Expression analysis of Entamoeba invadens profilins in encystation and excystation.

Cell motility by actin cytoskeleton is essential for differentiation processes of excystation and encystation of Entamoeba. We recently studied an actin depolymerizing factor (ADF)/cofilin (Cfl) of Entamoeba invadens (Ei), and demonstrated its contribution to the encystation and excystation of E. invadens through actin cytoskeletal reorganization. Profilin is also an actin-binding protein but its function is different from that of Cfl in actin assembly. This study investigated E. invadens profilins in relation to encystation and excystation which were induced in axenic culture systems. A homology search of the E. invadens genome database and molecular cloning identified four profilins of the parasite named EiPFN1, EiPFN2, EiPFN3, and EiPFN4. There were also multiple genes of profilin in Entamoeba histolytica (Eh) and Entamoeba dispar (Ed), each of which had three profilins. A search for conserved domains revealed that these profilins of Entamoeba had actin, phosphoinositide, and poly-proline binding sites. Phylogenetic analysis revealed that EiPFN3 and EiPFN4 formed the same clades including EhPFN3 and EdPFN3, and EhPFN2 and EdPFN2, respectively, while EiPFN1 and EiPFN2 were separated from EhPFN1 and EdPFN1. Rabbit anti-EiPFN1 serum reacted with recombinant EiPFN3 and EiPFN4 but not EiPFN2, and also reacted with EiPFN in lysates of cysts and trophozoites. Immunofluorescence staining with this antiserum showed co-localization of EiPFN with actin beneath the cell membrane through the life stages and also showed cytoplasmic localization. Both proteins proved to be rich in pseudopodia of trophozoites. Real-time RT-PCR showed that the mRNA level of EiPFN1 and EiPFN4 in trophozoites was comparable but that of EiPFN2 and EiPFN3 was very low. During encystation, the mRNA expression of EiPFN1 and EiPFN4 increased remarkably in the early phase much higher than that of EiPFN2 and EiPFN3. Then, the expression of all four PFNs sharply decreased in the later phase. This was in contrast to the sharp decrease in the mRNA level of EiCfl-2 during encystation in our previous study. In cysts, EiPFN1 was most abundantly expressed and EiPFN4 was at a lower level, while the expressions of EiPFN2 and EiPFN3 were virtually absent. Following the induction of excystation, mRNA levels of EiPFN1, EiPFN2, and EiPFN4 in cysts 5 h after induction were significantly higher than those in cysts before induction, while that of EiPFN3 was slightly higher than before induction. The mRNAs of EiPFN1 increased most extensively when the excystation was induced in the presence of cytochalasin D. Small interfering RNA (siRNA) to EiPFN1 inhibited both encystation and excystation but not growth. These findings demonstrate different expression of EiPFNs and the contribution of EiPFN to the encystation and excystation.

Different structure and mRNA expression of Entamoeba invadens chitinases in the encystation and excystation.

Entamoeba histolytica forms chitin-walled cysts during encystation process, where formation of the cyst wall needs not only chitin synthase but also chitinase. During excystation, quadruplet amoebae emerge from the chitin-walled cysts by dissolving the wall, so that chitinase may be necessary for excystation process as well. There is, however, no report on chitinase expression during excystation. In this study, we used Entamoeba invadens, a reptilian amoeba, as a model for encystation and excystation of E. histolytica, and studied chitinase mRNA expression in those processes. Although expression of three E. invadens chitinases designated EiChit1, EiChit2, and EiChit3 during encystation has been reported, we identified another enzyme named as EiChit4 in the E. invadens genome database. Therefore, we investigated the primary structure and mRNA expression of these four chitinases of Ei in the excystation as well as the encystation by real-time reverse transcription polymerase chain reaction (RT-PCR). Like EiChit1, EiChit4 had an 8 × Cys chitin-binding domain (CBD) and a hydrophilic spacer between the CBD and catalytic domain, and was also closer to EiChit1 than EiChit2 and EiChit3 in the phylogenetic tree. During encystation, the expression of all four chitinases increased in the early phase; the increase in EiChit1 and EiChit4 was much higher than in EiChit2 and EiChit3. Then, the expression of all four chitinases sharply decreased in the later phase. In cysts, EiChit1 was most abundantly expressed and EiChit4 was at a lower level, while the expressions of EiChit2 and EiChit3 were virtually absent. Following the induction of excystation, mRNA levels of EiChit1 and EiChit4 in cysts 5 h after induction were significantly lower than those in cysts before induction, while those of EiChit2 and EiChit3 were remarkably higher than before induction. The mRNAs of only EiChit2 and EiChit3 remarkably increased when the excystation was induced in the presence of cytochalasin D. These data demonstrate different structures and expressions of four chitinases in the differentiation of E. invadens.

