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Development and testing of markers for genotyping of 'Entamoeba histolytica'
Abstract
Only one in ten Entamoeba histolytica infections is invasive but they are responsible for an annual death toll of up to 100,000 people. A key question in amoebiasis is, therefore, what is responsible for the variable outcome of infection. To investigate whether it is linked to the genotype of the infecting strain, we developed 6 pairs of species-specific primers for genotyping after investigating 46 potentially polymorphic short tandem repeat loci adjacent to tRNA genes that were identified during the E. histolytica genome project. We tested the primers using E. histolytica samples from Bangladesh and 11 other countries. Results revealed that the genotypes present in 3 different clinical populations - asymptomatic, diarrhoeal/dysenteric and liver abscess - were different from each other. A few individual genotypes also showed links to the outcome of infection although their sample coverage was low and therefore they had little predictive value. Although the loci used as polymorphic markers are unlikely to be directly responsible for the outcome of infection, the results do suggest that parasite genetic factors are at least partly responsible. Our multilocus genotyping method is simple and reliable as it amplifies DNA extracted from axenic or xenic culture, stool samples, and liver abscess pus samples. We believe that these markers will help in studying the patterns of transmission of this important disease and the epidemiological links between individual infections.
... No such concerns have been raised about the other polymorphic loci being discussed. Indeed, where such observations have been possible it is clear from follow-up sampling that the PCR product sizes obtained remain stable over the course of the same infection (21,22). It is also the case that the patterns remain stable over a period of years in culture. ...
... The degree of variation detected among the six tRNAlinked loci varies from moderate to substantial, and so some resemble more the level of diversity seen in the chitinase gene while others resemble SREHP. In combination the six loci can distinguish a remarkable number of distinct variants-almost 100 combinations have been observed in Bangladesh alone (21). This raises a conundrum. ...
... The level of diversity seen in the tRNA-linked loci of E. dispar also appears to be largely similar to that observed in E. histolytica (24), with one exception. In E. dispar one of the six selected STR loci shows no polymorphism and sequence analysis has shown that it lacks the short tandem repeats (21). The degree of diversity at the other tRNAlinked loci is comparable to that in E. histolytica and they should be useful should anyone wish to study E. dispar molecular epidemiology. ...
The ability to distinguish variants of a species has many potential applications. In Entamoeba histolytica the first method to detect variation was based on isoenzyme analysis. However, this approach has been superseded by DNA-based analysis. In this review I discuss the basis of the variation detected in E. histolytica by the various molecular methods that have been published to date. Information on diversity in other species is mentioned where such information exists. (C) 2006 IMSS. Published by Elsevier Inc.
... No such concerns have been raised about the other polymorphic loci being discussed. Indeed, where such observations have been possible it is clear from follow-up sampling that the PCR product sizes obtained remain stable over the course of the same infection (Ali, 2005;Zaki et al, 2003). It is also the case that the patterns remain stable over a period of years in culture. ...
Entamoeba histolytica is an intestinal parasite and the causative agent of amoebiasis, which is a significant source of morbidity and mortality in developing countries. Here we present the genome of E. histolytica, which reveals a variety of metabolic adaptations shared with two other amitochondrial protist pathogens: Giardia lamblia and Trichomonas vaginalis. These adaptations include reduction or elimination of most mitochondrial metabolic pathways and the use of oxidative stress enzymes generally associated with anaerobic prokaryotes. Phylogenomic analysis identifies evidence for lateral gene transfer of bacterial genes into the E. histolytica genome, the effects of which centre on expanding aspects of E. histolytica's metabolic repertoire. The presence of these genes and the potential for novel metabolic pathways in E. histolytica may allow for the development of new chemotherapeutic agents. The genome encodes a large number of novel receptor kinases and contains expansions of a variety of gene families, including those associated with virulence. Additional genome features include an abundance of tandemly repeated transfer-RNA-containing arrays, which may have a structural function in the genome. Analysis of the genome provides new insights into the workings and genome evolution of a major human pathogen.
