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.     

Characteristic protein electrophoretic patterns of fourEntamoeba strains

Summary Total proteins from trophozoites of four axenized strains of the histolytica group ofEntamoeba (twoE. histolytica, oneE. invadens, and oneE. moshkovskii) were analyzed by disc-electrophoresis in polyacrylamide sodium dodecyl sulphate slab gels and by radial immunodiffusion. Each strain had a characteristic and reproducible electrophoretic pattern with about 30 major protein bands distributed between molecular weights 12,000 and 110,000. A striking similarity was found between the protein patterns of bothE. histolytica strains. Immunodiffusion also resulted in characteristic patterns, in which the greatest resemblance occurred in the same strains. The sensitivity and high resolution of the electrophoretic method for protein analysis and its correlation with antigenic characterization suggests its use as a taxonomic criterion of the histolytica group of the genusEntamoeba.     

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.     

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.     

<Emphasis Type="Italic">Entamoeba moshkovskii</Emphasis> infections in Sydney,Australia

Entamoeba moshkovskii is considered to be a free-living ameba, which is morphologically similar, but biochemically and genetically different, to Entamoeba histolytica and Entamoeba dispar. However, recent studies have suggested that E. moshkovskii may be a “potential” pathogen, with infections giving rise to diarrhea and other intestinal disorders. Microscopy and polymerase chain reaction (PCR) targeting the 18S ribosomal (r) DNA was performed on fecal samples collected from patients presenting with diarrhea and other gastrointestinal disorders (test group), as well as fecal samples collected from healthy controls (control group). Of the 110 patients microscopically positive for the Entamoeba species, 55/110 (50%) samples were positive for E. moshkovskii in the test group of patients presenting with diarrhea. E. moshkovskii was the only pathogen detected (including bacteria or viruses) in 3/55 (5.5%) of the test group of patients presenting with diarrhea and abdominal pain. The DNA of E. moshkovskii was not detected in the fecal samples collected from the healthy controls. These results suggest that E. moshkovskii may not simply be a commensal of the human gastrointestinal tract and provides evidence for E. moshkovskii as a “potential” pathogen in the case of diarrhea and other gastrointestinal disorders. Further studies are needed to determine the role of E. moshkovskii in causing diarrheal diseases in our population.     

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.     

Lectin-induced agglutination of trophozoites of different species and strains ofEntamoeba

In vitro agglutinability of trophozoites of threeEntamoeba histolytica strains, cultivated under axenic conditions in the presence of concanavalin A (Con A), was shown to be related to the degree of their pathogenicity for experimental animals and of the concentration of Con A. Seven strains ofE. invadens tested also agglutinated in the presence of Con A, and the degree of agglutination was proportional to the concentration of the lectin. Three strains ofE. histolytica-like Laredo-type amebae, a strain ofE. terrapinae, and a strain ofE. moshkovskii agglutinated very slightly, only in the presence of the highest concentration of Con A tested.     

DNA polymerase activity in encysting Entamoeba invadens

Using an axenic encystation system of Entamoeba invadens as a model for E. histolytica encystation, we examined the level of DNA polymerase activity in E. invadens during encystation induced in vitro. We first characterized the DNA polymerase activity of trophozoites of E. invadens, comparing it with that of E. histolytica, and found that the activity of E. invadens was lower than that of E. histolytica at pH 2, 4, and 6 and was higher at pH 8 and 10. The activity of E. invadens was completely inhibited by high concentrations of K⁺. Among inhibitors of mammalian DNA polymerases, aphidicolin and N-ethylmaleimide inhibited the activity, but 2′,3′-dideoxythymidine-5′-triphosphate did not. Thus, the sensitivity of the E. invadens activity to salt and inhibitors of mammalian DNA polymerases was basically the same as that recorded for E. histolytica in our previous results. The level of DNA polymerase activity in cysts decreased as encystation proceeded as compared with that of trophozoites. The results indicate that encystation is accompanied by a reduced level of DNA polymerase activity, which correlates with the previous finding that nuclear division occurs during cyst maturation in the absence of DNA synthesis. Received: 28 November 1998 / Accepted: 16 December 1998     

Effect of cytochalasin D on the growth,encystation, and multinucleation of <Emphasis Type="Italic">Entamoeba invadens</Emphasis>

