A stage-specific sialoglycoprotein in encysting cells of Entamoeba invadens

A novel sialoglycoprotein with an apparent molecular mass of approximately 250 kDa was detected on the surface of cysts of Entamoeba invadens. Sialic acid was identified in this glycoprotein by gas chromatography after methanolysis; N-acetyl- and N-glycolyl neuraminic acid were identified by thin layer chromatography in hydrolysates of partially purified preparations of the 250 kDa glycoprotein as well as in whole cysts. The sialoglycoprotein is stage-specific and could be detected by binding of wheat germ agglutinin and a specific monoclonal antibody (JAM3) only to precysts and mature cysts but not to trophozoites. A 250 kDa protein could be metabolically labeled with [35S]methionine. This, together with the absence of such a glycoprotein in the encystation medium, suggests that the 250 kDa sialoglycoprotein is not an adsorbed serum glycoprotein. Indirect evidence suggests that the parasite may utilize serum components as a source for sialic acid.     

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     

Chitinase activity in encysting Entamoeba invadens and its inhibition by allosamidin.

Chitinase activity was measured in extracts of Entamoeba invadens cells as a function of time of encystation in axenic conditions using 4-MU(Ch)3 as substrate. Encystment was paralleled by chitinase activity which showed a peak after about 72 h of cultivation where cysts accounted for 63% of cell population. Thereafter, activity fell off rapidly, whereas encystment continued, reaching 80% at the end of the experiment (96 h). Comparison of activity between cysts and the total cell population in 48- and 72-h-old encysting cultures suggested that chitinase may start to accumulate in the pre-cyst forms. About 70% of the enzyme was recovered in the supernatant following low-speed centrifugation of whole extracts. Most of this activity represented soluble chitinase since it was not sedimented by further centrifugation at 105,000 x g. A minor proportion of enzyme activity remained associated to the buffer-washed, high-speed sediment. In addition to 4-MU(Ch)3, chitinase activity was also measured following the hydrolysis of other substrates such as nascent, preformed or colloidal chitin. Like other chitinases, the cyst enzyme preferred nascent over preformed chitin as substrate. Digestion of the former yielded GlcNAc and minor amounts of (GlcNAc)2 as products. Allosamidin strongly inhibited hydrolysis of the fluorogenic substrate by the amebic chitinase in vitro with a Ki of 0.065 microM. IC50 values were 0.085 microM and 0.16 microM at 5 microM and 10 microM 4-MU(Ch)3, respectively. When added to the axenic medium, the drug markedly retarded encystment though it was partially recovered after longer periods of incubation.     

Appearance of sialoglycoproteins in encysting cells of Entamoeba histolytica.

Amoeba-bacterium cultures of Entamoeba histolytica transferred to a hypoosmotic medium depleted of nutrients changed morphologically and biochemically. The cells ejected grains of rice starch, rounded up, and formed a distinct cell wall that was resistant to detergent, bound the sialic acid-specific lectin from Limulus polyphemus, and became fluorescent with Calcofluor M2R. A subpopulation of these cells displayed more than one nucleus. All these signs are characteristic of encysting cells and were also observed in cysts obtained from a human patient. The morphological changes were accompanied by the appearance of two new glycoproteins with apparent molecular sizes of 100 and 150 kilodaltons which contained sialic acid. Sialic acid has been reported to be absent from trophozoites of Entamoeba species. The presence of this sugar residue on cyst-specific proteins parallels recently reported findings during the encystation of the related reptilian parasite Entamoeba invadens. This may indicate a basic role for sialic acid in the encystation of Entamoeba parasites.     

Ribonuclease of Entamoeba invadens

Two bands of RNAase activity were observed on gel electrophoresis of fractions obtained during partial purification of the RNAase of Entamoeba invadens. The faster migrating band which was responsible for most of the RNAase activity was isolated by gel electrophoresis. A pI of 5.5 was determined for this RNAase activity and estimates of its molecular weight and sedimentation coefficient gave values of about 25 000 and approximately 2.8 S. It exhibited maximum activity around pH 6, and digestion of RNA showed a pattern expected of an endoribonuclease.     