Entamoeba invadens: identification of ADF/cofilin and their expression analysis in relation to encystation and excystation.

The differentiation processes of excystation and encystation of Entamoeba are essential for infection and completion of their life-cycle, and the processes need cell motility and its control by actin cytoskeletal reorganization. This study investigated actin depolymerizing factor (ADF)/cofilin (Cfl) family proteins, which are important molecules in actin cytoskeletal reorganization, in Entamoeba invadens in relation to the encystation and excystation. Axenic culture systems were used to induce encystation and excystation. A homology search of the E. invadens genome database and molecular cloning identified three ADF/Cfl family proteins of the parasite (named for short as EiCfl-1, EiCfl-2, and EiCfl-3). This is different from other Entamoeba species, i.e. Entamoeba histolytica and Entamoeba dispar, each of which has only one ADF/Cfl family protein. These ADF/Cfl of E. invadens do not have Ser3 (serine locates third from first methionine), similar to E. histolytica, E. dispar, Saccharomyces cerevisiae and Schizosaccharomyces pombe, although the activity of ADF/Cfl is negatively regulated by phosphorylation of the Ser3 in metazoans. Phylogenetic analysis revealed that Entamoeba Cfl formed a distinctive clade that is separate from other organisms, and the branches of the tree were separated in two consistent with the presence and absence of Ser3. Rabbit anti-EiCfl-2 serum reacted with all recombinant EiCfls and EiCfl in lysates of cysts, trophozoites and metacystic amoebae. Immunofluorescence staining with this antiserum showed co-localization of EiCfl with actin beneath the cell membrane through the life stages. Both proteins proved to be rich in pseudopodia of trophozoites and metacystic amoebae. Real-time RT-PCR showed that mRNAs of EiCfl-2 and actins were highly expressed, but there were few mRNA of EiCfl-1 and EiCfl-3. Remarkably decreased mRNA levels were observed in EiCfl-2 and actins during encystation. All three EiCfls and actins became transcribed after the induction of excystation. The mRNAs of only EiCfl-1 and EiCfl-3 increased remarkably when the excystation was induced in the presence of cytochalasin D. These findings demonstrate that EiCfl-2 and actins co-localize beneath the cell membrane in trophozoites and cysts as well as metacystic amoebae being rich in pseudopodia, that EiCfl-1 and EiCfl-3 are expressed only after the induction of excystation, and that enhanced excystation by cytochalasin D is associated with high expression of EiCfl-1 and EiCfl-3.

Involvement of serine proteases in the excystation and metacystic development of Entamoeba invadens.

Although the functions of cysteine proteases involved in the pathogenicity and differentiation of Entamoeba histolytica have been demonstrated, little is known about the functions of serine proteases. We examined the involvement of serine proteases in amoebic excystation and metacystic development using inhibitors specific for serine proteases. Entamoeba invadens IP-1 strain was used as the model of excystation and metacystic development of E. histolytica. Four serine protease inhibitors, phenylmethanesulfonyl fluoride (PMSF), 4-(2-aminoethyl) bezensulfonylfluoride hydrochloride, 3, 4-dichloroisocoumarin, and N-tosyl-phe-chloromethylketone, decreased the number of metacystic amoebae in a dose-dependent manner, without showing cytotoxicity to cysts. PMSF inhibited not only the increase but also the development of metacystic amoebae as determined by the change of nucleus number from four- to one-nucleate amoebae. The protease activity in cyst lysates was also inhibited by PMSF and the band of protease on gelatin sodium dodecyl sulfate polyacrylamide gel electrophoresis was weaker than controls when treated with PMSF. Three serine protease families, S28 (three types), S9 (two), and S26 (one) were retrieved from the database of E. invadens. Phylogenetic analysis revealed that amebic enzymes from the serine protease families formed different clades from those from other organisms. The expression levels of these serine proteases in cysts 5 h after the induction of excystation as assessed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) were higher than those observed prior to induction assayed by real-time RT-PCR; the increase in one type of S9 (named S9-3) expression was the highest. The expression of S9 enzymes also increased from cysts to trophozoites higher than the other family serine proteases. Thus, the results show that Entamoeba uses their serine proteases in the excystation and metacystic development, which leads to successful infection.

