The dynamics of ultrastructural changes during Entamoeba invadens encystation.

Entamoeba histolytica infection causes amoebiasis, which is a global public health problem. The major route of infection is oral ingestion of E. histolytica cysts, cysts being the sole form responsible for host-to-host transmission. Cysts are produced by cell differentiation from proliferative trophozoites in a process termed 'encystation'. Therefore, encystation is an important process from a medical as well as a biological perspective. Previous electron microscopy studies have shown the ultrastructure of precysts and mature cysts; however, the dynamics of ultrastructural changes during encystation were ambiguous. Here, we analysed a series of Entamoeba invadens encysting cells by transmission electron microscopy. Entamoeba invadens is a model for encystation and the cells were prepared by short interval time course sampling from in vitro encystation-inducing cultures. We related sampled cells to stage conversion, which was monitored in the overall population by flow cytometry. The present approach revealed the dynamics of ultrastructure changes during E. invadens encystation. Importantly, the results indicate a functional linkage of processes that are crucial in encystation, such as glycogen accumulation and cyst wall formation. Hence, this study provides a reference for studying sequential molecular events during Entamoeba encystation.

Unique Features of Entamoeba Sulfur Metabolism; Compartmentalization, Physiological Roles of Terminal Products, Evolution and Pharmaceutical Exploitation.

Sulfur metabolism is essential for all living organisms. Recently, unique features of the Entamoeba metabolic pathway for sulfated biomolecules have been described. Entamoeba is a genus in the phylum Amoebozoa and includes the causative agent for amoebiasis, a global public health problem. This review gives an overview of the general features of the synthesis and degradation of sulfated biomolecules, and then highlights the characteristics that are unique to Entamoeba. Future biological and pharmaceutical perspectives are also discussed.

Uniqueness of Entamoeba sulfur metabolism: sulfolipid metabolism that plays pleiotropic roles in the parasitic life cycle.

Sulfur metabolism is ubiquitous and terminally synthesizes various biomolecules that are crucial for organisms, such as sulfur-containing amino acids and co-factors, sulfolipids and sulfated saccharides. Entamoeba histolytica, a protozoan parasite responsible for amoebiasis, possesses the unique sulfur metabolism features of atypical localization and its terminal product being limited to sulfolipids. Here, we present an overall scheme of E. histolytica sulfur metabolism by relating all sulfotransferases and sulfatases to their substrates and products. Furthermore, a novel sulfur metabolite, fatty alcohol disulfates, was identified and shown to play an important role in trophozoite proliferation. Cholesteryl sulfate, another synthesized sulfolipid, was previously demonstrated to play an important role in encystation, a differentiation process from proliferative trophozoite to dormant cyst. Entamoeba survives by alternating between these two distinct forms; therefore, Entamoeba sulfur metabolism contributes to the parasitic life cycle via its terminal products. Interestingly, this unique feature of sulfur metabolism is not conserved in the nonparasitic close relative of Entamoeba, Mastigamoeba, because lateral gene transfer-mediated acquisition of sulfatases and sulfotransferases, critical enzymes conferring this feature, has only occurred in the Entamoeba lineage. Hence, our findings suggest that sulfolipid metabolism has a causal relationship with parasitism.

Entamoeba Encystation: New Targets to Prevent the Transmission of Amebiasis.

Amebiasis is caused by Entamoeba histolytica infection and can produce a broad range of clinical signs, from asymptomatic cases to patients with obvious symptoms. The current epidemiological and clinical statuses of amebiasis make it a serious public health problem worldwide. The Entamoeba life cycle consists of the trophozoite, the causative agent for amebiasis, and the cyst, the form responsible for transmission. These two stages are connected by "encystation" and "excystation." Hence, developing novel strategies to control encystation and excystation will potentially lead to new measures to block the transmission of amebiasis by interrupting the life cycle of the causative agent. Here, we highlight studies investigating encystation using inhibitory chemicals and categorize them based on the molecules inhibited. We also present a perspective on new strategies to prevent the transmission of amebiasis.