Cell-surface carbohydrates of Entamoeba invadens

Cell-surface carbohydrates of Entamoeba invadens trophozoites were analyzed using (a) a panel of highly purified lectins specific for molecules containing N-acetylglucosamine or sialic acid, N-acetylgalactosamine, galactose, mannose-like residues, and fucose; (b) Escherichia coli K-12 with mannose-sensitive fimbria; (c) enzymatic digestion; and (d) scanning electron microscopy. The presence of galactose (D-Gal) and N-acetylgalactosamine (D-GalNAc) was detected in the amoeba. Previous trypsinization induced the appearance of Glycine max (SBA, specific for D-GalNAc residues)-binding sites, whereas such treatment completely abolished the ability of Ricinus communis (RCAI) and Axinalla polypoides (APP, specific for D-Gal) lectins and partially abolished that of Euonymus europaeus (EEL, specific for D-Gal) lectins to agglutinate the trophozoites. The agglutinating activity of E. coli K-12 adhesins with the amoeba was markedly increased after trypsin digestion, indicating that mannose units become exposed after enzyme treatment. These findings were essentially confirmed by scanning electron microscopy. After neuraminidase treatment the parasites became strongly agglutinated with SBA and Arachis hypogaea (PNA, specific for D-Gal) and the cell interaction with Wisteria floribunda (WFH, specific for D-GalNAc) was markedly increased. These results suggest that in E. invadens trophozoites, sialic acid residues are linked to D-Gal and D-GalNAc. Received: 24 March 1997 / Accepted: 10 April 1997     

Gene discovery in the Entamoeba invadens genome

Entamoeba invadens, a parasite of reptiles, is a model for the study of encystation by the human enteric pathogen Entamoeba histolytica, because E. invadens form cysts in axenic culture. With approximately 0.5-fold sequence coverage of the genome, we were able to get insights into E. invadens gene and genome features. Overall, the E. invadens genome displays many of the features that are emerging from ongoing genome sequencing efforts in E. histolytica. At the nucleotide level the E. invadens genome has on average 60% sequence identity with that of E. histolytica. The presence of introns in E. invadens was predicted with similar consensus (GTTTGT em leader A/TAG) sequences to those identified in E. histolytica and Entamoeba dispar. Sequences highly repeated in the genome of E. histolytica (rRNAs, tRNAs, CXXC-rich proteins, and Leu-rich repeat proteins) were found to be highly repeated in the E. invadens genome. Numerous proteins homologous to those implicated in amoebic virulence, (Gal/GalNAc lectins, amoebapores, and cysteine proteinases) and drug resistance (p-glycoproteins) were identified. Homologs of proteins involved in cell cycle, vesicular trafficking and signal transduction were identified, which may be involved in en/excystation and cell growth of E. invadens. Finally, multiple copies of a number of E. invadens genes coding for predicted enzymes involved in core metabolism and the targets of anti-amoebic drugs were identified.     

Preliminary characterization of ribosomes of Entamoeba invadens

The ribosomes of Entamoeba invadens trophozoites have sedimentation coefficients of 77, 53 and 36 S. Most of the ribosomal proteins are basic and their one- and two-dimensional electrophoretic patterns differ from the corresponding patterns of Escherichia coli and Saccharomyces cerevisiae. Two dozen bands were observed in the 10 000 to 100 000 molecular weight range following sodium dodecylsulfate-gel electrophoresis of amoebal ribosomal proteins. Long, thin pronase-sensitive structures were seen in electron micrographs of E. invadens ribosomal preparations.     

