with a molecular mass of at least 24.5 kDa. Alignment of EhRab7 with other Rab proteins demonstrated that EhRab7 shared significant homology at the amino acid level with Rab7-like proteins from a number of other eukaryotes, suggesting that EhRab7 is a Rab7 homolog for E. histolytica. Using immunofluorescence microscopy, EhRab7 was demonstrated to be associated with early fluid-phase endosomes (<30 min) and secretory vesicles. The association of EhRab7 with early endosomes disappeared 1 h after their formation. Immunofluorescence microscopy also revealed that this GTPase did not colocalize significantly with phagosomes nor with markers for other organelles including the endoplasmic reticulum, Golgi and late endosomes. These results, together with the known function for Rab7 in other systems, suggest that EhRab7 is bound to vesicles, and that it may participate in vesicle docking and fusion in secretory events, and in the early stages of fluid-phase endocytosis in E. histolytica.

Amino Acid Sequence↗