Effect of artificial gastrointestinal fluids on the excystation and metacystic development of Entamoeba invadens.

The effect of artificial gastric fluid (AGF), containing 0.5% pepsin and 0.6% hydrochloric acid, pH 1.8, in distilled water, on the excystation and metacystic development of Entamoeba invadens was examined. Excystation, which was assessed by counting the number of metacystic amoebae after inducing excystation, was enhanced by pretreatment of cysts with AGF for 30 to 60 min at 37 degrees C but not 26 degrees C. Longer exposure of cysts to AGF significantly reduced their viability. Significant enhancement of excystation was observed by pretreatment of cysts with distilled water only at 37 degrees C. In addition, 0.6% hydrochloric acid had a comparable enhancing effect on excystation to AGF. Metacystic development, when determined by the number of nuclei in amoeba, was slightly enhanced by pretreatment with AGF. An artificial intestinal fluid (AIF), containing 1% pancreatin, 1% sodium bicarbonate, and 5% ox bile, pH 8.0, in distilled water, had a significant toxic effect on cysts, where 1% pancreatin had neither an enhancing effect on excystation nor a toxic effect on cysts, whereas 5% ox bile had a toxic effect on cysts. Pretreatment of cysts with AGF followed by AIF had a similar toxic effect on cysts to that by AIF only. These results suggest that gastric fluid but not intestinal fluid at 37 degrees C contributes to enhancing excystation for Entamoeba infection.

Characterization of protein geranylgeranyltransferase I from the enteric protist Entamoeba histolytica.

Entamoeba histolytica is a unique protozoan parasite possessing both protein farnesyltransferase and geranylgeranyltrasferase I (GGT-I) for isoprenylation of small GTPases. In this study, we demonstrated unique enzymological properties of the amebic GGT-I (EhGGT-I), including substrate specificity and insensitivity to known mammalian inhibitors. Some of important residues of the catalytic beta subunit implicated in the specificity for GTPase acceptors and prenyl donors are substituted in EhGGT-I. Recombinant alpha and beta subunits of EhGGT-I, co-expressed in Escherichia coli, showed activity to transfer geranylgeranyl to both human wild-type (CVLS) and mutant (CVLL) H-Ras, while the mammalian GGT-I geranylgeranylated, but not farnesylated, only mutant H-Ras. All the representative amebic Ras and Rho/Rac small GTPases with phenylalanine, leucine, methionine, or alanine terminus were preferentially geranylgeranylated by EhGGT-I. This indicates that the acceptor specificity of the amebic GGT-I is remarkably broader than that of its mammalian counterpart. In contrast to EhFT, which farnesylates but not geranylgeranlylates solely EhRas4-CVVA, EhGGT-I also showed significant farnesyltransferase activity against Ras GTPase acceptors. EhGGT-I showed remarkable resistance to peptidomimetics known to inhibit mammalian GGT-I. Together with our previous observation that this parasite does not appear to depend on farnesylation for a majority of Ras and Rho/Rac, these data indicate that biological and biochemical advantages leading to the evolutional selection of this isoprenyl modification must exist uniquely in this parasitic protist. Finally, remarkable biochemical differences in binding to substrates and inhibitors between amebic and mammalian GGT-I highlight this enzyme as an attractive target for the development of new chemotherapeutics against amebiasis.

Entamoeba invadens: cysteine protease inhibitors block excystation and metacystic development.

We examined the effects of six cysteine protease inhibitors on the excystation and metacystic development of Entamoeba invadens. Excystation, which was assessed by counting the number of metacystic amoebae after the induction of excystation, was inhibited by the cysteine protease inhibitors Z-Phe-Ala-DMK and E-64d in a concentration-dependent manner during incubation compared to the controls. Neither inhibitor had a significant effect on cyst viability; thus, their inhibitory effects were not due to the toxic effect on cysts. Metacystic development, when determined by the number of nuclei in amoeba, was also inhibited by these protease inhibitors, because the percentage of 4-nucleate amoebae was higher than in the controls on Day 3 of incubation. Although other cysteine protease inhibitors, Z-Phe-Phe-DMK, E-64, ALLM, and cathepsin inhibitor III, had a weak or little effect on the excystation, they inhibited cysteine protease activity in the lysates of E. invadens cysts. Broad bands with gelatinase activity of metacystic amoebae, as well as cysts and trophozoites, were detected in the gelatin substrate gel electrophores and were inhibited by Z-Phe-Ala-DMK. There was a difference in the protease composition between cysts and trophozoites, and the protease composition of metacystic amoebae changed from cyst-type to trophozoite-type during development. These results strongly suggest that cysteine proteases contribute to the excystation and metacystic development of E. invadens, which leads to successful infection.

