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     

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     

Detection and characterization of DNA polymerase activity in Entamoeba histolytica

 DNA polymerase activity was detected and characterized in nuclear extracts from trophozoites of Entamoeba histolytica. The activity was high at pH 2 to pH 6, but at pH 8 and 10 the activity was very low. The presence of K⁺ was inhibitory for the activity and a higher concentration of K⁺ markedly inhibited the activity. Magnesium ions (Mg²⁺) were absolutely required for activity and its optimal concentration was 6 to 8 mM. The activity was markedly inhibited by aphidicolin which is an inhibitor of mammalian DNA polymerases α, δ, and ɛand also by N-ethylmaleimide which is an inhibitor of DNA polymerases, α, γ, δand ɛ. However, inhibition of the activity by 2’, 3’-dideoxythymidine-5’-triphosphate which is an inhibitor of DNA polymerases βand γwas relatively weak. Thus sensitivity of the E. histolytica enzyme to these inhibitors was similar to that of mammalian DNA polymerases (α, δand ɛ) of the αfamily. Monoclonal antibodies against human DNA polymerase αdid not bind to DNA polymerase of E. histolytica. Received: 19 April 1995 / Accepted: 16 June 1995     

Construction and rapid screening of a representative yeast artificial chromosome library from the Plasmodium falciparum strain Dd2

DNA polymerase activity was detected and characterized in nuclear extracts from trophozoites of Entamoeba histolytica. The activity was high at pH 2 to pH 6, but at pH 8 and 10 the activity was very low. The presence of K⁺ was inhibitory for the activity and a higher concentration of K⁺ markedly inhibited the activity. Magnesium ions (Mg⁺) were absolutely required for activity and its optimal concentration was 6 to 8 mM. The activity was markedly inhibited by aphidicolin which is an inhibitor of mammalian DNA polymerases α, 8, and ε and also by N-ethylmaleimide which is an inhibitor of DNA polymerases, α, γ δ and ε. However, inhibition of the activity by 2′, 3′-dideoxythymidine-5′-triphosphate which is an inhibitor of DNA polymerases β and γ was relatively weak. Thus sensitivity of the E. histolytica enzyme to these inhibitors was similar to that of mammalian DNA polymerases (α, δ and ε) of the α family. Monoclonal antibodies against human DNA polymerase α did not bind to DNA polymerase of E. histolytica.     

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.     

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.     

High prevalence of infection with Entamoeba dispar, but not E. histolytica, in captive macaques

A total of 268 nonhuman primates (20 species) kept in the Primate Research Institute, Kyoto University, Japan, were surveyed for intestinal amebas. Total positive rates as based on the presence of cysts in the stool following formalin-ether sedimentation were as follows: Entamoeba histolytica/E. dispar, 53%; E. coli, 34%; E. hartmanni, 34%; Iodamoeba buetschlii, 25%; Endolimax nana, 8%; and E. chattoni, 3%. Positive rates were higher in Old World monkeys and lower in New World monkeys. All the 141 E. histolytica/E. dispar-positive animals were Macaca monkeys. The E. histolytica/E. dispar-positive samples were analyzed by polymerase chain reaction (PCR) for identification of E. histolytica and E. dispar. E. dispar DNA was detected in 137 samples, whereas no E. histolytica DNA was seen. Zymodeme analysis and reactivity to monoclonal antibodies of cultured trophozoites also supported the presence of E. dispar and the absence of E. histolytica. When the sera of 93 macaques were examined by an indirect fluorescent antibody test, only 3 animals proved to be positive for E. histolytica, showing the lowest titer. These results demonstrate that infection with E. dispar, but not E. histolytica, is predominant in macaques. Received: 23 March 2000 / Accepted: 3 July 2000     

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.     

