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

ABSTRACT 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.

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Localization and Targeting of an Unusual Pyridine Nucleotide Transhydrogenase in Entamoeba histolytica

Eukaryotic Cell ◽

10.1128/ec.00011-10 ◽

2010 ◽

Vol 9 (6) ◽

pp. 926-933 ◽

Cited By ~ 25

Author(s):

Mohammad Abu Yousuf ◽

Fumika Mi-ichi ◽

Kumiko Nakada-Tsukui ◽

Tomoyoshi Nozaki

Keyword(s):

Entamoeba Histolytica ◽

Fluorescent Protein ◽

Physiological Role ◽

Protozoan Parasite ◽

Pyridine Nucleotide ◽

Immunofluorescence Assay ◽

Chemical Degradation ◽

Content Type ◽

Targeting Signal ◽

Pyridine Nucleotide Transhydrogenase

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.

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Knockdown of Five Genes Encoding Uncharacterized Proteins Inhibits Entamoeba histolytica Phagocytosis of Dead Host Cells

Infection and Immunity ◽

10.1128/iai.01325-15 ◽

2016 ◽

Vol 84 (4) ◽

pp. 1045-1053 ◽

Cited By ~ 6

Author(s):

Adam Sateriale ◽

Peter Miller ◽

Christopher D. Huston

Keyword(s):

Host Cell ◽

Entamoeba Histolytica ◽

Protozoan Parasite ◽

Host Cells ◽

Data Set ◽

Feed Forward ◽

Content Type ◽

Genes Encoding ◽

Relative Specificity ◽

Cell Engulfment

Entamoeba histolyticais the protozoan parasite that causes invasive amebiasis, which is endemic to many developing countries and characterized by dysentery and liver abscesses. The virulence ofE. histolyticacorrelates with the degree of host cell engulfment, or phagocytosis, andE. histolyticaphagocytosis alters amebic gene expression in a feed-forward manner that results in an increased phagocytic ability. Here, we used a streamlined RNA interference screen to silence the expression of 15 genes whose expression was upregulated in phagocyticE. histolyticatrophozoites to determine whether these genes actually function in the phagocytic process. When five of these genes were silenced, amebic strains with significant decreases in the ability to phagocytose apoptotic host cells were produced. Phagocytosis of live host cells, however, was largely unchanged, and the defects were surprisingly specific for phagocytosis. Two of the five encoded proteins, which we namedE. histolyticaILWEQ (EhILWEQ) andE. histolyticaBAR (EhBAR), were chosen for localization via SNAP tag labeling and localized to the site of partially formed phagosomes. Therefore, both EhILWEQ and EhBAR appear to contribute toE. histolyticavirulence through their function in phagocytosis, and the large proportion (5/15 [33%]) of gene-silenced strains with a reduced ability to phagocytose host cells validates the previously published microarray data set demonstrating feed-forward control ofE. histolyticaphagocytosis. Finally, although only limited conclusions can be drawn from studies using the virulence-deficient G3Entamoebastrain, the relative specificity of the defects induced for phagocytosis of apoptotic cells but not healthy cells suggests that cell killing may play a rate-limiting role in the process ofEntamoeba histolyticahost cell engulfment.

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Mass Spectrometric Analysis of l-Cysteine Metabolism: Physiological Role and Fate of l-Cysteine in the Enteric Protozoan Parasite Entamoeba histolytica

mBio ◽

10.1128/mbio.01995-14 ◽

2014 ◽

Vol 5 (6) ◽

Cited By ~ 22

Author(s):

Ghulam Jeelani ◽

Dan Sato ◽

Tomoyoshi Soga ◽

Haruo Watanabe ◽

Tomoyoshi Nozaki

Keyword(s):