Molecular cloning and characterization of a protein farnesyltransferase from the enteric protozoan parasite Entamoeba histolytica.

Genes encoding alpha- and beta-subunits of a putative protein farnesyltransferase (FT) from the enteric protozoan parasite Entamoeba histolytica were obtained and their biochemical properties were characterized. Deduced amino acid sequences of the alpha- and beta-subunit of E. histolytica FT (EhFT) were 298- and 375-residues long with a molecular mass of 35.6 and 42.6 kDa, and a pI of 5.43 and 5.65, respectively. They showed 24% to 36% identity to and shared common signature domains and repeats with those from other organisms. Recombinant alpha- and beta-subunits, co-expressed in Escherichia coli, formed a heterodimer and showed activity to transfer farnesyl using farnesylpyrophosphate as a donor to human H-Ras possessing a C-terminal CVLS, but not a mutant H-Ras possessing CVLL. Among a number of small GTPases that belong to the Ras superfamily from this parasite, we identified EhRas4, which possesses CVVA at the C terminus, as a sole farnesyl acceptor for EhFT. This is in contrast to mammalian FT, which utilizes a variety of small GTPases that possess a C-terminal CaaX motif, where X is serine, methionine, glutamine, cysteine, or alanine. EhFT also showed remarkable resistance against a variety of known inhibitors of mammalian FT. These results suggest that remarkable biochemical differences in binding to substrates and inhibitors exist between amebic and mammalian FTs, which highlights this enzyme as a novel target for the development of new chemotherapeutics against amebiasis.

Involvement of signaling through protein kinase C and phosphatidylinositol 3-kinase in the excystation and metacystic development of Entamoeba invadens.

Using an axenic excystation system in vitro, we examined the effect of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K), which are signaling molecules responsible for numerous cellular responses, on the excystation and metacystic development of Entamoeba invadens. Excystation, which was assessed by counting the number of metacystic amoebae after the induction of excystation, was inhibited by the PKC inhibitors staurosporine, chelerythrine chloride and calphostin C in a concentration-dependent manner during incubation, compared with the controls. As cyst viability was not affected by these inhibitors, reduced excystation was not due to their direct toxic effects on cysts. Metacystic development, when determined by the number of nuclei in the amoebae, was delayed by these PKC inhibitors, because the percentage of 1-nucleate amoebae was lower than in controls at day 3 of incubation. Wortmannin, a potent inhibitor of PI3K, also inhibited excystation and metacystic development of E. invadens in a concentration-dependent manner, compared with the controls. These results indicate that signaling through PKC and PI3K contributes to the excystation and metacystic development of E. invadens.

Entamoeba invadens: inhibition of excystation and metacystic development by aphidicolin.

The effect of aphidicolin, a specific inhibitor of the replicative DNA polymerases, on the excystation and metacystic development of Entamoeba invadens was examined. The protein profile of metacystic amoebae and their immunogenicity in the presence and absence of aphidicolin were also examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Excystation, which was assessed by counting the number of metacystic amoebae after the induction of excystation, was inhibited by aphidicolin in a concentration-dependent manner during incubation compared to the controls. Metacystic development, when determined by the number of nuclei in amoeba, was also inhibited by aphidicolin, because the percentage of 4-nucleate amoebae in cultures with aphidicolin during incubation was higher than that in cultures without the drug. The addition of aphidicolin to cultures at day 1 of incubation reduced the number of metacystic amoebae thereafter compared to cultures without the drug. The inhibitory effect of aphidicolin on excystation and metacystic development was reversed by removal of the drug. Pretreatment of cysts with aphidicolin before transfer to a growth medium containing the drug had no further effect on the excystation and metacystic development. Cellular proteins of metacystic amoebae with 4 nuclei, which were predominant even at day 3 in the cultures with aphidicolin, reacted strongly with rabbit anticyst serum absorbed with trophozoite proteins. In contrast, those of metacystic amoebae with 1 nucleus, which were predominant at day 3 in cultures without aphidicolin, no longer reacted with the absorbed anticyst serum, suggesting change in the expression of proteins during metacystic development.

Inhibition of excystation and metacystic development of Entamoeba invadens by the dinitroaniline herbicide oryzalin.

