Molecular biology of the hexokinase isoenzyme pattern that distinguishes pathogenic Entamoeba histolytica from nonpathogenic Entamoeba dispar

The electrophoretic patterns of hexokinase and phosphoglucomutase have been widely used to distinguish Entamoeba histolytica from Entamoeba dispar isolates. Although E. histolytica and E. dispar, previously called pathogenic and nonpathogenic Entamoeba histolytica, differ clearly in sequences of many homologous genes, a conversion between the two has been reported by several laboratories, in each case showing the conversion of hexokinase (ATP, d-hexose 6-phosphotransferase, EC 2.7.1.1) isoenzyme patterns. An apparent mobility shift of this enzyme may either be due to posttranslational modification or processing, or to the appearance of a new isoform encoded by a second gene. In this study we observed that the four observed bands in the isoenzyme patterns of pathogenic and nonpathogenic forms of Entamoeba were correlated with four different cDNAs, and that the four recombinant hexokinases produced in Escherichia coli comigrated with their natural counterparts. Polymerase chain reaction (PCR) experiments did not reveal hidden genes which might be responsible for conversion phenomena. These results strongly support the redefinition of pathogenic and nonpathogenic Entamoeba histolytica as two closely related species Entamoeba histolytica and Entamoeba dispar.     

Differences in protein profiles of the isolates of <Emphasis Type="Italic">Entamoeba histolytica</Emphasis> and <Emphasis Type="Italic">E. dispar</Emphasis> by surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS) ProteinChip assays

Surface-enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF MS) ProteinChip assays with weak cationic exchange chips were used for protein profiling of different isolates of Entamoeba histolytica and E. dispar. When SELDI-TOF MS spectra of cell lysates from E. histolytica strain HM-1:IMSS were compared with those from four other laboratory strains (200:NIH, HK-9, DKB, and SAW755CR) grown under the same culture conditions, different peak patterns of SELDI-TOF MS were observed among these strains, independent of their zymodeme types. Similarly, five Japanese isolates of E. histolytica grown under the same culture conditions revealed different peak patterns among themselves. The SELDI-TOF MS spectra of cell lysates from two isolates of E. dispar strain AS16IR and CYNO 09:TPC showed the presence of peaks specific for E. dispar isolates and the absence of peaks common to E. histolytica isolates. This is not only the first use of SELDI-TOF MS ProteinChip technology for protein profiling of different strains of Entamoeba but also the use for parasitic protozoa. The SELDI-TOF MS spectra show a realistic view of proteins with a biological status of E. histolytica and E. dispar isolates, contributing to show their phenotypic differences of proteins and provide a unique means of distinguishing them.     

Differential identification of Entamoeba spp. based on the analysis of 18S rRNA

Entamoeba histolytica is known to cause intestinal and extra-intestinal disease while the other Entamoeba species are not considered to be pathogenic. However, all Entamoeba spp. should be reported when identified in clinical samples. Entamoeba polecki, Entamoeba coli, and Entamoeba hartmanii can be differentiated morphologically from E. histolytica, but some of their diagnostic morphologic features overlap. E. histolytica, Entamoeba dispar, and Entamoeba moshkovskii are morphologically identical but can be differentiated using molecular tools. We developed a polymerase chain reaction (PCR) procedure followed by DNA sequencing of specific regions of 18S rRNA gene to differentiate the Entamoeba spp. commonly found in human stools. This approach was used to analyze 45 samples from cases evaluated for the presence of Entamoeba spp. by microscopy and a real-time PCR method capable of differential detection of E. histolytica and E. dispar. Our results demonstrated an agreement of approximately 98% (45/44) between the real-time PCR for E. histolytica and E. dispar and the 18S rRNA analysis described here. Five previously negative samples by microscopy revealed the presence of E. dispar, E. hartmanii, or E. coli DNA. In addition, we were able to detect E. hartmanii in a stool sample that had been previously reported as negative for Entamoeba spp. by microscopy. Further microscopic evaluation of this sample revealed the presence of E. hartmanii cysts, which went undetected during the first microscopic evaluation. This PCR followed by DNA sequencing will be useful to refine the diagnostic detection of Entamoeba spp. in stool and other clinical specimens.     