Inhibition of encystation of Entamoeba invadens by wortmannin

Using an axenic encystation system in vitro, we examined the effect of wortmannin, a potent inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), which is a signaling molecule responsible for numerous cellular responses, on the encystation of Entamoeba invadens. Wortmannin inhibited both encystation and growth of E. invadens strain IP-1 in a dose-dependent manner, the former being more resistant to the drug than the latter. There was little decrease in the number of trophozoites after 3 days of culture in encystation medium containing wortmannin; and the cells remained motile, suggesting that the inhibitory effect of the drug on encystation was not due to its toxic effect on trophozoites. The addition of wortmannin after the induction of encystation was also inhibitory for encystation. Trophozoites incubated for 1 day in encystation medium with wortmannin did not encyst after removal of the drug, suggesting that the drug effect was not reversible in encystation medium. In contrast, trophozoites cultured in growth medium with wortmannin did encyst after their transfer to encystation medium without the drug. Encystation with wortmannin was more strongly inhibited among trophozoites grown in the presence of the drug than among those grown in the absence of the drug. The process of cyst maturation was slightly affected by wortmannin. These results suggest a possible role for PI 3-kinase in the signaling involved in the encystation of E. invadens.     

Entamoeba invadens contains the components of a classical adrenergic signaling system

Epinephrine (Epi) was previously found to bypass the need for galactose ligands during early steps in the initiation of Entamoeba encystment. Epinephrine is presumed to act on amoebae through a classical adrenergic signaling pathway that results in the increased production of cyclic adenosine monophosphate (cAMP). The object of this study was to verify the existence of an adrenergic like pathway and its response to Epi in both whole Entamoeba trophozoites and purified plasma membrane preparations. Whole trophozoite and purified membrane preparations from Entamoeba invadens responded to the presence of Epi by increasing the production of cAMP. The modulators of heterotrimeric G protein signaling, forskolin (FK), pertussis toxin (PTX) and cholera toxin (CTX), also increased cAMP levels in whole cells and membrane fragments. All of these increases in cAMP were inhibited by specific inhibitors of adenylyl cyclase (AC). Treatment of membrane fragments with epinephrine caused an increased binding of non-hydrolysable GTP analogs. Entamoeba trophozoites therefore appear to contain G-protein-regulated adenylyl cyclase that functions downstream of an adrenergic ligand receptor.     

Ultrastructural study of Entamoeba invadens encystation and excystation

In the life cycle of Entamoeba species, the cyst and all the processes associated to it have been poorly studied. Entamoeba invadens, a serpent's parasite, has been commonly accepted as a model for the study of encystation and excystation. Here we analyzed through scanning and transmission electron microscopy the in vitro morphological differentiation of both processes. During encystation, the formation of an irregular net of fibrillar material on the surface of precysts was observed. In thin sections of cryofixed and cryosubstituted specimens, abundant vacuoles containing a microfibrillar material of similar appearance to the structural components of the cyst wall were found in the cytoplasm. Assays with a calcofluor probe on cryosections of encysting trophozoites and precysts showed the presence of fluorescent circular cytoplasmic structures. In the cyst stage, the fluorescence was located on the surface. During excystation, the detachment of the metacyst from the cyst wall was observed through scanning electron microscopy. Metacysts endocyting amorphous material which may correspond to cyst wall residues were commonly found. By transmission electron microscopy the formation of a crescent-shaped space between the plasma membrane and the cyst wall was observed. Abundant small electrondense bodies were found in the cytoplasm. Many of them were in close apposition to the plasma membrane and frequently some of them were seen projecting towards this newly formed space. Our results suggest that the microfibrillar content of the vacuoles corresponds to the cyst wall material, that the electrondense bodies may be involved in the excystation process, and that part of the cyst wall residues may be endocyted by the parasite.     

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.     

Transient and stable transfection in the protozoan parasite Entamoeba invadens

Entamoeba histolytica is an important human pathogen and a major health problem worldwide. Many aspects of parasite biology can be studied with the exception of stage conversion, which cannot be reproduced adequately in E. histolytica. The reptile parasite Entamoeba invadens is a vital model system for studying stage conversion since it can be induced to undergo both encystation and excystation with high efficiency in vitro. However, functional studies using E. invadens have been limited by the lack of genetic tools in this species. Here, we report a new method for both transient and stable transfection of E. invadens. These new tools will greatly enhance research into Entamoeba development.     

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.     