Entamoeba histolytica causes amebic dysentery and amebic liver abscess, major causes of morbidity and mortality worldwide. We have used differential hybridization screening to isolate an E. histolytica-specific cDNA clone. The cDNA was found to encode a serine-rich E. histolytica protein (SREHP) containing multiple tandem repeats. The structural motif of SREHP resembles some of the repetitive antigens of malarial species, especially the circumsporozoite proteins. A recombinant trpE fusion protein containing the tandem repeats of SREHP was recognized by immune serum from a patient with amebiasis, demonstrating that SREHP is a naturally immunogenic protein. An antiserum raised against the recombinant fusion protein specifically bound to two distinct bands with apparent molecular masses of 46 and 52 kDa in a crude preparation of E. histolytica trophozoite membranes. This antiserum also inhibited E. histolytica trophozoite adhesion to Chinese hamster ovary cells in vitro. The ability to isolate E. histolytica-specific genes, and to express those genes in Escherichia coli, may be important in studying the molecular basis of E. histolytica pathogenesis and for the future development of vaccines.
An invasive strain-specific monoclonal antibody against Entamoeba histolytica has been used in a capture enzyme-linked immunosorbent assay (ELISA) for the detection of invasive E. histolytica fecal antigen in clinical specimens and for the diagnosis of amebic dysentery in patients from Bangladesh. The fecal antigen capture ELISA (FAC-ELISA) did not cross-react with other parasite species in the clinical specimens or with noninvasive E. histolytica present in those specimens and in experimentally seeded stools. The limit of detection of the assay for invasive E. histolytica crude antigen diluted in phosphate-buffered saline or in stools was 0.58 and 3.9 micrograms/ml, respectively, which is the equivalent of approximately 72 and 487 E. histolytica trophozoites per well, respectively. The sensitivity, specificity, and efficiency of the FAC-ELISA were 87, 100, and 98%, respectively, for the detection of invasive E. histolytica antigens and 100, 100, and 100%, respectively, for the diagnosis of amebic dysentery. The FAC-ELISA is a potential alternative for the field diagnosis of amebic dysentery and for epidemiological studies to define the distribution of invasive E. histolytica.
The epidemiological implications of the recent separation of "Entamoeba histolytica" into two separate species, pathogenic E. histolytica sensu stricto and commensal E. dispar, will not become apparent without methods of distinguishing between them which are applicable to large numbers of specimens. We have modified a PCR-based method to produce such a technique which may be completed in 1 day while still identifying 10(-1) E. histolytica and 1 to 10 E. dispar trophozoites per g of feces when present separately and 10 E. histolytica and 100 E. dispar trophozoites per g in the presence of 10(6) trophozoites per g of the other species. Applied to fecal specimens from 18 patients from which E. histolytica or E. dispar had been grown and identified to the species level by hexokinase isoenzyme analysis, the method in every case yielded the correct result. Positive and negative results are easily distinguished by eye, and we are now applying this technique to a large-scale epidemiological study of amebiasis in the eastern Mediterranean region.
We report here a simple and economical slide agglutination test, the co-agglutination (Co-A) test, for the detection of circulating amebic antigen in sera for the diagnosis of amebic liver abscess. Fifty serum specimens from cases of amebic liver abscess, 25 from other individuals with parasitic and miscellaneous infections, and 25 from healthy controls were tested for the presence of serum antigen by the Co-A test. Forty-five (90%) amebic liver abscess sera were found to be amebic-antigen positive by the Co-A test. None of 25 sera from healthy controls were positive for the antigen. However, false-positive results were seen with two sera from those with other parasitic and miscellaneous infection controls. These results show that the Co-A test can be used as a sensitive and specific rapid slide agglutination test for the detection of amebic antigen in the sera for diagnosis of cases of amebic liver abscess in a routine parasitology laboratory.
