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Two major zymodemes from different Entamoeba histolytica strains were detected, despite different growth conditions, using isoelectrofocusing in ultrathin gels. One was associated with strains cultured from symptomatic patients, while the other was associated with microorganisms obtained from clinically symptomatic persons.
The action of antiamoebic drugs on the incorporation of (14C) 1-DL-valine and (14C)-8 adenine in TCA insoluble fraction of axenic Entamoeba histolytica was investigated. Adenine showed greater sensitivity to all the drugs tested viz. camoquin, chloroquine, diodoquin, metronidazole, entobex and diloxanide furoate except enterovioform.
The development of a vaccine against Entamoeba histolytica, the causative agent of amebic colitis and liver abscess, would reduce childhood mortality in countries such as Bangledesh where community-based studies have demonstrated a high prevalence of E. histolytica. Immunological studies from this population have shown that protection from amebiasis is associated with mucosal anti-E. histolytica Gal/GalNAc lectin antibodies, suggesting that a vaccine is an achievable goal. However, garnering resources for vaccine development is a challenge when the vaccine is targeted to poor people living in developing countries.
The human pathogenic protozoan Entamoeba histolytica is a motile cell polarized into a front pseudopod and a rear uroid. The amoebic Gal/GalNAc surface lectin is a major adhesion molecule composed of an immunodominant 170-kDa heavy subunit, mostly extracellular except for a short cytoplasmic tail, and of an extracellular light subunit. The binding of multivalent ligands triggers lectin capping and recruitment to the uroid. The properties of the Gal/GalNAc lectin and its role in amoeba adhesion and uroid polarization are reviewed in the context of the molecular mechanisms underlying cell polarization and locomotion.
Gastrointestinal protozoa, viz. Entamoeba histolytica and Giardia, are able to survive in a microaerobic environment. The role of parasitic factors, particularly cysteine, cysteine-rich proteins, superoxide dismutase, and certain alternative mechanisms, has been described in the defence against oxidative stress. The role of the host-derived factors, particularly the phagocytosis of bacteria or host erythrocytes (intact or their enzymatic/non enzymatic components), in the detoxification of reactive oxygen metabolites in E. histolytica may provide novel approaches for the chemotherapy of invasive amoebiasis.
Recent studies have increased our knowledge of Entamoeba histolytica cell biology and gene regulation. In the ameba, dominant-negative mutations in the Gal/GalNAc lectin affect adhesion and cytolysis, whereas mutations in meromyosin affect cytoskeletal function. Studying these mutant proteins has improved our understanding of the role of these proteins in E. histolytica virulence. The characterization of the CP5 cysteine protease and the induction of apoptosis in host target cells has led to a better comprehension of the mechanisms by which trophozoites can lyse target cells.
One of the major soluble antigenic proteins of Entamoeba histolytica was purified to homogeneity and identified on a molecular basis. Its recombinant protein was expressed in Escherichia coli as a fusion protein with Shistosoma japonicum glutathione S-transferase. Apparent molecular weight of the purified antigenic protein was estimated to be 40-kDa and molecular-based analysis indicated that the purified protein was NADP+-dependent alcohol dehydrogenase (EhADH1). The application of the purified protein for the serodiagnosis of amebiasis was evaluated using an enzyme-linked immunosorbent assay applied to sera obtained from patients with amebiasis and healthy human controls. The purified protein was well recognized by the sera from asymptomatic amebiasis humans (22/22, 100%), whereas, it was less recognized by the sera from symptomatic amebiasis patients (5/16, 31%) with amebic colitis or liver abscess. To confirm the antigenicity of EhADH1, the recombinant glutathione S-transferase-EhADH1 fusion protein was also evaluated by the enzyme-linked immunosorbent assay using the same sera. The recombinant protein was also recognized by the sera from asymptomatic amebiasis humans (14/22, 64%) and less recognized by the sera from symptomatic amebiasis patients (2/16, 13%). These results suggest that the purified protein is applicable antigen for serodiagnostic screening of asymptomatic amebiasis humans.
Genes encoding putative mitochondrial-type heat shock protein 70 (mit-hsp70) were isolated and sequenced from amitochondriate protists, Giardia intestinalis, Entamoeba histolytica, and two microsporidians, Encephalitozoon hellem and Glugea plecoglossi. The deduced mit-hsp70 sequences were analyzed by sequence alignments and phylogenetic reconstructions. The mit-hsp70 sequence of these four amitochondriate protists were divergent from other mit-hsp70 sequences of mitochondriate eukaryotes. However, all of these sequences were clearly located within a eukaryotic mitochondrial clade in the tree including various type hsp70 sequences, supporting the emerging notion that none of these amitochondriate lineages are primitively amitochodrial, but lost their mitochondria secondarily in their evolutionary past.