Entamoeba mitosomes play an important role in encystation by association with cholesteryl sulfate synthesis.

Hydrogenosomes and mitosomes are mitochondrion-related organelles (MROs) that have highly reduced and divergent functions in anaerobic/microaerophilic eukaryotes. Entamoeba histolytica, a microaerophilic, parasitic amoebozoan species, which causes intestinal and extraintestinal amoebiasis in humans, possesses mitosomes, the existence and biological functions of which have been a longstanding enigma in the evolution of mitochondria. We previously demonstrated that sulfate activation, which is not generally compartmentalized to mitochondria, is a major function of E. histolytica mitosomes. However, because the final metabolites of sulfate activation remain unknown, the overall scheme of this metabolism and the role of mitosomes in Entamoeba have not been elucidated. In this study we purified and identified cholesteryl sulfate (CS) as a final metabolite of sulfate activation. We then identified the gene encoding the cholesteryl sulfotransferase responsible for synthesizing CS. Addition of CS to culture media increased the number of cysts, the dormant form that differentiates from proliferative trophozoites. Conversely, chlorate, a selective inhibitor of the first enzyme in the sulfate-activation pathway, inhibited cyst formation in a dose-dependent manner. These results indicate that CS plays an important role in differentiation, an essential process for the transmission of Entamoeba between hosts. Furthermore, we show that Mastigamoeba balamuthi, an anaerobic, free-living amoebozoan species, which is a close relative of E. histolytica, also has the sulfate-activation pathway in MROs but does not possess the capacity for CS production. Hence, we propose that a unique function of MROs in Entamoeba contributes to its adaptation to its parasitic life cycle.

Evidence that the Entamoeba histolytica Mitochondrial Carrier Family Links Mitosomal and Cytosolic Pathways through Exchange of 3'-Phosphoadenosine 5'-Phosphosulfate and ATP.

Entamoeba histolytica, a microaerophilic protozoan parasite, possesses mitosomes. Mitosomes are mitochondrion-related organelles that have largely lost typical mitochondrial functions, such as those involved in the tricarboxylic acid cycle and oxidative phosphorylation. The biological roles of Entamoeba mitosomes have been a long-standing enigma. We previously demonstrated that sulfate activation, which is not generally compartmentalized to mitochondria, is a major function of E. histolytica mitosomes. Sulfate activation cooperates with cytosolic enzymes, i.e., sulfotransferases (SULTs), for the synthesis of sulfolipids, one of which is cholesteryl sulfate. Notably, cholesteryl sulfate plays an important role in encystation, an essential process in the Entamoeba life cycle. These findings identified a biological role for Entamoeba mitosomes; however, they simultaneously raised a new issue concerning how the reactions of the pathway, separated by the mitosomal membranes, cooperate. Here, we demonstrated that the E. histolytica mitochondrial carrier family (EhMCF) has the capacity to exchange 3'-phosphoadenosine 5'-phosphosulfate (PAPS) with ATP. We also confirmed the cytosolic localization of all the E. histolytica SULTs, suggesting that in Entamoeba, PAPS, which is produced through mitosomal sulfate activation, is translocated to the cytosol and becomes a substrate for SULTs. In contrast, ATP, which is produced through cytosolic pathways, is translocated into the mitosomes and is a necessary substrate for sulfate activation. Taking our findings collectively, we suggest that EhMCF functions as a PAPS/ATP antiporter and plays a crucial role in linking the mitosomal sulfate activation pathway to cytosolic SULTs for the production of sulfolipids.

Links between cholesteryl sulfate-dependent and -independent processes in the morphological and physiological changes of Entamoeba encystation.