The apicomplexan parasite Cryptosporidium parvum is a major cause of serious diarrheal disease in both humans and animals. No efficacious chemo- or immunotherapies have been identified for cryptosporidiosis, but certain antibodies directed against zoite surface antigens and/or proteins shed by gliding zoites have been shown to neutralize infectivity in vitro and/or to passively protect against, or ameliorate, disease in vivo. We previously used monoclonal antibody 11A5 to identify a 15-kDa surface glycoprotein that was shed behind motile sporozoites and was recognized by several lectins that neutralized parasite infectivity for cultured epithelial cells. Here we report the cloning and sequence analysis of the gene encoding this 11A5 antigen. Surprisingly, the gene encoded a 330-amino-acid, mucin-like glycoprotein that was predicted to contain an N-terminal signal peptide, a homopolymeric tract of serine residues, 36 sites of O-linked glycosylation, and a hydrophobic C-terminal peptide specifying attachment of a glycosylphosphatidylinositol anchor. The single-copy gene lacked introns and was expressed during merogony to produce a 60-kDa precursor which was proteolytically cleaved to 15- and 45-kDa glycoprotein products that both localized to the surface of sporozoites and merozoites. The gp15/45/60 gene displayed a very high degree of sequence diversity among C. parvum isolates, and the numerous single-nucleotide and single-amino-acid polymorphisms defined five to six allelic classes, each characterized by additional intra-allelic sequence variation. The gp15/45/60 single-nucleotide polymorphisms will prove useful for haplotyping and fingerprinting isolates and for establishing meaningful relationships between C. parvum genotype and phenotype.
Entamoeba histolytica causes amebic colitis and liver abscess in developing countries such as Mexico and India. Entamoeba dispar is morphologically identical but is not associated with disease. Here we determined the ploidy of E. histolytica and developed PCR-based methods for distinguishing field isolates of E. histolytica or E. dispar. Fluorescence in situ hybridization showed that E. histolytica trophozoites are diploid for five "single-copy" probes tested. Intergenic sequences between superoxide dismutase and actin 3 genes of clinical isolates of E. histolytica from the New and Old Worlds were identical, as were those of E. dispar. These results suggest a bottleneck or demographic sweep in entamoebae which infect humans. In contrast, E. histolytica and E. dispar genes encoding repeat antigens on the surface of trophozoites (Ser-rich protein) or encysting parasites (chitinase) were highly polymorphic. chitinase alleles suggested that the early axenized strains of E. histolytica, HM-1 from Mexico City, Mexico, and NIH-200 from Calcutta, India, are still present and that similar E. dispar parasites can be identified in both the New and Old Worlds. Ser-rich protein alleles, which suggested the presence of the HM-1 strain in Mexico City, included some E. histolytica genes that predicted Ser-rich proteins with very few repeats. These results, which suggest diversifying selection at chitinase and Ser-rich protein loci, demonstrate the usefulness of these alleles for distinguishing clinical isolates of E. histolytica and E. dispar.
An important gap in our understanding of the epidemiology of amebiasis is what determines the outcome of Entamoeba histolytica infections. To investigate the possible existence of invasive and noninvasive strains as one factor, the ability to differentiate
individual isolates of E. histolytica is necessary. Two new loci containing internal repeats, locus 1-2 and locus 5-6, have been isolated. Each contains a single
repeat block with two types of related direct repeats arranged in tandem. Southern blot analysis suggests that both loci are
multicopy and may themselves be arranged in tandem arrays. Three other previously reported, internally repetitive loci containing
at least two repeat blocks each with one or more related repeat units were also investigated. PCR was used to study polymorphism
at each of these loci, which was detected to various degrees in each case. Variation was seen in the total number of bands
obtained per isolate and their sizes. Nucleotide sequence comparison of loci 1-2 and 5-6 in five axenic isolates revealed
differences in the number of repeat units, which correlated with the observed PCR product size variation, and in repeat sequence.