Appearance of a stage-specific immunodominant glycoprotein in encysting Entamoeba invadens

The appearance of cyst-specific proteins in encysting Entamoeba invadens and their immunogenicity were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting using an axenic encystation system in vitro. A rabbit antiserum against trophozoites of E. invadens reacted with a number of proteins of cysts after 1–4 days of incubation. Thus, a number of cyst proteins remained antigenically unchanged as common antigens of the two forms after transformation from trophozoites to cysts. A rabbit antiserum against cysts also reacted with the trophozoite proteins as well as the cyst proteins. The most interesting result was that the rabbit anticyst serum reacted predominantly with an 88-kDa protein of cysts after 1 day of incubation. The 88-kDa protein reacted with the anticyst serum absorbed with trophozoite proteins and was thus cyst-specific. The reactivity of the 88-kDa protein of cysts with the absorbed anticyst serum decreased as encystation proceeded. When soluble and particulate fractions prepared from cysts after 1 day of incubation were examined by electrophoresis and immunoblotting, the 88-kDa protein that had reacted with the absorbed anticyst serum was found to be present in the particulate fraction, which was rich in cell-wall fragments, and stained with periodic acid-Schiff 's reagent, indicating that it is a glycoprotein. The results indicate that encystation is accompanied by appearance of the cyst-specific 88-kDa glycoprotein, which is immunodominant and most abundantly expressed in cysts after 1 day of incubation and appears to be associated with the cyst wall. Received: 31 May 1999 / Accepted: 20 July 1999     

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.     

Protection of hamsters from amebic liver abscess formation by a monoclonal antibody to a 150-kDa surface lectin of Entamoeba histolytica

We examined the effects of passive immunization with a monoclonal antibody (EH3015) that recognizes a 150-kDa surface lectin of Entamoeba histolytica on amebic liver-abscess formation in hamsters. The hamsters were inoculated i.p with 0.1, 1.0, or 10 mg of EH3015 at 24 h prior to an intrahepatic challenge with 10⁵ trophozoites of E. histolytica. In hamsters treated with 1.0 and 10 mg of EH3015 the incidence of liver abscesses was significantly reduced. These results demonstrate that monoclonal antibody EH3015 can prevent the development of amebic liver abscesses and that the 150-kDa lectin may be a protective antigen on the surface of E. histolytica. Received: 5 May 1998 / Accepted: 28 May 1998     

Entamoeba histolytica : production of nitric oxide and in situ activity of NADPH diaphorase in amebic liver abscess of hamsters

Entamoeba histolytica trophozoites were inoculated into the liver of hamsters and serum nitrate/nitrite levels [expressed as nitric oxide (NO) production] were determined at different times during amebic liver abscess (ALA) development. We also tested the effects of NO synthase (NOS) inhibitors such as N ^G -nitro-L-arginine methyl ester (L-NAME), aminoguanidine, and dexamethasone during ALA production. Since NOS activity has been correlated with expression of reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) in tissues, we performed histochemistry studies to determine the activity of the latter in livers infected with E. histolytica trophozoites. Production of NO in serum was directly proportional to the size of ALAs, and NOS inhibitors caused low levels of NO and smaller ALAs. Our data suggest that NO does not have any lytic effect on E. histolytica trophozoites and is therefore incapable of providing protection against the amebic liver infection. In addition, NADPHd activity was detected histochemically in hepatocytes and inflammatory cells associated with focal necrosis containing trophozoites. The positive reactivity observed in these parasites may be attributable to a close biochemical similarity of NADPHd to the NADPH:flavin oxidoreductase described in E. histolytica by other investigators. Received: 16 February 2000 / Accepted: 19 June 2000     

Detection of Entamoeba dispar DNA in macaque feces by polymerase chain reaction

We used the polymerase chain reaction (PCR) to determine the prevalence of Entamoeba histolytica and E. dispar in the wild population of macaque monkeys (Macaca fuscata) in Mt. Takasaki, Oita Prefecture, Japan. Of the 101 samples collected, 41 (42.57%) were found to be positive for E. dispar. However, no E. histolytica was detected from the collected samples. The results of this survey demonstrate the high prevalence of E. dispar in macaque monkeys in the study area. Moreover, they provide additional baseline information on naturally acquired infectious agents of macaque monkeys and offer an accurate tool for detection of E. histolytica and E. dispar, which are needed for biomedical research using nonhuman primate models. Received: 25 September 1998 / Accepted: 15 December 1998     

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.     