In this review we discuss the mechanisms and molecules involved in the multidrug resistance (MDR) of the protozoan parasite Entamoeba histolytica. Drug resistant mutants exhibited the main characteristics presented by the MDR mammalian cells. They showed cross-resistance to several unrelated drugs that is reverted by calcium channel blockers. MDR phenotype in E. histolytica is regulated at a transcriptional level by the EhPgp1 gene, which is constitutively expressed and by the EhPgp5 gene, whose expression is induced in the presence of the drug. Transcription factors participate in the expression regulation of these genes. After over transcription, the EhPgp genes are amplified, cooperating to produce the MDR phenotype. Post-transcriptional mechanisms such as mRNA stability seem to be involved in this phenomenon. As for other mdr gene products, the EhPGP5 protein functions as a chloride current inductor or as a regulator of cellular regulatory volume decrease.
Upon analysis of 304 expressed sequence tags derived from the protozoan parasite Entamoeba histolytica, a number of novel protein encoding amoeba sequences were isolated. In addition, two unrelated, abundantly expressed transcripts were identified, and designated, ehapt1 and ehapt2. Although these transcripts do not contain any overt open reading frame, both are polyadenylated and together represent about 19% of total polyA+-RNA(11.6% for ehapt1 and 7.5% for ehapt2), thus being the most highly expressed polyA-containing transcripts so far identified in E. histolytica trophozoites. Northern blot and primer extension analyses revealed single-sized transcripts of 0.5 and 0.6 kb for ehapt1 and ehapt2, respectively, and Southern blot analysis suggests that both are encoded by multiple genes, which are distributed throughout the amoeba genome. Comparison between various ehapt1- and ehapt2-derived cDNAs indicated that both transcripts are highly polymorphic. Whereas nucleotide substitutions in ehapt2 are distributed throughout the sequence, variations in ehapt1 are mainly restricted to two regions, one of which comprises a deletion of variable length within an 8 nt tandem repeat unit. At present there is no convincing explanation for the possible role of ehapt1 and ehapt2 in E. histolytica, and analogous sequences have not been described so far for any other organism. Most likely they might represent regulatory RNAs or transcribed transposable elements.
A conference on Amebiasis and the Biology of Entamoeba histolytica was held in Agra, India, from 11 to14 February 2002.
A novel tetrahydroquinolinyl ester, quinfamide, administered orally in multiple doses for 3 days had an ED50 of 0.25 mg/kg/day (total dose 0.75 mg/kg) for eradicating Entamoeba criceti in hamsters in several tests. It was significantly more active by direct comparison than 3 commercially available amoebicides and at least as active as 2 other esters of the parent compound, 1-(dichloroacety)-1,2,3,4-tetrahydro-6-quinolinol. After administration of a single dose, ED50 calculations for quinfamide averaged 0.9 mg/kg. Quinfamide was considerably more active than the other tetrahydroquinolinols, diloxanide furoate and teclozan, and it was approximately 1.5 times more active than etofamide; a statistical significance between the latter 2 drugs could be demonstrated in one of 4 tests. Administered prophylactically, quinfamide was shown to protect hamsters from re-infection with E. criceti. It also inhibited propagation of E. histolytica in vitro at a concentration of 20 microgram/ml. No adverse effects were noted in rodents after a single dose as high as 10 g/kg. Daily administration to monkeys of doses up to 500 mg/kg for as long as 37 days produced no pharmacological aberrations during or after medication; haematological studies and urine analyses were normal and no gross or microscopical tissue changes attributable to quinfamide were observed. No toxicity was revealed following acute (2 g/kg) and chronic (500 mg/kg/day x 31 days) administration of the drug to dogs and rats, respectively.
Recombinant ribosomal DNA sequences were amplified by PCR and used as probes to perform a fingerprint analysis of total DNA from different Entamoeba histolytica isolates. RFLPs obtained with one of the probes, R-1, support previous proposals that pathogenic and non-pathogenic E. histolytica are closely related, yet genotypically distinct. Another probe, R-2, while not distinguishing between the two forms of E. hystolytica, was able to differentiate between them and E. moshkovskii, which has morphologically identical cysts and trophozoites. A third probe, BR-1, identified strain-specific RFLPs.