Use of multiple loci collectively allowed differentiation of a majority of the 13 isolates studied, and we believe that these
loci have the potential to be used as polymorphic molecular markers for investigating the epidemiology ofE. histolytica and the potential existence of genetically distinct invasive and noninvasive strains.
Twelve human infections with Entamoeba spp. producing uninucleated cysts were studied. DNA was extracted from infected feces and used to amplify part of the ameba
small-subunit rRNA gene. Sequence analysis identified four distinct types ofEntamoeba, all of which are related toEntamoeba polecki and E. chattoni and two of which have not been reported previously. Whether these genetic types represent different species is unclear. We
propose that the agent of all human infections with uninucleated cyst-producingEntamoeba species be reported as “E. polecki-like.”
Sequences corresponding to some of the polymorphic loci previously reported from Entamoeba histolytica have been detected in Entamoeba dispar. Comparison of nucleotide sequences of two loci between E. dispar strain SAW760 and E. histolytica strain HM-1:IMSS revealed significant differences in both repeat and flanking regions. The tandem repeat units varied not
only in sequence but also in number and arrangement between the two species at both the loci. Using the sequences obtained,
primer pairs aimed at amplifying species-specific products were designed and tested on a variety of E. histolytica and E. dispar samples. Amplification results were in complete agreement with the original species classification in all cases, and the
PCR products displayed discernible size and pattern variations among the isolates.
OBJECTIVE:The majority of individuals infected by the protozoan parasite Entamoeba histolytica experience subclinical infections. However, a small proportion of parasitized individuals develop severe invasive disease such as amebic dysentery or amebic liver abscess. Invasive amebiasis affects predominantly men; the usual explanation for this has been that men have a higher rate of asymptomatic infections and therefore experience a higher rate of invasive disease. To date, there is no convincing evidence of an increased rate of asymptomatic infection of men as compared with women. The purpose of this study was to evaluate the evidence supporting the hypothesis that men have higher rates of asymptomatic infection and thus an increased frequency of invasive amebiasis.METHODS:We reviewed published reports of invasive amebiasis and population-based parasitological studies from 1929–1997 to compare the gender ratio of asymptomatic and symptomatic E. histolytica infection. Infections with E. histolytica were differentiated from the nonpathogenic E. dispar whenever possible.RESULTS:The reports of invasive amebiasis (dysentery, liver abscess, colonic perforation, peritonitis, appendicitis, and ameboma) showed a higher proportion of men than women (ratio, male:female = 3.2:1, p < 0.05). This contrasts with the epidemiological surveys, where the rate of asymptomatic infection with E. histolytica was the same (1:1) for both genders (p > 0.05).CONCLUSIONS:Asymptomatic E. histolytica infection is equally distributed between the genders. The high proportion of men with invasive amebiasis may be due to a male-related susceptibility to invasive disease.
The genome of the lower eukaryote
A 3-year-old boy with hepatopulmonary amebiasis treated with chloroquine phosphate for 20 days subsequently was found to have three brain abscesses and responded to prolonged combined therapy with metronidazole and chloroquine phosphate.
Cerebral amebiasis should be considered in any patient with amebic dysentery, hepatic abscess, or pulmonary involvement in whom signs or symptoms of central nervous system involvement appear. A prolonged course of appropriate therapy with periodic brain scanning for evidence of improvement is suggested.
Previously a cloned emetine-resistant mutant of the protozoal parasite Entamoeba histolytica was shown to overexpress a gene
for an ameba homolog of the mammalian P-glycoprotein, a plasma membrane pump that removes hydrophobic drugs from multidrug-resistant
tumor cells. Three sets of experiments were performed to better characterize the multidrug-resistant phenotype of the emetine-resistant
amebae. First, the emetine resistance of the mutant amebae was reversed by concentrations of calcium and sodium channel blockers
effective in reversing drug resistance by multidrug-resistant tumor cells, but it was reversed only in the presence of very
high concentrations of the tricyclic antidepressants. Second, the mutant amebae showed cross-resistance to antiamebic drugs
used to treat luminal infection (iodoquinol and diloxanide) but were not cross-resistant to drugs used to treat invasive disease
(chloroquine and metronidazole). Third, when amebae were loaded with radiolabeled emetine, the mutant parasites released the
drug at approximately 1.6 times the rate of the wild-type organisms. We conclude that the emetine-resistant E. histolytica
parasites have some but not all the features of the multidrug-resistant phenotype.