The effect of cytochalasin D, a specific inhibitor of microfilaments, on the growth, encystation, and multinucleation of Entamoeba invadens was examined. Cytochalasin D blocked the growth of axenic E. invadens strain IP-1 in a dose-dependent manner, which suggests that the drug is effective against this species of Entamoeba as well as against E. histolytica strain HM1: IMSS as previously demonstrated. Encystation of E. invadens as induced in vitro was also inhibited by cytochalasin D. This is the first evidence of the participation of microfilaments in the encystation process. Concentrations of cytochalasin D effective for the inhibition of encystation were lower than those effective for the inhibition of growth. Trophozoites grown with cytochalasin D became multinucleate; more than three nuclei per cell were observed in 71% of trophozoites grown in the presence of the drug as opposed to only 5% of those grown in the absence of the drug. Also, trophozoites grown with cytochalasin D produced multinucleate cysts following their transfer to encystation medium. Encystation with cytochalasin D was more strongly inhibited among trophozoites grown in the presence of the drug than among those grown in the absence of the drug. Also, encystation without cytochalasin D was less frequently observed among trophozoites grown in the presence of the drug than among those grown in the absence of the drug. Thus, the multinucleation of trophozoites induced by cytochalasin D had an inhibitory effect on their encystation. Received: 28 September 1999 / Accepted: 25 November 1999     

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     

Development of RNA Interference Trigger-Mediated Gene Silencing in Entamoeba invadens

Entamoeba histolytica, a protozoan parasite, is an important human pathogen and a leading parasitic cause of death. The organism has two life cycle stages, trophozoites, which are responsible for tissue invasion, and cysts, which are involved in pathogen transmission. Entamoeba invadens is the model system to study Entamoeba developmental biology, as high-grade regulated encystation and excystation are readily achievable. However, the lack of gene-silencing tools in E. invadens has limited the molecular studies that can be performed. Using the endogenous RNA interference (RNAi) pathway in Entamoeba, we developed an RNAi-based trigger gene-silencing approach in E. invadens. We demonstrate that a gene''s coding region that has abundant antisense small RNAs (sRNAs) can trigger silencing of a gene that is fused to it. The trigger fusion leads to the generation of abundant antisense sRNAs that map to the target gene, with silencing occurring independently of trigger location at the 5′ or 3′ end of a gene. Gene silencing is stably maintained during development, including encystation and excystation. We have used this approach to successfully silence two E. invadens genes: a putative rhomboid protease gene and a SHAQKY family Myb gene. The Myb gene is upregulated during oxidative stress and development, and its downregulation led, as predicted, to decreased viability under oxidative stress and decreased cyst formation. Thus, the RNAi trigger silencing method can be used to successfully investigate the molecular functions of genes in E. invadens. Dissection of the molecular basis of Entamoeba stage conversion is now possible, representing an important technical advance for the system.     

Increase of DNA content in tissue stages ofEntamoeba histolytica strain SFL 3

The DNA content of culture forms and tissue stages of pathogenicE. histolytica strain SFL 3 were measured photometrically after the nuclei had been stained with the fluorochrome BAO. As a control, the DNA guartity ofE. histolytica strain HK 9 andE. invadens were determined by the same method and compared with reference values. Tissue stages were obtained from hamsters experimentally infected by intrahepatic injection of SFL 3 amoebae. Further studies concerning possible changes in the DNA content of tissue stages involved the following methods: (a) isolation of tissue stages from the liver, followed by distinct suspension periods. (b) Infected liver pieces were directly transferred into culture medium; amoebae emigrating therefrom were cultivated. The study demonstrated that tissue stages contained up to 4 times more DNA than did culture forms. After 3 h cultivation, the DNA content of tissue stages decreased to the level of culture forms. Possible reasons for this change are discussed.Supported by Deutsche Forschungsgemeinschaft (DFG)     

Effects of Panmede and various horse serum concentrations on the axenic cultivation ofEntamoeba histolytica strains in TPS-1 medium

We analyzed the influence of Panmede and horse serum concentrations on the growth of fiveEntamoeba histolytica strains (HK9, HM1, HM2, HM3 and HM38) axenically cultivated in TPS-1 medium. Panmede was evaluated by comparing the growth of strain HM1 in medium prepared with each of 15 Panmede lots; the yields ofE. histolytica trophozoites depended on the lot quality of Panmede, and their maximal values ranged from 8×10³ to 8.9×10⁴ amoebae/ml. The growth-promoting effect of eight lots of horse serum on strains HK9 and HM1 were studied using a single Panmede lot of good quality. Yields obtained with strain HK9 ranged from 8×10⁴ to 1.8×10⁵ amoebae/ml, whereas yields obtained with HM1 ranged from 3×10⁴ to 1.2×10⁵ amoebae/ml. Thus, the optimal serum concentration in TPS-1 medium that caused maximal growth ofE. histolytica cultures depended on the quality of the serum lot and proved to be specific for each of the fiveE. histolytica strains investigated. It ranged from 18% (v/v) for strain HM2 to 28% (v/v) for strain HM1. Our results reveal that the growth ofE. histolytica trophozoites in TPS-1 medium can be distinctly improved by selecting appropriate lots of Panmede and horse serum and using optimal serum concentrations.     

