Kumiko Nakada-Tsukui

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 α-proteobacterial, δ-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.

Bacterial-type oxygen detoxification and iron-sulfur cluster assembly in amoebal relict mitochondria.

The assembly of vital reactive iron‐sulfur (Fe‐S) cofactors in eukaryotes is mediated by proteins inherited from the original mitochondrial endosymbiont. Uniquely among eukaryotes, however, Entamoeba and Mastigamoeba lack such mitochondrial‐type Fe‐S cluster assembly proteins and possess instead an analogous bacterial‐type system acquired by lateral gene transfer. Here we demonstrate, using immunomicroscopy and biochemical methods, that beyond their predicted cytosolic distribution the bacterial‐type Fe‐S cluster assembly proteins NifS and NifU have been recruited to function within the relict mitochondrial organelles (mitosomes) of Entamoeba histolytica. Both Nif proteins are 10‐fold more concentrated within mitosomes compared with their cytosolic distribution suggesting that active Fe‐S protein maturation occurs in these organelles. Quantitative immunoelectron microscopy showed that amoebal mitosomes are minute but highly abundant cellular structures that occupy up to 2% of the total cell volume. In addition, protein colocalization studies allowed identification of the amoebal hydroperoxide detoxification enzyme rubrerythrin as a mitosomal protein. This protein contains functional Fe‐S centres and exhibits peroxidase activity in vitro. Our findings demonstrate the role of analogous protein replacement in mitochondrial organelle evolution and suggest that the relict mitochondrial organelles of Entamoeba are important sites of metabolic activity that function in Fe‐S protein‐mediated oxygen detoxification.

Phosphatidylinositol-phosphates mediate cytoskeletal reorganization during phagocytosis via a unique modular protein consisting of RhoGEF/DH and FYVE domains in the parasitic protozoon Entamoeba histolytica.

To understand the roles of phosphoinositides [PtdIns] in phagocytosis of parasitic eukaryotes, we examined the interaction of phosphatidylinositol‐3‐phosphate [PtdIns(3)P] and putative PtdIns‐P‐binding proteins during phagocytosis in the enteric protozoan parasite Entamoeba histolytica. It was previously shown that phagocytosis in E. histolytica is indispensable for virulence and is inhibited by PtdIns 3‐kinase inhibitors. We demonstrated by time‐lapse live imaging that during the initiation of phagocytosis, the PtdIns(3)P biomarker GFP–Hrs–FYVE, was translocated to the phagocytic cup, phagosome, and to tunnel‐like structures connecting the plasma membrane and phagosomes. E. histolytica possesses 12 FYVE domain‐containing proteins (EhFP1‐12), 11 of which also contain the RhoGEF/DH domain. Among them EhFP4 was shown to be recruited to the tunnel‐like structures and to the proximal region of the phagosome. We further demonstrated that EhFP4 physically interacted with 4 of 10 predominant Rho/Rac small GTPases. Phosphoinositide binding assay showed that EhFP4 unexpectedly bound to PtdIns(4)P via the carboxyl‐terminal domain and that the FYVE domain modulates the binding specificity of EhFP4 to PtdIns‐P. Expression of the FYVE domain from EhFP4 inhibited phagocytosis while enhancement was observed when mammalian Hrs–FYVE domain was expressed. Altogether, we demonstrated that PtdIns(3)P, PtdIns(4)P and EhFP4 coordinately regulate phagocytosis and phagosome maturation in this parasitic eukaryote.

Autophagy during Proliferation and Encystation in the Protozoan Parasite Entamoeba invadens

ABSTRACT 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 lipoprotein receptor-related protein-1 (LRP) adapter protein GULP mediates trafficking of the LRP ligand prosaposin, leading to sphingolipid and free cholesterol accumulation in late endosomes and impaired efflux.