Diloxanide furoate is used for treating asymptomatic or mildly symptomatic persons who are passing cysts of Entamoeba histolytica. The Centers for Disease Control (Atlanta) released this drug for 4,371 treatment courses from 1977 through 1990. Of the
2,815 report forms (64%) returned, 656 adverse effects were reported for 390 treatment courses (14%); they included flatulence
(260), diarrhea or cramping (100), nausea (93), headache (17), disorientation or dizziness (9), and diplopia (4). During 1984–1990
uniform collection of data allowed more detailed analysis of toxicity and efficacy; fewer adverse effects were reported for
persons aged 20 months to 10 years than for persons aged >10 years (6 of 206 [3%] vs. 89 of 763 [12%], relative risk = 0.27,
95% confidence interval = 0.12 < relative risk < 0.61). Parasitological cures were achieved during 497 (86%) of the 575 treatment
courses (52%) administered to asymptomatic persons who were passing cysts, who had received a full l0-day treatment course,
and for whom results of a follow-up stool examination (⩾14 days post-treatment) were available. Diloxanide furoate is safe
and effective for treating asymptomatic persons who are passing E. histolytica cysts and may be particularly well tolerated in children.
Since the application of isoenzyme electrophoresis to the study of Entamoeba histolytica, the prevalence and natural history of asymptomatic intestinal colonization in patients with amebic liver abscess (ALA) has
not been addressed. We prospectively evaluated this enteric phase in 50 patients with ALA, using two dosage regimens of metronidazole.
The overall prevalence of asymptomatic colonization was 72% (36/50). All these isolates, without exception, proved to express
pathogenic zymodemes. Despite a 100% clinical response of the hepatic lesions, failure to eradicate the organism from the
bowel occurred in 20 of these 36 subjects. During longitudinal posttreatment surveillance, three carriers returned with second
bouts of invasive disease: one with dysentery and two with liver abscesses. Thus, in patients with ALA, there is a high prevalence
of intestinal colonization with exclusively pathogenic strains, and treatment with metronidazole frequently results in a continued
carrier state. These carriers have a propensity for developing recurrent invasive disease and constitute a public health hazard.
We previously reported the identification of homologous cDNA clones derived from a pathogenic isolate and a nonpathogenic isolate of Entamoeba histolytica, which had been designated cEh-P1 and cEh-NP1, respectively. Sequence analysis of both clones had revealed 10% nucleic acid substitutions, which were dispersed over the entire sequence. This genetic difference had been found to be conserved between all four pathogenic and all five nonpathogenic laboratory strains of E. histolytica tested. On the basis of nucleic acid substitutions, we have now developed a sensitive assay to distinguish pathogenic from nonpathogenic forms of E. histolytica by using fresh clinical isolates. Comparing the sequence of cEh-P1 and cEh-NP1, we identified a 482-bp segment that contained identical 5' and 3' ends but differed in internal cleavage sites for restriction endonucleases. By using oligonucleotide primers corresponding to the 5' and 3' ends of this segment, the corresponding gene was amplified by the polymerase chain reaction. Endonuclease digestion of the amplified DNA yielded restriction fragments that are characteristic for pathogenic and nonpathogenic forms. This assay allows the detection and classification of fewer than 10 amoebae within a few hours. The differentiation of 48 isolates into pathogenic and nonpathogenic strains by using this method corresponded to the clinical status of the infected individuals and to the classification obtained by isoenzyme determination. The results further support the concept that pathogenic and nonpathogenic strains of E. histolytica constitute distinct subspecies.