Entamoeba invadens: Identification of a SERCA protein and effect of SERCA inhibitors on encystation

Calcium has an important role on signaling of different cellular processes, including growth and differentiation. Signaling by calcium also has an essential function in pathogenesis and differentiation of the protozoan parasites Entamoeba histolytica and Entamoeba invadens. However, the proteins of these parasites that regulate the cytoplasmic concentration of this ion are poorly studied. In eukaryotic cells, the calcium-ATPase of the SERCA type plays an important role in calcium homeostasis by catalyzing the active efflux of calcium from cytoplasm to endoplasmic reticulum. Here, we reported the identification of SERCA of E. invadens (EiSERCA). This protein contains a putative sequence for endoplasmic reticulum retention and all domains involved in calcium transport identified in mammalian SERCA. By immunofluorescence assays, an antibody against SERCA of E. histolytica detected EiSERCA in a vesicular network in the cytoplasm of E. invadens trophozoites, co-localizing with calreticulin. Interestingly, EiSERCA was redistributed close to plasma membrane during encystation, suggesting that this pump could participate in regulate the calcium concentration during this process. In addition, thapsigargin and cyclopiazonic acid, both specific inhibitors of SERCA, affected the number and structure of cysts, supporting the hypothesis that calcium flux mediated by SERCA has an important role in the life cycle of Entamoeba.     

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.     

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.     

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.     

Prevalence and genetic diversity of Entamoeba histolytica in an institution for the mentally retarded in the Philippines

A total of 113 mentally retarded patients residing in a mental institution in Metropolitan Manila, Philippines, were screened for the presence of Entamoeba histolytica based on microscopy and polymerase chain reaction (PCR). Anti-E. histolytica antibodies were also screened in 97 serum samples collected using immunofluorescence antibody (IFA) test. Parasitological examination showed E. histolytica/Entamoeba dispar in 43 cases (38.05%), while PCR detected 74 cases (65.48%) positive for E. histolytica and 6 cases (5.30%) positive for E. dispar. Interestingly, these 6 samples were coinfected with E. histolytica. IFA test revealed that 80.41% (78/97) of the respondents possessed significant antibody titers for intestinal infection of E. histolytica. Of this number, there were 5 patients negative in IFA test but positive in PCR. The genetic diversity of E. histolytica isolates was also investigated by analyzing polymorphism in the serine-rich gene by nested PCR on DNA directly extracted from stool specimens. A combination of the nested PCR results and the AluI digestion of the PCR products examined yielded six distinct DNA banding patterns among the 74 stool isolates. An apparent clustering of E. histolytica strains was observed in patients living in different residential cottages of the institution. These results indicate the high prevalence of E. histolytica in an institution for the mentally retarded in the Philippines.     

Effect of the antitubulin drug oryzalin on the encystation of Entamoeba invadens

We have recently demonstrated that the antimicrotubule drug oryzalin inhibits the growth of Entamoeba invadens as well as E. histolytica, the former being more resistant to the drug than the latter, and that effective doses of oryzalin are higher for Entamoeba than for the other parasitic protozoa examined thus far. The aim of the present study was to examine the effect of oryzalin on the encystation of E. invadens using an axenic encystation system in vitro. Oryzalin inhibited the encystation of E. invadens strain IP-1 in a dose-dependent manner. The addition of oryzalin after the induction of encystation was also inhibitory for encystation and cyst maturation. Trophozoites incubated for 1 day in encystation medium with oryzalin did not encyst after removal of the drug. Although trophozoites grown in the presence of 300 μM oryzalin for 2 days did not encyst after their transfer to encystation medium containing the same concentration of drug, a number of trophozoites survived for at least 3 days. In contrast, trophozoites grown in the absence of oryzalin neither survived nor encysted after their transfer to encystation medium supplemented with the drug, which suggests that pretreatment of trophozoites with oryzalin contributes to their continued survival as trophozoites, i.e., without their transforming into cysts, in encystation medium. Trophozoites grown with oryzalin did encyst after their transfer to encystation medium without the drug. Accumulation of trophozoites in the mitotic phase was observed after culture with oryzalin. When cysts prepared at day 1 of encystation, most of which were mononucleate, were reincubated in the presence of oryzalin for an additional 2 days, inhibition of their maturation was observed. Thus, oryzalin is a potent mitotic-phase inhibitor of E. invadens and may become a useful tool for studies on the relationship between the cell cycle and encystation of this parasite. Received: 29 November 1999 / Accepted: 28 January 2000