The protozoan parasite Entamoeba histolytica causes amoebiasis, a global public health problem. Amoebiasis is solely transmitted by cysts that are produced from proliferative trophozoites by encystation in the large intestine of humans. During encystation, various metabolites, pathways, and cascades sequentially orchestrate the morphological and physiological changes required to produce cysts. Cholesteryl sulfate (CS) has recently been revealed to be among the key molecules that control the morphological and physiological changes of encystation by exerting pleiotropic effects. CS promotes the rounding of encysting Entamoeba cells and maintains this spherical morphology as encysting cells are surrounded by the cyst wall, a prerequisite for resistance against environmental stresses. CS is also involved in the development of membrane impermeability, another prerequisite for resistance. The initiation of cyst wall formation is, however, CS-independent. Here, we overview CS-dependent and -independent processes during encystation and discuss their functional linkage. We also discuss a potential transcriptional cascade that controls the processes necessary to produce dormant Entamoeba cysts.

Unique features of Entamoeba histolytica glycerophospholipid metabolism; has the E. histolytica lipid metabolism network evolved through gene loss and gain to enable parasitic life cycle adaptation?

Entamoeba histolytica, a protozoan parasite, causes amoebiasis, which is a global public health problem. During the life cycle of this parasite, the properties of the cell membrane are changed markedly. To clarify the mechanism of membrane lipid changes, we exploited state-of-the-art untargeted lipidomic analysis, and atypical features of glycerophospholipids, lysoglycerophospholipids, and sphingolipids were observed compared with human equivalents. Here, we overview an entire E. histolytica glycerophospholipid metabolic pathway based on re-evaluated whole lipidome and genome along with the results of metabolic labeling experiments. We also discuss whether the E. histolytica lipid metabolism network, including the glycerophospholipid metabolic pathway, has unique features necessary for parasitic life cycle adaptation through gene loss and/or gain, and raise important questions involving biochemistry, molecular cell biology, and physiology underlying this network. Answering these questions will advance the understanding of Entamoeba physiology and will provide potential targets to develop new anti-amoebiasis drugs.

Mitosomes in Entamoeba histolytica contain a sulfate activation pathway.

Hydrogenosomes and mitosomes are mitochondrion-related organelles in anaerobic/microaerophilic eukaryotes with highly reduced and divergent functions. The full diversity of their content and function, however, has not been fully determined. To understand the central role of mitosomes in Entamoeba histolytica, a parasitic protozoon that causes amoebic dysentery and liver abscesses, we examined the proteomic profile of purified mitosomes. Using 2 discontinuous Percoll gradient centrifugation and MS analysis, we identified 95 putative mitosomal proteins. Immunofluorescence assay showed that 3 proteins involved in sulfate activation, ATP sulfurylase, APS kinase, and inorganic pyrophosphatase, as well as sodium/sulfate symporter, involved in sulfate uptake, were compartmentalized to mitosomes. We have also provided biochemical evidence that activated sulfate derivatives, adenosine-5'-phosphosulfate and 3'-phosphoadenosine-5'-phosphosulfate, were produced in mitosomes. Phylogenetic analysis showed that the aforementioned proteins and chaperones have distinct origins, suggesting the mosaic character of mitosomes in E. histolytica consisting of proteins derived from alpha-proteobacterial, delta-proteobacterial, and ancestral eukaryotic origins. These results suggest that sulfate activation is the major function of mitosomes in E. histolytica and that E. histolytica mitosomes represent a unique mitochondrion-related organelle with remarkable diversity.

Pleiotropic Roles of Cholesteryl Sulfate during Entamoeba Encystation: Involvement in Cell Rounding and Development of Membrane Impermeability.