Interaction of antibodies with Entamoeba histolytica trophozoites from experimental amebic liver abscess: an immunocytochemical study

Using immunocytochemical techniques, we studied the interaction of antibodies with Entamoeba histolytica trophozoites present during the development of amebic liver abscess. Hamsters were intrahepatically inoculated with HM1-IMSS axenic amebas and sacrificed at different days post-inoculation. IgG of rabbit anti-E. histolytica and IgG of rabbit anti-IgG of hamster were used, both labeled with peroxidase. With the rabbit anti-E. histolytica, all trophozoites present in hepatic lesions from 1–7 days post-inoculation were highly labeled. The IgG of rabbit anti-IgG of hamster intensively stained only those trophozoites present in lesions from 1–2 days post-inoculation. From day 3, the intensity and number of labeled trophozoites decreased progressively. The results suggest that the interaction between the amebas and the IgG of hamster is non-specific during the first 2 days. The absence of labeling in the chronic stages could be due to changes in the membrane antigens of the parasite or to alterations in the bloodstream around necrosis. Also, the anti-E. histolytica antibodies produced in the serum during the development of the hepatic disease are apparently incapable of reaching and interacting with the trophozoites present on the liver abscess. This can explain in part why antibodies do not have an important role in the defense of the host. Received: 26 September 1999 / Accepted: 24 November 1999     

Protection of hamsters from amebic liver abscess formation by immunization with the 150- and 170-kDa surface antigens of Entamoeba histolytica

A monoclonal antibody, EH3015, prevents in vitro adherence of Entamoeba histolytica trophozoites to mammalian cells and inhibits amebic liver abscess formation in hamsters. By immunoaffinity chromatography with the monoclonal antibody, purified E. histolytica antigens with molecular masses of 150 and 170 kDa under non-reduced conditions were obtained. Hamsters were immunized with these antigens (group I) or with fractions further purified by polyacrylamide gel electrophoresis (group II). Pooled immune sera from the two groups inhibited in vitro amebic adherence to Chinese hamster ovary cells by 98% at 1:10 dilutions. The immunized hamsters were challenged by the intrahepatic injection of E. histolytica trophozoites. Complete protection from abscess formation was observed in 38% of hamsters in group I and 67% in group II, whereas all control hamsters inoculated only with adjuvant developed amebic liver abscesses. In the immunized hamsters, the abscesses in the two groups were significantly smaller than in the controls. These results demonstrate that the E. histolytica antigens are possible vaccine candidates for amebiasis. Received: 10 August 2000 / Accepted: 5 September 2000     

Trophozoites of <Emphasis Type="Italic">Entamoeba histolytica</Emphasis> express a CD59-like molecule in human colon

In vitro studies have proved the presence of epitopes of CD59 in the surface of trophozoites of Entamoeba histolytica (E. histolytica). However, it has not been proved if CD59 molecules are expressed in the surface during the trophozoites’ tissue invasion. The aim of the present study was to determine whether the complement-regulatory protein CD59 is present on trophozoites of E. histolytica in human colon. Eleven specimens of amoebic colitis were studied by immunohistochemistry and electron microscopy techniques with a monoclonal antibody against human CD59 molecule. Our results show that a CD59-like molecule is expressed in trophozoites of E. histolytica found in colonic amebic lesions. Also, a CD59-like molecule was detected by western blot analysis in whole lysate of E. histolytica as well as on the plasma membrane by immunocytochemistry. These results suggest that E. histolytica can use CD59-like protein against the lytic action of membrane attack complex.     

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.