The effect of oryzalin on excystation and metacystic development of Entamoeba invadens strain IP-1 was examined by transfer of cysts to a growth medium containing the drug. Excystation, which was assessed by counting the number of metacystic amoebae after induction of excystation, was inhibited by oryzalin in a concentration-dependent manner. Metacystic development, which was determined by the number of nuclei in metacystic amoebae, was also inhibited by oryzalin because the percentage of 4-nucleate amoebae at day 1 remained unchanged at day 3. The addition of oryzalin after the induction of excystation decreased the number of metacystic amoebae, compared with control cultures. When cysts were incubated for 1 day in growth medium plus oryzalin, little increase in the number of metacystic amoebae was observed after removal of the drug. Excystation and metacystic development were further inhibited when the cysts were incubated for 30 min in encystation medium containing oryzalin before transfer to growth medium with the drug. When cysts were incubated for 30 min in encystation medium before transfer to growth medium without the drug, metacystic amoebae decreased in number. Pretreatment of cysts with oryzalin for 30 min in phosphate-buffered saline markedly reduced viability and prevented excystation in growth medium with or without the drug. The results indicate that oryzalin inhibits excystation and metacystic development of E. invadens, suggesting that it may be an inhibitor of Entamoeba infection.

Possible role of calcium ions, calcium channels and calmodulin in excystation and metacystic development of Entamoeba invadens.

The effect of calcium ions (Ca(2+)) and calmodulin (CaM) on the excystation and metacystic development of Entamoeba invadens was examined by transfer of cysts to a growth medium containing calcium antagonists and CaM inhibitors. Excystation, which was assessed by counting the number of metacystic amoebae after induction of excystation, was inhibited by the calcium chelators ethyleneglycol bis (beta-aminoethyl ether)- N,N'-tetraacetate (EGTA) and ethylene-diaminetetraacetate (EDTA), with EDTA being more potent than EGTA. The inhibitory effect of higher concentrations of these chelators on excystation was associated with reduced viability of cysts. Metacystic development, when determined by the number of nuclei in an amoeba, was delayed by EGTA, because the percentage of four-nucleate amoebae was higher than in controls at day 3 of incubation. EDTA made metacystic development unusual by producing a large number of metacystic amoebae with more than ten nuclei. The inhibition of excystation by these chelators was partially abrogated by their removal. A putative antagonist of intracellular calcium flux, 8-( N,N-diethylamino) octyl-3,4,5-trimethoxybenzoate (TMB-8) also inhibited the excystation and metacystic development, but had little effect on cyst viability. The slow Na(+)-Ca(2+) channel blocker bepridil but not verapamil inhibited the excystation and metacystic development, associating with reduced cyst viability at higher concentrations. The inhibitory effect of bepridil on excystation was abrogated by removal of the drug. The CaM inhibitor trifIuoperazine (TFP) but not W-7 [ N-(6-aminohexyl)-chloro-1-naphtalene sulphonamide] inhibited the excystation and metacystic development. The inhibitory effect of TFP on excystation was also abrogated by removal of the drug. These results indicate that extracellular calcium ions, amoebic intracellular calcium flux, calcium channels, and a CaM-dependent process contribute to the excystation and metacystic development of E. invadens.

Effect of proteasome inhibitors on the growth, encystation, and excystation of Entamoeba histolytica and Entamoeba invadens.

The effect of three proteasome inhibitors, lactacystin, clasto-lactacystin beta-lactone, and MG-132, on the growth, encystation, and excystation of Entamoeba histolytica and Entamoeba invadens was examined. All of these drugs blocked E. histolytica growth in a concentration-dependent manner; lactacystin was most potent for the inhibition and MG-132 showed the inhibitory effect only at higher concentrations. E. invadens was more resistant to these drugs than E. histolytica. Encystation of E. invadens was also inhibited and was more sensitive to the drugs than was growth. Beta-lactone was the most potent encystation inhibitor. The inhibitory effect of lactacystin and the beta-lactone on encystation was slightly and little abrogated by the removal of the drug, respectively. Multinucleation occurred in E. histolytica trophozoites treated with these drugs, being most marked with lactacystin. In contrast, no multinucleation was observed in E. invadens treated with the drugs. Electron microscopy revealed that the treatment of E. histolytica trophozoites with lactacystin led to an increase in the number of cells with many glycogen granules in the cytoplasm. Lactacystin, beta-lactone and MG-132 had no or little effect on the excystation and metacystic development of E. invadens. These results suggest that proteasome function plays an important role for Entamoeba growth and encystation, but has no obvious effect on excystation or metacystic development.