Entamoeba histolytica autochthonous isolates from mentally retarded Italian patients

A total of 77 mentally retarde male inpatients residing in a psychiatric institution in northern Italy were screened for the presence of stool parasites,Entamoeba histolytica particularly. Parasitological stool examination showedEntamoeba spp. (E. histolytica and/orE. dispar) in 26 cases (33.7%). In vitro culture on Robinson's medium was positive in 16 cases (61.1%); in 11 cases we could stabilize and clone the isolates and proceed to electrophoretic assays. In all cases, patterns of pathogenic zymodemes were found (zymodeme II, 3 isolates; zymodeme XII, 4 isolates; zymodeme XIV, 4 isolates). All isolates were therefore identified asE. histolytica.     

Trophozoite and nuclear size,DNA base composition,and nucleotide sequence homology of several Entamoeba strains in axenic culture

We carried out a comparative study of nuclear and trophozoite diameters and of DNA thermal denaturation in eight Entamoeba strains cultured axenically (four of them E. histolytica, two initially designated as E. invadens, one E. moshkovskii, and one E. histolytica-like), as well as an analysis of the overall DNA sequence homology of the non-E. histolytica strains. The average nuclear (N) and trophozoite (T) diameters (in m) were, respectively: global averages ±SD for E. histolytica strains, 6.5±2.5 and 28.8±3.7; E. invadens IP101, 5.8 and 27.5, and PZ, 7.8 and 33.6; E. moshkovskii FIC, 4.1 and 12.9; E. histolytica- like Laredo strain, 5.0 and 20.6. The GC content of DNA, estimated by thermal elution in hydroxyapatite, was around 23% in HK9 and its clone HK9-1 and around 27% in the HM2 and HM3 E. histolytica strains; it was 37% in the Laredo strain, 26% in IP101, 35% in PZ, and 33% in FIC. The reassociation kinetics of PZ strain DNA showed that it consists of 40% repeated sequences and 60% unique sequences. By means of DNA association experiments in which one of each pair of DNAs tested had been labeled in vitro with ¹²⁵I, we found the following overall sequence homology among the strains tested: PZ-FIC, 38%; IP101-Laredo, 38%; IP101-FIC, 47%; PZ-Laredo, 49%; Laredo-FIC, 69%; and IP101-PZ, 83%. We conclude that trophozoites of different E. histolytica strains have similar nuclear size and GC content, whereas these parameters and the nucleotide sequences are clearly different in every other Entamoeba species. Our data also suggest that PZ and IP101 strains do not belong to the same species.     

Differential detection of Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii in fecal samples by nested PCR in the United Arab Emirates (UAE)

Amoebiasis is one of the most important infectious diseases afflicting mainly tropical and subtropical countries. This study was carried out in the Sharjah Emirate, UAE in order to accurately detect and differentiate Entamoeba histolytica, Entamoeba dispar and E. moshkovskii in fecal samples collected from the Sharjah municipality public health clinic by ELISA and nested polymerase chain reaction (PCR). One hundred and twenty specimens were examined and the PCR was positive for E. histolytica, E. dispar and E. moshkovskii (collectively referred to as Entamoeba complex) in 19.2% (23 out of 120). Of those, 10% (12/120) were mono — infection with E. histolytica; 2.5% (3/120) with E. dispar; and 2.5% (3/120) E. moshkovskii. The nested PCR also detected mixed infections by both E. histolytica and E. dispar in 3.3% (4/120) and E. dispar and E. moshkovskii in 0.8% (1/120). The TechLab ELISA kit failed to detect E. histolytica in any of the E. histolytica PCR positive samples. Overall, the percentage of E. histolytica including those found in mixed infections was 13.3% (16/120). Compared to nested PCR, microscopy was found to have an overall sensitivity of 52.2% and a specificity of 75.2% for detection of Entamoeba complex. The present study indicates that E. histolytica is present in the UAE with an average incidence rate of 13.3%. However, larger studies need to be conducted in order to confirm these findings. We propose the use of PCR in both the routine diagnosis of amoebiasis and epidemiological survey in the UAE.     

Entamoeba histolytica and Entamoeba dispar infections in cynomolgus monkeys imported into Japan for research

Three hundred and three stool samples of cynomolgus monkeys (Macaca fascicularis) imported from China and the Philippines were examined for Entamoeba histolytica/Entamoeba dispar infections. Microscopy detected E. histolytica/E. dispar cysts in 41 samples. Positive rates were higher in the monkeys from China (37.5%) than in the monkeys from the Philippines (3.7%). PCR analysis of 25 samples successfully cultured from the cysts demonstrated that 24 were E. dispar, one of the samples from China was E. histolytica. The one sample was also identified as E. histolytica by an antigen detection kit, although the monkey was asymptomatic and serology was negative. To our knowledge, this is the first report of E. histolytica isolation from cynomolgus monkeys based on the discrimination between E. histolytica and E. dispar.     