Entamoeba histolytica, a protozoan parasite, causes amoebiasis, which is a global public health problem. The major route of infection is oral ingestion of cysts, the only form that is able to transmit to a new host. Cysts are produced by cell differentiation from proliferative trophozoites in a process termed "encystation." During encystation, cell morphology is markedly changed; motile amoeboid cells become rounded, nonmotile cells. Concomitantly, cell components change and significant fluctuations of metabolites occur. Cholesteryl sulfate (CS) is a crucial metabolite for encystation. However, its precise role remains uncertain. To address this issue, we used in vitro culture of Entamoeba invadens as the model system for the E. histolytica encystation study and identified serum-free culture conditions with CS supplementation at concentrations similar to intracellular CS concentrations during natural encystation. Using this culture system, we show that CS exerts pleiotropic effects during Entamoeba encystation, affecting cell rounding and development of membrane impermeability. CS dose dependently induced and maintained encysting cells as spherical maturing cysts with almost no phagocytosis activity. Consequently, the percentage of mature cysts was increased. CS treatment also caused time- and dose-dependent development of membrane impermeability in encysting cells via induction of de novo synthesis of dihydroceramides containing very long N-acyl chains (≥26 carbons). These results indicate that CS-mediated morphological and physiological changes are necessary for the formation of mature cysts and the maintenance of the Entamoeba life cycle. Our findings also reveal important morphological aspects of the process of dormancy and the control of membrane structure. IMPORTANCE Entamoeba histolytica causes a parasitic infectious disease, amoebiasis. Amoebiasis is a global public health problem with a high occurrence of infection and inadequate clinical options. The parasite alternates its form between a proliferative trophozoite and a dormant cyst that enables the parasite to adapt to new environments. The transition stage in which trophozoites differentiate into cysts is termed "encystation." Cholesteryl sulfate is essential for encystation; however, its precise role remains to be determined. Here, we show that cholesteryl sulfate is a multifunctional metabolite exerting pleiotropic roles during Entamoeba encystation, including the rounding of cells and the development of membrane impermeability. Such morphological and physiological changes are required for Entamoeba to produce cysts that are transmissible to a new host, which is essential for maintenance of the Entamoeba life cycle. Our findings are therefore relevant not only to Entamoeba biology but also to general cell and lipid biology.

Metabolome analysis revealed increase in S-methylcysteine and phosphatidylisopropanolamine synthesis upon L-cysteine deprivation in the anaerobic protozoan parasite Entamoeba histolytica.

L-cysteine is ubiquitous in all living organisms and is involved in a variety of functions, including the synthesis of iron-sulfur clusters and glutathione and the regulation of the structure, stability, and catalysis of proteins. In the protozoan parasite Entamoeba histolytica, the causative agent of amebiasis, L-cysteine plays an essential role in proliferation, adherence, and defense against oxidative stress; however, the essentiality of this amino acid in the pathways it regulates is not well understood. In the present study, we applied capillary electrophoresis time-of-flight mass spectrometry to quantitate charged metabolites modulated in response to L-cysteine deprivation in E. histolytica, which was selected as a model for examining the biological roles of L-cysteine. L-cysteine deprivation had profound effects on glycolysis, amino acid, and phospholipid metabolism, with sharp decreases in the levels of L-cysteine, L-cystine, and S-adenosylmethionine and a dramatic accumulation of O-acetylserine and S-methylcysteine. We further demonstrated that S-methylcysteine is synthesized from methanethiol and O-acetylserine by cysteine synthase, which was previously considered to be involved in sulfur-assimilatory L-cysteine biosynthesis. In addition, L-cysteine depletion repressed glycolysis and energy generation, as it reduced acetyl-CoA, ethanol, and the major nucleotide di- and triphosphates, and led to the accumulation of glycolytic intermediates. Interestingly, L-cysteine depletion increased the synthesis of isopropanolamine and phosphatidylisopropanolamine, and it was confirmed that their increment was not a result of oxidative stress but was a specific response to L-cysteine depletion. We also identified a pathway in which isopropanolamine is synthesized from methylglyoxal via aminoacetone. To date, this study represents the first case where L-cysteine deprivation leads to drastic changes in core metabolic pathways, including energy, amino acid, and phospholipid metabolism.

Localization and targeting of an unusual pyridine nucleotide transhydrogenase in Entamoeba histolytica.