One of the conserved functional pathways linked to engulfment of apoptotic corpses involves two membrane proteins low density lipoprotein receptor-related protein-1 (LRP) and ABCA1 and the LRP adapter protein GULP. Because LRP and ABCA1 play roles in cellular lipid trafficking and efflux, here we addressed whether the third member, the LRP adapter protein GULP, also affects cellular lipid transport. Several lines of evidence show that overexpression of GULP causes glycosphingolipid and free cholesterol accumulation in the late endosome/lysosome compartment that is accompanied by down-regulation of ABCA1 and decreased efflux. Conversely, knockdown of endogenous GULP expression promoted cholesterol flux through the late endosomes and up-regulation of ABCA1, even in the context of a disease state such as Niemann-Pick Type C disease. Mechanistically, we were able to show that trafficking of the LRP ligands α2-macroglobulin and prosaposin, a protein cofactor necessary for glycosphingolipid degradation, are impaired in cells expressing full-length GULP protein, resulting in glycosphingolipid and free cholesterol accumulation in the late endosome/lysosome compartment. On the other hand, knockdown of endogenous GULP results in enhanced targeting of prosaposin and enhanced clearance of glycosphingolipids and cholesterol from the late endosomes. Taken together, these data reveal that GULP/LRP/ABCA1 represents a triad of molecules involved in engulfment and cellular lipid homeostasis.

Transcriptome analysis of encystation in Entamoeba invadens.

Encystation is an essential differentiation process for the completion of the life cycle of a group of intestinal protozoa including Entamoeba histolytica, the causative agent of intestinal and extraintestinal amebiasis. However, regulation of gene expression during encystation is poorly understood. To comprehensively understand the process at the molecular level, the transcriptomic profiles of E . invadens , which is a related reptilian species that causes an invasive disease similar to that of E. histolytica, was investigated during encystation. Using a custom-generated Affymetrix platform microarray, we performed time course (0.5, 2, 8, 24, 48, and 120 h) gene expression analysis of encysting E . invadens . ANOVA analysis revealed that a total of 1,528 genes showed ≥3 fold up-regulation at one or more time points, relative to the trophozoite stage. Of these modulated genes, 8% (116 genes) were up-regulated at the early time points (0.5, 2 and 8h), while 63% (962 genes) were up-regulated at the later time points (24, 48, and 120 h). Twenty nine percent (450 genes) are either up-regulated at 2 to 5 time points or constitutively up-regulated in both early and late stages. Among the up-regulated genes are the genes encoding transporters, cytoskeletal proteins, proteins involved in vesicular trafficking (small GTPases), Myb transcription factors, cysteine proteases, components of the proteasome, and enzymes for chitin biosynthesis. This study represents the first kinetic analysis of gene expression during differentiation from the invasive trophozoite to the dormant, infective cyst stage in Entamoeba . Functional analysis on individual genes and their encoded products that are modulated during encystation may lead to the discovery of targets for the development of new chemotherapeutics that interfere with stage conversion of the parasite.

A novel class of cysteine protease receptors that mediate lysosomal transport

The transport of lysosomal proteins is, in general, mediated by mannose 6‐phosphate receptors via carbohydrate modifications. Here, we describe a novel class of receptors that regulate the transport of lysosomal hydrolases in the enteric protozoan Entamoeba histolytica, which is a good model organism to investigate membrane traffic. A novel 110 kDa cysteine protease (CP) receptor (CP‐binding protein family 1, CPBF1) was initially discovered by affinity co‐precipitation of the major CP (EhCP‐A5), which plays a pivotal role in the pathogenesis of E. histolytica. We demonstrated that CPBF1 regulates EhCP‐A5 transport from the endoplasmic reticulum to lysosomes and its binding to EhCP‐A5 is independent of carbohydrate modifications. Repression of CPBF1 by gene silencing led to the accumulation of the unprocessed form of EhCP‐A5 in the non‐acidic compartment and the mis‐secretion of EhCP‐A5, suggesting that CPBF1 is involved in the trafficking and processing of EhCP‐A5. The CPBF represents a new class of transporters that bind to lysosomal hydrolases in a carbohydrate‐independent fashion and regulate their trafficking, processing and activation and, thus, regulate the physiology and pathogenesis of E. histolytica.