Role of leukocytes and amebic proteinases in experimental rat testicular necrosis produced byEntamoeba histolytica

To investigate the role of amebic proteinases and host leukocytes, we studied amebiasis experimentally in the rat testis. The degree of inflammation and necrosis produced by different strains was correlated with proteinase activity and with zymograms. Intratesticular injection of axenically grown trophozoites of a pathogenic strain (HM-1 ofEntamoeba histolytica) produced indistinguishable lesions in normal animals and leukopenic rats (<1000 leukocytes/mm³), suggesting that granulocytes do not contribute to the formation of lesions in this model. Testicular lesions produced by five different strains ofE. histolytica ranging from highly virulent to almost nonpathogenic were proportional to the proteinase activity of each amebic strain. Inhibition of amebic proteinases in vitro and subsequent injection into the rat testis markedly reduced the inflammatory lesions resulting from highly virulentE. histolytica. The pathogenicity of three other amebae (E. laredo, E. moshkovskii, andE. invadens) was generally proportional to their proteinase activity; however,E. laredo showed high proteinase activity and caused minimal tissue damage. These results suggest that the pathogenic potential ofEntamoeba spp. in the rat testis may be related to the type as well as the level of their proteinase activity.     

Comparison of <Emphasis Type="Italic">Entamoeba histolytica</Emphasis> DNA isolated from a cynomolgus monkey with human isolates

Three protein-coding loci in DNA of an Entamoeba histolytica strain (EHMfas1) isolated from cynomolgus monkey (Macaca fascicularis) were sequenced; these loci corresponded to the genes for chitinase, the serine-rich E. histolytica protein (SREHP), and the 16 S-like small subunit ribosomal RNA (16S-like SSUrRNA). The nucleotide and deduced amino-acid sequences of chitinase and SREHP were compared with sequences from human isolates. EHMfas1 had several specific mutations in units in the polymorphic regions of the chitinase and SREHP loci, with some repetition of these mutated units. The sequence of the 16S-like SSUrRNA gene (16S-like SSUrDNA) was compared with other Entamoeba species. In phylogenetic analysis, EHMfas1 was not categorized in the E. histolytica cluster but between E. histolytica and E. dispar. To our knowledge, this is the first molecular characterization of E. histolytica isolated from cynomolgus monkey, and our results indicate that EHMfas1 may be a subspecies of E. histolytica that infects cynomolgus monkey.     

Genetic differentiation of pathogenic and nonpathogenic strains ofEntamoeba histolytica by random amplified polymorphic DNA polymerase chain reaction

The random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) method was used to compare pathogenic and nonpathogenic strains ofEntamoeba histolytica. DNA polymorphisms were detected among the different strains and dendrograms were constructed by PHYLIP and PAUP analyses to study the relationship of the strains. Both analyses resulted in identical results, which indicated that pathogenic strains ofE. histolytica are closely related and clearly separated from the nonpathogenic strains. The results of this study agree with classification of the strains based on isoenzyme analyses. This suggests that RAPD-PCR is a valuable method in differentiating between strains of this parasite, and the results are consistent with the concept that pathogenic and nonpathogenicEntamoeba represent two different species.Abbreviations DNA Deoxyribonucleic acid - PAUP phylogenetic analysis using parsimony - RAPD random amplified polymorphic DNA - PCR polymerase chain reaction - PHYLIP phylogeny inference package - UPGMA unweighted pair-group method with arithmetic mean     

High prevalence of Entamoeba infections in captive long-tailed macaques in China

Long-tailed macaques (Macaca fascicularis) are bred in China for export and for use in experiments. Entamoeba infections in captive long-tailed macaques were surveyed in one of the biggest colonies located in Guangxi Province, China. One stool sample was obtained from each of the 152 different cages representing >3,000 macaques in the colony. The samples were examined by PCR for five Entamoeba species. The number of detected Entamoeba coli infections comprised 94% of the samples, 93% for Entamoeba chattoni, and 83% for Entamoeba dispar. In contrast, Entamoeba histolytica and Entamoeba nuttalli were not detected. Six isolates of E. dispar were obtained by culture in Tanabe–Chiba medium. Analysis of serine-rich protein genes in these isolates showed two genotypes, one of which is identical to that of the E. dispar SAW760 strain in humans. This suggests transmission of E. dispar between humans and nonhuman primates. These results demonstrate that Entamoeba infections are common, but virulent Entamoeba species are absent in this colony. This work also confirms the need for monitoring with PCR-based identification of Entamoeba species for captive macaques in breeding colonies to ensure animal health and protection of humans from zoonotic hazards.     