Several repetitive DNA families were identified in Entamoeba histolytica DNA digested with Sau3AI. Characterisation of one of these repetitive DNA families showed the presence of multiple copies of Saccharomyces cerevisiae autonomously replicating sequence (ARS) core consensus sequences. The E. histolytica ARS consensus sequences allowed a yeast-integrating plasmid, YIP5, to replicate autonomously in S. cerevisiae. A 'bent DNA' fragment was located in one member of this E. histolytica repetitive DNA family.
We report the first human case of Entamoeba polecki infection found in Japan. Cysts of E. histolytica-like amoeba were detected in a stool sample from a female Cambodian refugee. The cysts were morphologically investigated after stained by the modified Kohn's method. The amoeba was identified as E. polecki by the findings that the cyst had frequently an inclusion mass, usually one or very rarely two nuclei, frequently a darkly stained nucleus, and some other characteristics. This protozoan parasite is essentially non-pathogenic to humans, but it is morphologically similar to and often confused with E. histolytica, a pathogenic species. Therefore, it is very important in laboratory diagnosis to differentiate the former from the latter.
Major problems with a wide array of imperfect tests for diagnosis of amebiasis severely limit the understanding of its magnitude
and epidemiology. A greater hindrance is the varied, inconsistent application of existing methods in different areas of the
world. The best estimates suggest that probably 480 million people were infected with Entamoeba histolytica and 36 million developed disabling colitis or extraintestinal abscesses in 1981. At least 40 thousand deaths are attributable
to amebiasis, and on a global scale, amebiasis likely ranks third among parasitic causes of death, behind only malaria and
schistosomiasis. Much remains to be learned of its frequency of occurrence and epidemiology as needed improved diagnostic
tools are developed.
The demonstration of a new zymodeme of Entamoeba histolytica produced in culture from cloned isolates suggested possible genetic exchange in this parasite. We have attempted to substantiate that finding by using rats as biological hosts. Clones were made from 3 separate isolates of E. histolytica, each established in culture from liver pus or faeces. After enzyme characterization these clones, of zymodemes II, XIV and XIX, were paired in each of the 3 possible combinations and the mixtures injected into the caecum of rats. Clones of new or hybrid zymodemes were produced as well as the original parents, with one exception, the mixture of XIV and XIX, from which only one of the parents was recovered. The hybrids produced included zymodeme XX, observed previously, and zymodeme XI, a naturally occurring zymodeme that has in the past been recovered only from subjects with invasive amoebiasis.
A case of multiple amoebic lung abscesses without indication of direct extension from a subclinical liver abscess, which delayed correct diagnosis, is reported. Severe constitutional symptoms, life-threatening haemoptysis and large pulmonary lesions were the prominent clinical manifestations. The response to metronidazole was dramatic. It is postulated that haematogenous spread was responsible. The rarity of this form of amoebiasis is evident on published reports.
A method for culturing amoebae, suitable for a clinical laboratory, is given and assessed by recording the results of combined
microscopical and cultural diagnosis on 4,236 specimens from tropical patients of the Dreadnought Seamen's Hospital, London.
Of the total specimens examined 17·7% were positive microscopically for one or more species, and 2/9 of these (4·1% of total
specimens) were missed culturally, while 26·2% were positive culturally for one or more species, and 1/3 of these (8·5%) were
missed microscopically. The figures for Entamoeba histolytica alone are 2·8% missed microscopically but none missed culturally, out of 7·3%. High rates for Nigerian amoebic carriers and
Goan amoebic abscesses are recorded.
Immunization of hamsters by intramuscular injections of an amebic extract and of two of its fractions, emulsified with complete Freund's adjuvant, resulted in protection against the development of amebic liver abscess, following intrahepatic inoculation of axenic trophozoites of Entamoeba histolytica such that Fraction I, Fraction II, and the whole antigen conferred protection to 18 out of 18, 4 out of 18, and 11 out of 18 hamsters, respectively. Splenomegaly was found to accompany the development of hepatic liver abscesses in this experimental system. There was a very high degree of correlation (r = 0.95) between the weights of the abscesses and the spleens. On the other hand, there was no correlation between the anti-amebic antibody titers (determined by the indirect hemagglutination test) and the development of the liver abscesses within the time-span of the experimental protocol used in this study.