Pyridine nucleotide transhydrogenase (PNT) catalyzes the direct transfer of a hydride-ion equivalent between NAD(H) and NADP(H) in bacteria and the mitochondria of eukaryotes. PNT was previously postulated to be localized to the highly divergent mitochondrion-related organelle, the mitosome, in the anaerobic/microaerophilic protozoan parasite Entamoeba histolytica based on the potential mitochondrion-targeting signal. However, our previous proteomic study of isolated phagosomes suggested that PNT is localized to organelles other than mitosomes. An immunofluorescence assay using anti-E. histolytica PNT (EhPNT) antibody raised against the NADH-binding domain showed a distribution to the membrane of numerous vesicles/vacuoles, including lysosomes and phagosomes. The domain(s) required for the trafficking of PNT to vesicles/vacuoles was examined by using amoeba transformants expressing a series of carboxyl-terminally truncated PNTs fused with green fluorescent protein or a hemagglutinin tag. All truncated PNTs failed to reach vesicles/vacuoles and were retained in the endoplasmic reticulum. These data indicate that the putative targeting signal is not sufficient for the trafficking of PNT to the vesicular/vacuolar compartments and that full-length PNT is necessary for correct transport. PNT displayed a smear of >120 kDa on SDS-PAGE gels. PNGase F and tunicamycin treatment, chemical degradation of carbohydrates, and heat treatment of PNT suggested that the apparent aberrant mobility of PNT is likely attributable to its hydrophobic nature. PNT that is compartmentalized to the acidic compartments is unprecedented in eukaryotes and may possess a unique physiological role in E. histolytica.

Entamoeba Chitinase is Required for Mature Round Cyst Formation.

Entamoeba histolytica, a protozoan parasite, causes amoebiasis in humans. Amoebiasis transmission is solely mediated by chitin-walled cysts, which are produced in the large intestine of humans from proliferative trophozoites by a cell differentiation process called encystation. Resistance to environmental stresses, an essential characteristic for transmission, is attributed to the cyst wall, which is constructed from chitin and several protein components, including chitinase. Chitinase may play a key role in cyst wall formation; however, this has not been confirmed. Here, to elucidate the physiological role of chitinase during Entamoeba encystation, we identified a new chitinase inhibitor, 2,6-dichloro-4-[2-(1-piperazinyl)-4-pyridinyl]-N-(1,3,5-trimethyl-1H-pyrazol-4-yl)-benzenesulfonamide, by recombinant-Entamoeba chitinase-based screening of 400 Pathogen Box chemicals. This compound dose dependently inhibited native chitinase associated with Entamoeba invadens encystation, a model for E. histolytica encystation, with an 50% inhibitory concentration (IC50) of ∼0.6 µM, which is comparable to the IC50s (0.2 to 2.5 µM) for recombinant E. histolytica and E. invadens chitinases. Furthermore, the addition of this compound to E. invadens encystation-inducing cultures increased the generation of cyst walls with an abnormal shape, the most characteristic of which was a "pot-like structure." A similar structure also appeared in standard culture, but at a far lower frequency. These results indicate that chitinase inhibition increases the number of abnormal encysting cells, thereby significantly reducing the efficiency of cyst formation. Transmission electron microscopy showed that compound-treated encysting cells formed an abnormally loose cyst wall and an unusual gap between the cyst wall and cell membrane. Hence, Entamoeba chitinase is required for the formation of mature round cysts. IMPORTANCE Amoebiasis is caused by Entamoeba histolytica infection and is transmitted by dormant Entamoeba cells or cysts. Cysts need to be tolerant to severe environmental stresses faced outside and inside a human host. To confer this resistance, Entamoeba parasites synthesize a wall structure around the cell during cyst formation. This cyst wall consists of chitin and several protein components, including chitinase. The physiological roles of these components are not fully understood. Here, to elucidate the role of chitinase during cyst formation, we identified a new chitinase inhibitor by screening a library of 400 compounds. Using this inhibitor, we showed that chitinase inhibition causes the formation of abnormal cyst walls, the most characteristic of which is a "pot-like structure." This results in decreased production of mature cysts. Chitinase is therefore required for Entamoeba to produce mature cysts for transmission to a new host.

Stage-Specific De Novo Synthesis of Very-Long-Chain Dihydroceramides Confers Dormancy to Entamoeba Parasites.