DNA characterization of simian <Emphasis Type="Italic">Entamoeba histolytica</Emphasis>-like strains to differentiate them from <Emphasis Type="Italic">Entamoeba histolytica</Emphasis>

Two simian Entamoeba histolytica-like strains, EHMfas1 and P19-061405, have been suggested to represent a new species based on genetic characterization. Sequence analyses of the hexokinase, glucose phosphate isomerase, and phosphoglucomutase genes supported the previous findings of isoenzyme analyses demonstrating a new zymodeme pattern. Phylogenetic studies of 18S rDNA, 5.8S rDNA, the chaperonin 60 gene, and the pyridine nucleotide transhydrogenase gene showed original clusters of simian E. histolytica-like strains below or near E. histolytica, respectively. Comparative studies of the chitinase and the serine-rich E. histolytica protein genes and locus 1–2 region revealed that most mutated units were shared among the simian E. histolytica-like strains. The similarities of each of the repeating units within the simian E. histolytica-like strains or E. histolytica and the differences of those between the both might be generated by concerted evolution. Our results indicate that EHMfas1 and P19-061405 should be considered to be the same species, despite that they were isolated from different monkey species and different habitats. Simian E. histolytica-like amebas may be endemic to macaque monkeys, as a counterpart to E. histolytica in humans, and should be differentiated from E. histolytica by the revival name Entamoeba nuttalli, as proposed for P19-061405.     

HLA characterization in adult asymptomatic cyst passers of Entamoeba histolytica/E. dispar

The present work aimed at studying the possible association of HLA antigens with Entamoeba histolytica/E. dispar asymptomatic infection in a Mexican mestizo population. A case-control design was selected for evaluation of the role of genetic markers in parasite infection. For this purpose the HLA-A, HLA-B, and HLA-DR profiles of a population of asymptomatic E. histolytica/E. dispar adult cyst passers (cases) and a corresponding nonparasitized adult group (controls) followed for 12 months were identified. Entamoeba species were identified through zymodeme patterns and/or amplification of species-specific DNA sequences. A healthy, nonparasitized group of individuals was included as a control. Our results show that apparently, no specific HLA marker is associated with the asymptomatic cyst passers' condition. These findings have to be added to previous results in which, in contrast to a demonstrated association between HLA-DR3 and amebic liver abscess in Mexican mestizo adults and infants, no significant association with amebic rectocolitis was found. Received: 18 March 1999 / Accepted: 16 April 1999     

Comparison of serine-rich protein genes of Entamoeba histolytica isolates obtained from institutions for the mentally retarded in Kanagawa and Shizuoka Prefectures, Japan

In Japan, amebiasis has been observed in homosexual men, in institutionalized persons, and in overseas travelers. We have previously reported an outbreak of amebiasis that occurred from 1986 to 1994 in institutions for the mentally retarded in Kanagawa and Shizuoka Prefectures in Eastern Japan. Entamoeba histolytica but not Entamoeba dispar was identified in Entamoeba cultures obtained from cyst passers in four institutions located in different municipalities in this region. In the present study, serine-rich protein genes of eight isolates from the four institutions were sequenced, and their polymorphism was analyzed. The results showed that all the sequences from the E. histolytica isolates were identical. This retrospective study led us to conclude that the outbreak of amebiasis in different municipalities was derived from a single source of E. histolytica.     

Adherence of pathogenic and non-pathogenicEntamoeba histolytica strains to neutrophils

The adherence of polymorphonuclear leukocytes (PMNs) to eight pathogenic and nine nonpathogenic strains ofEntamoeba histolytica was examined. No difference between pathogenic and nonpathogenic strains was found. The addition of different carbohydrates confirmed the importance of the 170-kDa lectin ofE. histolytica in binding to PMNs, corroborated by the finding that treatment of PMNs with galactosidase inhibited adherence. Inhibition of the microfilament system ofE. histolytica using cytochalasin B resulted in a loss of adherence to PMNs. Inhibition of the microtubule system using nocodazole did not affect adherence. Preincubation of the trophozoites with serum resulted in enbanced adherence, but the serum factor responsible for this effect could not be identified. Fibronectin, vitronectin, integrins (CD11/CD18 molecules), complement, and mannose-binding protein did not seem to mediate adherence betweenE. histolytica and PMNs. In summary, these results indicate that defective adherence mechanisms are not a common feature of nonpathogenicE. histolytica strains.     