The clinical, histopathological, and serological features of 35 homosexual men with infection with Entamoeba histolytica were studied and compared with a group of 35 non-infected homosexual men. Each isolate was of Zymodeme type I. Although there was no significant difference in the numbers of infected and non-infected men with gastrointestinal symptoms (48.6% and 28.6% respectively), the mean duration of symptoms was greater in with amoebiasis (p less than 0.05). The histology of the rectal mucosa was abnormal in 17 (63.0%) of the 27 men with amoebic infection only and in two (7.4%) of the 27 control subjects (p less than 0.001). Serum antibodies reactive with E. histolytica were not shown in any patient.
The intestinal protozoan parasite Entamoeba histolytica causes amebic dysentery, a major cause of morbidity worldwide. The induction of a mucosal antibody response capable of blocking amebic adhesion to intestinal cells could represent an approach to preventing E. histolytica infection and disease. Here we describe the expression of a chimeric protein containing an immunogenic dodecapeptide derived from the serine-rich E. histolytica protein (SREHP), fused to the cholera toxin B subunit (CtxB). The CtxB-SREHP-12 chimeric protein was purified from Escherichia coli lysates and retained the critical GM1 ganglioside-binding activity of the CtxB moiety. Mice fed the CtxB-SREHP-12 fusion protein along with a subclinical dose of cholera toxin developed mucosal immunoglobulin A and immunoglobulin G and systemic antibody responses that recognized recombinant and native SREHP. Our study confirms the feasibility of inducing mucosal immune responses to immunogenic peptides by their genetic fusion to the CtxB subunit and identifies the CtxB-SREHP-12 chimeric protein as a candidate oral vaccine to prevent E. histolytica infection.
A genomic library of Entamoeba histolytica (pathogenic strain HM-1:IMSS) was screened to detect repetitive DNA clones other than those from the highly abundant ribosomal DNA (rDNA). One such clone (HMc) had a 2.3 kb insert which hybridized with the main genome and not the rDNA circle. Southern hybridization of E. histolytica genomic DNA, digested with EcoR I and probed with HMc, showed multiple bands. The banding pattern was identical in all axenic pathogenic strains tested. Differences, however, existed when the banding pattern of a pathogenic strain was compared with that of a non-pathogenic strain. HMc was present in about 25-30 copies per genome in strain HM-1:IMSS. Nucleotide sequence analysis of HMc revealed a partial open reading frame which hybridized with a 1.35 kb poly A+ transcript in Northern blots. The deduced amino acid sequence did not, however, show significant homology with known proteins. The HMc sequence was found only in E. histolytica as it hybridized with 5 different axenic strains of E. histolytica but did not recognize other closely related species of Entamoeba. It has thus the potential to be used as a species-specific DNA probe.
Sera from patients with invasive amebiasis were used to identify a cDNA clone (K2p-1) encoding a commonly recognized, repeat-containing antigen of the pathogenic Entamoeba histolytica HM-1:IMSS. K2p-1 was used to isolate 3 cDNA clones (K2 clones); one K2p-1 related clone from the same pathogenic E. histolytica strain and 2 from the nonpathogenic E. histolytica strain SAW-142. The nucleotide sequence and predicted amino acid sequence revealed a closely related family of transcripts differing mainly in the extent and arrangement of an internal region consisting of tandemly arranged repetitive elements. The repetitive units encoding either 12 or 8 amino acids were found to be highly conserved in all the K2 clones analyzed so far, suggesting that the repeat motifs perform functions common to both pathogenic and nonpathogenic E. histolytica. The genomic organization of the K2 genes was different when compared in pathogenic and nonpathogenic E. histolytica and may therefore be used to discriminate between pathogenic and nonpathogenic E. histolytica strains.