Amoebiasis is a parasitic disease caused by Entamoeba histolytica infection and is a serious public health problem worldwide due to ill-prepared preventive measures as well as its high morbidity and mortality rates. Amoebiasis transmission is solely mediated by cysts. Cysts are produced by the differentiation of proliferative trophozoites in a process termed "encystation." Entamoeba encystation is a fundamental cell differentiation process and proceeds with substantial changes in cell metabolites, components, and morphology, which occur sequentially in an orchestrated manner. Lipids are plausibly among these metabolites that function as key factors for encystation. However, a comprehensive lipid analysis has not been reported, and the involved lipid metabolic pathways remain largely unknown. Here, we exploited the state-of-the-art untargeted lipidomics and characterized 339 molecules of 17 lipid subclasses. Of these, dihydroceramide (Cer-NDS) was found to be among the most induced lipid species during encystation. Notably, in encysting cells, amounts of Cer-NDS containing very long N-acyl chains (≥26 carbon) were more than 30-fold induced as the terminal product of a de novo metabolic pathway. We also identified three ceramide synthase genes responsible for producing the very-long-chain Cer-NDS molecules. These genes were upregulated during encystation. Furthermore, these ceramide species were shown to be indispensable for generating membrane impermeability, a prerequisite for becoming dormant cyst that shows resistance to environmental assault inside and outside the host for transmission. Hence, the lipid subclass of Cer-NDS plays a crucial role for Entamoeba cell differentiation and morphogenesis by alternating the membrane properties.IMPORTANCEEntamoeba is a protozoan parasite that thrives in its niche by alternating its two forms between a proliferative trophozoite and dormant cyst. Cysts are the only form able to transmit to a new host and are differentiated from trophozoites in a process termed "encystation." During Entamoeba encystation, cell metabolites, components, and morphology drastically change, which occur sequentially in an orchestrated manner. Lipids are plausibly among these metabolites. However, the involved lipid species and their metabolic pathways remain largely unknown. Here, we identified dihydroceramides (Cer-NDSs) containing very long N-acyl chains (C26 to C30) as a key metabolite for Entamoeba encystation by our state-of-the-art untargeted lipidomics. We also showed that these Cer-NDSs are critical to generate the membrane impermeability, a prerequisite for this parasite to show dormancy as a cyst that repels substances and prevents water loss. Hence, ceramide metabolism is essential for Entamoeba to maintain the parasitic lifestyle.

Hypericum erectum alcoholic extract inhibits Toxoplasma growth and Entamoeba encystation: an exploratory study on the anti-protozoan potential.

Hypericum erectum is an important ethnobotanical medicine in East Asian tradition. To explore the anti-parasitic potential of H. erectum, inhibitory effects on the growth of intracellular parasite Toxoplasma and on the encystation of intestinal parasite Entamoeba were examined. The constituents in H. erectum alcoholic extracts and fractions separated by solvent-partitioning were analysed by high resolution LC-MS. Toxoplasma gondii growth inhibition assay was performed using GFP-labelled T. gondii strain PTG-GFP by measuring the fluorescence intensity. Anti-Toxoplasma drug pyrimethamine was used as a positive control. T. gondii-induced immune reaction was assessed by quantitative PCR and fluorescence microscopy, using co-culture of PTG-GFP and monocyte-macrophage cell line Raw264. The inhibitory effect on the encystation of Entamoeba invadens was measured by flow-cytometry, where paromomycin was used as a positive control. H. erectum methanol (MeOH) extract (50 µg/mL) and ethyl acetate (EtOAc) fraction (50 µg/mL) inhibited the growth of T. gondii, while 50%MeOH extract and hydrophilic fractions were ineffective. Co-culture with T. gondii reduced the viability of macrophages, however macrophages were protected in the presence of H. erectum MeOH extract or EtOAc fraction (above 10 µg/mL). The MeOH extract and EtOAc fraction also effectively suppressed the encystation of E. invadens at 1 mg/mL. Hypericine, a major constituent in MeOH extract and EtOAc fraction, inhibited T. gondii growth and E. invadens encystation. Our results demonstrated that H. erectum effectively inhibited Toxoplasma growth and Entamoeba encystation. These activities are partly mediated by hypericin. In addition, it was suggested the extract and fraction may protect innate immune cells from Toxoplasma-induced damages, thereby enhancing parasite clearance. Further investigation is warranted to address the in vivo effectiveness of H. erectum as an anti-protozoal medicine.