Establishment of a transient transfection system and expression of firefly luciferase in Entamoeba invadens

Entamoeba invadens is used as a model system to study trophozoite to cyst differentiation since Entamoeba histolytica, the causative agent of amoebiasis cannot encyst in culture. However, a system for introduction of cloned genes in E. invadens is not available. Here we report an electroporation-based method for transfection of E. invadens tophozoites and demonstrate the expression of firefly luciferase reporter gene driven from the E. invadens ribosomal protein L3 promoter. The efficiency of luciferase expression driven from the promoters of three different E. invadens genes (rpl3, rps10 and h2b) was tested and found to correlate with the in vivo expression levels of the respective gene. This system will permit the analysis of regulatory elements required for gene expression in E. invadens.     

Stable transfection and continuous expression of heterologous genes in Entamoeba invadens

Amoebiasis is spread by the ingestion of dormant Entamoeba histolytica cysts. Intervention of encystation could break the transmission cycle, thereby reducing disease burden. The model system used to study trophozoite to cyst differentiation is Entamoeba invadens. Here we describe an electroporation-based method for stable transfection of E. invadens with a plasmid pEiNEO-LUC containing the neomycin phosphotransferase gene under the control of E. invadens ribosomal protein gene S10 promoter. The plasmid also contains luciferase reporter gene expressed from the promoter of ribosomal protein gene L3. After electroporation, cells receiving the plasmid were selected by growth in 10μgml(-1) G418 and stable lines were obtained in four to five weeks. The plasmid was replicated episomally to ～10 copies per haploid genome. In the absence of drug selection 50% of the plasmid copies were lost in 9-10 days. In cells growing under drug selection the reporter gene was continuously expressed throughout the differentiation process from trophozoite to cyst and back, making this system suitable for analysis of genes involved in differentiation.     

Autophagy during proliferation and encystation in the protozoan parasite Entamoeba invadens

Autophagy is one of the three systems responsible for the degradation of cytosolic proteins and organelles. Autophagy has been implicated in the stress response to starvation, antigen cross-presentation, the defense against invading bacteria and viruses, differentiation, and development. Saccharomyces cerevisiae Atg8 and its mammalian ortholog, LC3, play an essential role in autophagy. The intestinal protozoan parasite Entamoeba histolytica and a related reptilian species, Entamoeba invadens, possess the Atg8 conjugation system, consisting of Atg8, Atg4, Atg3, and Atg7, but lack the Atg5-to-Atg12 conjugation system. Immunofluorescence imaging revealed that polymorphic Atg8-associated structures emerged in the logarithmic growth phase and decreased in the stationary phase and also increased in the early phase of encystation in E. invadens. Immunoblot analysis showed that the increase in phosphatidylethanolamine-conjugated membrane-associated Atg8 was also accompanied by the emergence of Atg8-associated structures during the proliferation and differentiation mentioned above. Specific inhibitors of class I and III phosphatidylinositol 3-kinases simultaneously inhibited both the growth of trophozoites and autophagy and also both encystation and autophagy in E. invadens. These results suggest that the core machinery for autophagy is conserved and plays an important role during proliferation and differentiation in Entamoeba.     

The cell cycle of Entamoeba invadens during vegetative growth and differentiation

The cell division cycle of Entamoeba invadens was studied during vegetative growth of trophozoites and during their differentiation into cysts. During vegetative growth of trophozoites, it was observed that DNA synthesis typically continued after one genome content had been duplicated. During encystation, DNA synthesis was arrested after 4n genome content had been synthesised. Using multi-parameter flow cytometry, the light scattering properties of cysts and trophozoites were studied. The cytoplasmic granularity, reflected by the side scatter of light, was proportional to DNA content of trophozoites, whereas cysts with similar DNA contents showed heterogeneity in their cytoplasmic granularity. Dynamic changes in the intracellular calcium pools were observed during differentiation of trophozoites to cysts. Comparison of E. invadens and Entamoeba histolytica cell cycles suggest that both organisms may have similar regulatory processes during cell division and differentiation. Since E. histolytica cannot be induced to encyst in axenic culture, analysis of the E. invadens cell cycle during encystation may be useful for identifying homologous processes in E.histolytica.