Attenuated salmonellae represent attractive candidates for the delivery of foreign antigens by oral vaccination. In this report, we describe the high-level expression of a recombinant fusion protein containing the serine-rich Entamoeba histolytica protein (SREHP), a protective antigen derived from virulent amebae, and a bacterially derived maltose-binding protein (MBP) in an attenuated strain of Salmonella typhimurium. Mice and gerbils immunized with S. typhimurium expressing SREHP-MBP produced mucosal immunoglobulin A antiamebic antibodies and serum immunoglobulin G antiamebic antibodies. Gerbils vaccinated with S typhimurium SREHP-MBP were protected against amebic liver abscess, the most common extraintestinal complication of amebiasis. Our findings indicate that the induction of mucosal and immune responses to the amebic SREHP antigen is dependent on the level of SREHP-MBP expression in S. typhimurium and establish that oral vaccination with SREHP can produce protective immunity to invasive amebiasis.
Advancements in our understanding of amebiasis have been rapid over the decade that I have followed this field. What was identified morphologically for years as Entamoeba histolytica has been redescribed with modern techniques as a complex of two species, the commensal parasite E. dispar and the pathogenic parasite E. histolytica that is the cause of colitis and liver abscess. Antigen detection tests are now available for the rapid detection in stool of the pathogenic species E. histolytica. New understandings of the importance of luminal as well as tissue-active antimebic medications in the treatment of invasive disease have been reached. The groundwork is being laid for an understanding of the protective immune responses to infection, and at the lab bench DNA transfection of the parasite has opened studies of pathogenesis to genetic analysis. While necessarily an incomplete sketch of the field, I have attempted here to highlight some recent and important developments of interest to clinicians and microbiologists.
Shotgun sequencing of cDNA clones is now an established approach to gain insight into the expressed nucleotide (nt) sequences in a given cell. We analysed 100 randomly picked cDNA clones of the protozoan parasite, Entamoeba histolytica, by nt sequencing, with a view to obtain novel gene sequences not detected so far by biochemical and genetic analyses. About 56% of the analysed clones showed significant homology with other genes in the database, including a number of genes whose presence may not be suspected in E. histolytica owing to its unusual subcellular organization. The results suggest that this approach can provide important clues to understand unique biochemical mechanisms in this parasite.
Characterization of YS-27, an axenic Entamoeba strain, was performed by three different laboratory methods. Zymodeme analysis using starch gel electrophoresis and PCR with species-specific primers showed that YS-27 is a pathogenic Entamoeba which belongs to the group II zymodeme. Pathogenicity of YS-27 was further confirmed by observing the formation of liver abscess in Mongolian gerbils. These results showed that YS-27 is E. hisolytica.
The varied organ tropisms and clinical presentations of infection by Entamoeba histolytica have stimulated interest in the role of parasite genetic diversity in virulence. We investigated genetic diversity among 54 E. histolytica isolates from Bangladesh by analyzing polymorphism in the serine-rich gene by nested PCR on DNA extracted from stool and liver aspirate pus. We detected both size and restriction site polymorphisms among the isolates within this endemic area. A combination of the nested PCR results and the AluI digestion of the PCR products examined yielded 25 distinct DNA banding patterns among the 42 stool isolates and an additional 9 distinct patterns among the 12 liver abscess isolates. Approximately half of the isolates had unique polymorphisms. Interestingly, the majority of E. histolytica from the liver had polymorphisms which were not present in intestinal isolates from the same geographic area. These data are consistent with the existence of genetic differences between E. histolytica which cause intestinal and those which cause hepatic disease. We conclude that there is genetic diversity within E. histolytica isolates from an endemic population as reflected in serine-rich E. histolytica protein gene polymorphism. The correlation of genetic differences with the pathogenic potential of E. histolytica strains and the implications of genetic diversity for the immunoprophylaxis of amebiasis require further study.