Comparison of two immunoassays for detection of Entamoeba histolytica

Effective diagnostic tools are essential in order to combat disease caused by the parasite Entamoeba histolytica. In this study, we compared the commercially available Ridascreen Entamoeba test (R-Biopharm) and the E. histolytica II test (Techlab), and we found that the E. histolytica II test detects E. histolytica infections more accurately.     

The pyruvate:ferredoxin oxidoreductase enzyme is located in the plasma membrane and in a cytoplasmic structure inEntamoeba

This work investigated the cellular location of the pyruvate:ferredoxin oxidoreductase (PFO) enzyme inEntamoeba. A 1.9 kb fragment located at the 3 end of theEhpfogene was cloned in the pRSETB vector and expressed. The recombinant peptide was purified and inoculated in rabbits. By Western blot assays the antibodies detected a single 130 kDa band in allE. histolyticastrains tested and inE. moshkovskii. By immunofluorescence, the antibodies showed the presence of PFO in the plasma membrane and in a cytoplasmic structure that appeared as a ring or as a compact small body inE. histolyticastrains. InE. invadensandE. moshkovskii(strains FIC and Laredo) PFO was located in the plasma membrane showing different fluorescence patterns. Immunofluorescence onE. histolyticasynchronized cultures showed that the cytoplasmic structure appeared in 85, 60, 20 and 10% of the trophozoites in mitosis, G1, S and G2 phases, respectively. Byin situhibridization theEhpfogene was found in the nuclei and the trophozoites of the clone A, strain HM1:IMSS, differed in theEhpfogene content.     

Knockdown of Five Genes Encoding Uncharacterized Proteins Inhibits Entamoeba histolytica Phagocytosis of Dead Host Cells

Entamoeba histolytica is the protozoan parasite that causes invasive amebiasis, which is endemic to many developing countries and characterized by dysentery and liver abscesses. The virulence of E. histolytica correlates with the degree of host cell engulfment, or phagocytosis, and E. histolytica phagocytosis alters amebic gene expression in a feed-forward manner that results in an increased phagocytic ability. Here, we used a streamlined RNA interference screen to silence the expression of 15 genes whose expression was upregulated in phagocytic E. histolytica trophozoites to determine whether these genes actually function in the phagocytic process. When five of these genes were silenced, amebic strains with significant decreases in the ability to phagocytose apoptotic host cells were produced. Phagocytosis of live host cells, however, was largely unchanged, and the defects were surprisingly specific for phagocytosis. Two of the five encoded proteins, which we named E. histolytica ILWEQ (EhILWEQ) and E. histolytica BAR (EhBAR), were chosen for localization via SNAP tag labeling and localized to the site of partially formed phagosomes. Therefore, both EhILWEQ and EhBAR appear to contribute to E. histolytica virulence through their function in phagocytosis, and the large proportion (5/15 [33%]) of gene-silenced strains with a reduced ability to phagocytose host cells validates the previously published microarray data set demonstrating feed-forward control of E. histolytica phagocytosis. Finally, although only limited conclusions can be drawn from studies using the virulence-deficient G3 Entamoeba strain, the relative specificity of the defects induced for phagocytosis of apoptotic cells but not healthy cells suggests that cell killing may play a rate-limiting role in the process of Entamoeba histolytica host cell engulfment.     

A gene highly homologous to ACP1 encoding cysteine proteinase 3 in Entamoeba histolytica is present and expressed in E. dispar

 We have cloned the complete gene encoding cysteine proteinase 3 in E. histolytica as well as a cDNA encoding a highly homologous protein in E. dispar. In addition, we have demonstrated the presence and expression of the CP3 gene in various E. histolytica and E. dispar isolates. Since the expression of the gene is rather similar within both Entamoeba species, we assume that it does not constitute the proposed virulence factor of E. histolytica. Received: 25 May 1995 / Accepted: 29 August 1995