Characterization of Entamoeba histolytica adenosine 5'-phosphosulfate (APS) kinase; validation as a target and provision of leads for the development of new drugs against amoebiasis.

BACKGROUND: Amoebiasis, caused by Entamoeba histolytica infection, is a global public health problem. However, available drugs to treat amoebiasis are currently limited, and no effective vaccine exists. Therefore, development of new preventive measures against amoebiasis is urgently needed. METHODOLOGY/PRINCIPAL FINDINGS: Here, to develop new drugs against amoebiasis, we focused on E. histolytica adenosine 5'-phosphosulfate kinase (EhAPSK), an essential enzyme in Entamoeba sulfolipid metabolism. Fatty alcohol disulfates and cholesteryl sulfate, sulfolipids synthesized in Entamoeba, play important roles in trophozoite proliferation and cyst formation. These processes are closely associated with clinical manifestation and severe pathogenesis of amoebiasis and with disease transmission, respectively. We validated a combination approach of in silico molecular docking analysis and an in vitro enzyme activity assay for large scale screening. Docking simulation ranked the binding free energy between a homology modeling structure of EhAPSK and 400 compounds. The 400 compounds were also screened by a 96-well plate-based in vitro APSK activity assay. Among fifteen compounds identified as EhAPSK inhibitors by the in vitro system, six were ranked by the in silico analysis as having high affinity toward EhAPSK. Furthermore, 2-(3-fluorophenoxy)-N-[4-(2-pyridyl)thiazol-2-yl]-acetamide, 3-phenyl-N-[4-(2-pyridyl)thiazol-2-yl]-imidazole-4-carboxamide, and auranofin, which were identified as EhAPSK inhibitors by both in silico and in vitro analyses, halted not only Entamoeba trophozoite proliferation but also cyst formation. These three compounds also dose-dependently impaired the synthesis of sulfolipids in E. histolytica. CONCLUSIONS/SIGNIFICANCE: Hence, the combined approach of in silico and in vitro-based EhAPSK analyses identified compounds that can be evaluated for their effects on Entamoeba. This can provide leads for the development of new anti-amoebic and amoebiasis transmission-blocking drugs. This strategy can also be applied to identify specific APSK inhibitors, which will benefit research into sulfur metabolism and the ubiquitous pathway terminally synthesizing essential sulfur-containing biomolecules.

A Flow Cytometry Method for Dissecting the Cell Differentiation Process of Entamoeba Encystation.

Amoebiasis is caused by Entamoeba histolytica infection, a protozoan parasite belonging to the phylum Amoebozoa. This parasite undergoes a fundamental cell differentiation process from proliferative trophozoite to dormant cyst, termed "encystation." The cysts formed by encystation are solely responsible for the transmission of amoebiasis; therefore, Entamoeba encystation is an important subject from both biological and medical perspectives. Here, we have established a flow cytometry strategy for not only determining the percentage of formed cysts but also for monitoring changes in cell populations during encystation. This strategy together with fluorescence microscopy enables visualization of the cell differentiation process of Entamoeba encystation. We also standardized another flow cytometry protocol for counting live trophozoites. These two different flow cytometry techniques could be integrated into 96-well plate-based bioassays for monitoring the processes of cyst formation and trophozoite proliferation, which are crucial to maintain the Entamoeba life cycle. The combined two systems enabled us to screen a chemical library, the Pathogen Box of the Medicine for Malaria Venture, to obtain compounds that inhibit either the formation of cysts or the proliferation of trophozoites, or both. This is a prerequisite for the development of new drugs against amoebiasis, a global public health problem. Collectively, the two different 96-well plate-based Entamoeba bioassay and flow cytometry analysis systems (cyst formation and trophozoite proliferation) provide a methodology that can not only overcome the limitations of standard microscopic counting but also is effective in applied as well as basic Entamoeba biology.