Conservation and function of Rab small GTPases in Entamoeba: annotation of E. invadens Rab and its use for the understanding of Entamoeba biology.

Entamoeba invadens is a reptilian enteric protozoan parasite closely related to the human pathogen Entamoeba histolytica and a good model organism of encystation. To understand the molecular mechanism of vesicular trafficking involved in the encystation of Entamoeba, we examined the conservation of Rab small GTPases between the two species. E. invadens has over 100 Rab genes, similar to E. histolytica. Most of the Rab subfamilies are conserved between the two species, while a number of species-specific Rabs are also present. We annotated all E. invadens Rabs according to the previous nomenclature [Saito-Nakano, Y., Loftus, B.J., Hall, N., Nozaki, T., 2005. The diversity of Rab GTPases in Entamoeba histolytica. Experimental Parasitology 110, 244-252]. Comparative genomic analysis suggested that the fundamental vesicular traffic machinery is well conserved, while there are species-specific protein transport mechanisms. We also reviewed the function of Rabs in Entamoeba, and proposed the use of the annotation of E. invadens Rab genes to understand the ubiquitous importance of Rab-mediated membrane trafficking during important biological processes including differentiation in Entamoeba.

Membrane trafficking as a virulence mechanism of the enteric protozoan parasite Entamoeba histolytica

Vesicular trafficking plays an essential role in the expression of virulence competence of the intestinal protozoan parasite E. histolytica that causes amebic dysentery, colitis, and liver abscess in humans, and is responsible for an estimated 50 million cases of amebiasis and 40–100 thousand deaths annually (Haque et al. 2003; Huston 2004). Phagocytosis does not only play a housekeeping role for nutrient uptake, but also participates in various processes essential for colonization and virulence. It has been shown that phagocytosis is involved in the removal of necrotic and apoptotic host cells for colonization and immune evasion (Huston et al. 2003). Indeed, phagocytosis-deficient E. histolytica mutants were defective in the destruction of tissue-cultured mammalian cells in vitro and the formation of hepatic abscesses in vivo (Orozco et al. 1985). In addition to phagocytosis, secretion of digestive proteins including cysteine proteases (CPs) and membrane-permeabilizing peptide amoebapores (APs) has been considered to be responsible for cytopathic activity, i.e., the degradation of host cells and destruction of tissues (Que and Reed 2000; Zhai and Saier 2000). The premise that CPs play a key role in in vitro and in vivo virulence was recently verified by reverse genetic approaches. Overexpression of CP2 caused augmentation of monolayer destruction, but no change in liver abscess formation (Hellberg et al. 2001). In contrast, antisense inhibition of CP5, a putative membrane-bound CP isotype, resulted in a reduced capacity of liver abscess formation (Ankri et al. 1999). These results imply that both secreted and surfacebound CPs are involved in pathogenesis in vitro and in vivo. Interestingly, the above-mentioned phagocytosis-deficient E. histolyticamutants contained a lower level of CPs than the wild-type amebae (Carpeniseanu et al. 2000), suggesting a defect in a pathway shared by phagocytosis and CP secretion, e.g., in trafficking or cytoskeleton, in these mutants. Phagocytosis consists of a number of steps including cell surface binding to ligands and the activation of a signaling pathway leading to F-actin polymerization. In addition, membrane trafficking plays an important role in the controlled maturation of phagosomes. The phagosome maturation is accompanied by sequential fusion with the endocytic and biosynthetic compartments to form a phagolysosome (Stuart and Ezekowitz 2005) and orchestrated by small GTPase, Rab proteins, which act as molecular switches regulating the fusion of vesicles with target membranes through the conformational change between active (GTP-bound) and inactive (GDP-bound) forms (Stenmark and Olkkonen 2001; Takai et al. 2001). Secretion of hydrolytic enzymes is also triggered by a specific ligand– receptor interaction, leading to dynamic vesicular trafficking as well as cytoskeletal reorganization.

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

ABSTRACT 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.