We describe a new, improved test for the detection of intestinal infection by Entamoeba histolytica. The test depends upon immunoadsorption of the E. histolytica cysteine proteinase, histolysain, from faecal samples, and subsequent visual detection of the enzyme by a colour reaction. With samples from 200 volunteers, results agreed closely with those obtained by the conventional microscopic technique, and there were no false positive reactions with samples containing other parasites. The test is suitable for use either in the laboratory or in the field.
A solid-phase enzyme immunoassay (EIA) was used to detect circulating antibodies to histolysain, the major cysteine proteinase of Entamoeba histolytica. Serum samples from 40 healthy controls, 33 asymptomatic E. histolytica cyst passers and 22 patients with amoebic liver abscess were tested. Antibodies to histolysain were found in 72.7% of cases of amoebic liver abscess, 18.1% of the cyst passers and 2.5% of healthy controls, which suggests that a humoral immune response is induced by histolysain during amoebic liver abscess.
Invasive properties of pathogenic Entamoeba histolytica have been postulated to depend on the secretion or release of cysteine proteinases and pore-forming peptides (amoebapores) by trophozoites. To establish whether such toxic molecules are released by viable trophozoites or upon cellular disintegration, amoebae were maintained in various culture media, and activities in supernatants were monitored over time in correlation to cellular integrity. By measuring the release of the cytoplasmic marker enzyme NADP(+)-alcohol dehydrogenase, it became apparent that release of amoebapore was accompanied by cellular disintegration. In contrast, considerable quantities of cysteine proteinases were found to be present in culture supernatants also when amoebae remained intact. Treatment of amoebae with concanavalin A, bacterial lipopolysaccharides or the calcium ionophore A23187 did not result in amoebapore secretion suggesting that here target cell contact is required as an essential stimulus.
Among the variety of virulence factors of Entamoeba histolytica, an adherence lectin (Gal/GalNAc, 260 kDa) is known to mediate colonization and subsequent host responses. Gal/GalNAc lectin is universally recognized by the immune sera of patients with amoebic liver abscess. It plays a crucial role in cytolysis and phagocytosis of human and rat colonic mucin glycoproteins. The objective of the present study was to elucidate the role of antioxidants in E. histolytica Gal/GalNAc lectin-induced signals in the target epithelial cells. We have attempted to define a pathway in target cells, Henle-407 cells (human intestinal epithelial cell line), that could link this immunodominant antigen to a known biological pathway for target cell activation and triggering of subsequent disease pathology/parasite survival. Since several workers have demonstrated that cAMP and cGMP may act as important cellular signals for altering ion transport, so in the present study, cAMP and cGMP levels were measured in Henle-407 cells which showed significant increase at 15 min after stimulation. Elevated cAMP and cGMP levels are implicated in altered electrolyte transport and conductance. Results showed that there were increased levels of ROS and RNI which led to reduced activities of antioxidant enzymes--catalase, superoxide dismutase and glutathione peroxidase. Despite the increased glutathione (reduced) levels, the enzymes were not able to combat the damage caused by ROS and RNI. Thus, there was an increased local concentration of the free radicals and reduced activities of all the three enzymes which could damage the target cell in terms of cytoskeleton and permeability changes.
The bacterial flora of the intestine plays an important role in the virulence caused by Entamoeba histolytica. Cysteine proteinase (CP), an amoebic virulence factor, plays a major role in host cell destruction. The mechanism of increased virulence following bacterial co-association is not understood. We studied CP of E. histolytica HM1:IMSS which was co-associated with Escherichia coli K12 strain pre-incubated with GalNAc or CP specific inhibitor E 64. Co-association of E. histolytica with bacteria enhanced CP activity 3-6-fold as assessed by azocasein assay and substrate gel electrophoresis showed bands at molecular weights of 28, 35 and 56 kDa. Northern and Western blot analysis showed increase in ehcp2 and ehcp5 gene expression. Trophozoites co-associated with E. coli showed greater cytotoxicity of BHK cells by a 51Cr release assay than trophozoites that had not been co-associated; this enhancement was abolished by E-64 treatment. The killing of BHK 21 targets by E. histolytica was characterized by DNA laddering which was not inhibited with E-64. GalNAc pre-incubation of trophozoites reduced cytotoxicity and DNA laddering, while E. coli co-associated E. histolytica showed smearing with faint laddering of BHK implicating both necrosis and apoptosis. Hence, bacterial co-association increases CP activity and CP gene expression and contributes to the necrosis of the target cell.