Apaf1 plays a negative regulatory role in T cell responses by suppressing activation of antigen-stimulated T cells.

Apaf1 is a critical component of the apoptosome and initiates apoptosis downstream mitochondrial damages. Although the importance of Apaf1 in embryonic development was shown, the role of Apaf1 in immune responses, especially T cell responses, has yet to be elucidated. We generated T cell-specific Apaf1-deficient mice (Lck-Cre-Apaf1f/f mice) and examined the antigen-specific delayed-type hypersensitivity (DTH). Lck-Cre-Apaf1f/f mice exhibited exacerbation of DTH responses as compared with Apaf1-sufficient control mice. In Lck-Cre-Apaf1f/f mice, antigen-specific T cells proliferated more, and produced more inflammatory cytokines than control T cells. Apaf1-deficient T cells from antigen-immunized mice showed higher percentages of activation phenotypes upon restimulation in vitro. Apaf1-deficient T cells from naive (non-immunized) mice also showed higher proliferation activity and cytokine production over control cells. The impact of Apaf1-deficiency in T cells, however, was not restored by a pan-caspase inhibitor, suggesting that the role of Apaf1 in T cell responses was caspase-independent/non-apoptotic. These data collectively demonstrated that Apaf1 is a negative regulator of T cell responses and implicated Apaf1 as a potential target for immunosuppressive drug discovery.

Mitochondria and apicoplast of Plasmodium falciparum: behaviour on subcellular fractionation and the implication.

The mitochondrion and the apicoplast of the malaria parasite, Plasmodium spp. is microscopically observed in a close proximity to each other. In this study, we tested the suitability of two different separation techniques--Percoll density gradient centrifugation and fluorescence-activated organelle sorting--for improving the purity of mitochondria isolated from the crude organelle preparation of Plasmodium falciparum. To our surprise, the apicoplast was inseparable from the plasmodial mitochondrion by each method. This implies these two plasmodial organelles are bound each other. This is the first experimental evidence of a physical binding between the two organelles in Plasmodium.

PfPDE1, a novel cGMP-specific phosphodiesterase from the human malaria parasite Plasmodium falciparum.

This is the first report of molecular characterization of a novel cyclic nucleotide PDE (phosphodiesterase), isolated from the human malaria parasite Plasmodium falciparum and designated PfPDE1. PfPDE1 cDNA encodes an 884-amino-acid protein, including six putative transmembrane domains in the N-terminus followed by a catalytic domain. The PfPDE1 gene is a single-copy gene consisting of two exons and a 170 bp intron. PfPDE1 transcripts were abundant in the ring form of the asexual blood stages of the parasite. The C-terminal catalytic domain of PfPDE1, produced in Escherichia coli, specifically hydrolysed cGMP with a K(m) value of 0.65 microM. Among the PDE inhibitors tested, a PDE5 inhibitor, zaprinast, was the most effective, having an IC50 value of 3.8 microM. The non-specific PDE inhibitors IBMX (3-isobutyl-1-methylxanthine), theophylline and the antimalarial chloroquine had IC50 values of over 100 microM. Membrane fractions prepared from P. falciparum at mixed asexual blood stages showed potent cGMP hydrolytic activity compared with cytosolic fractions. This hydrolytic activity was sensitive to zaprinast with an IC50 value of 4.1 microM, but insensitive to IBMX and theophylline. Furthermore, an in vitro antimalarial activity assay demonstrated that zaprinast inhibited the growth of the asexual blood parasites, with an ED50 value of 35 microM. The impact of cyclic nucleotide signalling on the cellular development of this parasite has previously been discussed. Thus this enzyme is suggested to be a novel potential target for the treatment of the disease malaria.