Oxidative Stress ◽

Amino Acid ◽

Carboxylic Acid ◽

Stable Isotope ◽

Carboxylic Acids ◽

Entamoeba Histolytica ◽

Protozoan Parasite ◽

Protozoan Parasites ◽

Metabolic Fate ◽

Content Type

ABSTRACTl-Cysteine is essential for virtually all living organisms, from bacteria to higher eukaryotes. Besides having a role in the synthesis of virtually all proteins and of taurine, cysteamine, glutathione, and other redox-regulating proteins,l-cysteine has important functions under anaerobic/microaerophilic conditions. In anaerobic or microaerophilic protozoan parasites, such asEntamoeba histolytica,l-cysteine has been implicated in growth, attachment, survival, and protection from oxidative stress. However, a specific role of this amino acid or related metabolic intermediates is not well understood. In this study, using stable-isotope-labeledl-cysteine and capillary electrophoresis-time of flight mass spectrometry, we investigated the metabolism ofl-cysteine inE. histolytica. [U-13C3,15N]l-cysteine was rapidly metabolized into three unknown metabolites, besidesl-cystine andl-alanine. These metabolites were identified as thiazolidine-4-carboxylic acid (T4C), 2-methyl thiazolidine-4-carboxylic acid (MT4C), and 2-ethyl-thiazolidine-4-carboxylic acid (ET4C), the condensation products ofl-cysteine with aldehydes. We demonstrated that these 2-(R)-thiazolidine-4-carboxylic acids serve for storage ofl-cysteine. Liberation ofl-cysteine occurred when T4C was incubated with amebic lysates, suggesting enzymatic degradation of thesel-cysteine derivatives. Furthermore, T4C and MT4C significantly enhanced trophozoite growth and reduced intracellular reactive oxygen species (ROS) levels when it was added to cultures, suggesting that 2-(R)-thiazolidine-4-carboxylic acids are involved in the defense against oxidative stress.IMPORTANCEAmebiasis is a human parasitic disease caused by the protozoan parasiteEntamoeba histolytica. In this parasite,l-cysteine is the principal low-molecular-weight thiol and is assumed to play a significant role in supplying the amino acid during trophozoite invasion, particularly when the parasites move from the anaerobic intestinal lumen to highly oxygenated tissues in the intestine and the liver. It is well known thatE. histolyticaneeds a comparatively high concentration ofl-cysteine for its axenic cultivation. However, the reason for and the metabolic fate ofl-cysteine in this parasite are not well understood. Here, using a metabolomic and stable-isotope-labeled approach, we investigated the metabolic fate of this amino acid in these parasites. We found thatl-cysteine inside the cell rapidly reacts with aldehydes to form 2-(R)-thiazolidine-4-carboxylic acid. We showed that these 2-(R)-thiazolidine-4-carboxylic derivatives serve as anl-cysteine source, promote growth, and protect cells against oxidative stress by scavenging aldehydes and reducing the ROS level. Our findings represent the first demonstration of 2-(R)-thiazolidine-4-carboxylic acids and their roles in protozoan parasites.

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Functional Characterization of Entamoeba histolytica Argonaute Proteins Reveals a Repetitive DR-Rich Motif Region That Controls Nuclear Localization

mSphere ◽

10.1128/msphere.00580-19 ◽

2019 ◽

Vol 4 (5) ◽

Cited By ~ 2

Author(s):

Hanbang Zhang ◽

Vy Tran ◽

Dipak Manna ◽

Gretchen Ehrenkaufer ◽

Upinder Singh

Keyword(s):

Gene Silencing ◽

Entamoeba Histolytica ◽

Nuclear Localization ◽

Functional Characterization ◽

Protozoan Parasite ◽

Rnai Pathway ◽

Content Type ◽

Localization Signal ◽

Response To Stress ◽

Paz Domain

ABSTRACT The RNA interference (RNAi) pathway regulates gene expression in many eukaryotic organisms. Argonaute (Ago) proteins, together with bound small RNAs (sRNAs), are key effectors that mediate gene silencing function. However, there is limited knowledge of Ago proteins and their functions in nonmodel systems. In the protozoan parasite Entamoeba histolytica, RNAi is a robust means for stable gene silencing mediated via large populations of antisense sRNAs. Here, we report functional characterization of three Ago proteins in E. histolytica (EhAgo2-1, EhAgo2-2, and EhAgo2-3). Our data show that each EhAgo protein has a distinct subcellular localization and binds 27-nucleotide (nt) sRNAs and that the localization of EhAgo proteins is altered in response to stress conditions. Via mutagenesis analyses, we demonstrated that the Ago PAZ (Piwi/Argonaute/Zwille) domain in all three EhAgos is essential for sRNA binding. With mutation of the PAZ domain in EhAgo2-2, there was no effect on the nuclear localization of the protein but a strong phenotype and a growth defect. We further show that EhAgo2-2 contains an unusual repetitive DR-rich (aspartic acid, arginine-rich) motif region which functions as a nuclear localization signal (NLS) and is both necessary and sufficient to mediate nuclear localization. Overall, our data delineate the localization and sRNA binding features of the three E. histolytica Ago proteins and demonstrate that the PAZ domain is necessary for sRNA binding. The repetitive DR-rich motif region in EhAgo2-2 has not previously been defined in other systems, which adds to the novel observations that can be made when studies of the RNAi pathway are extended to nonmodel systems. IMPORTANCE The protozoan parasite Entamoeba histolytica, which causes amebiasis and affects over 50 million people worldwide, contains an important RNAi pathway for gene silencing. Gene silencing via the RNAi pathway is mediated by the Argonaute (Ago) proteins. However, we lack knowledge on Ago function(s) in this nonmodel system. In this paper, we discovered that three E. histolytica Ago proteins (EhAgo2-1, EhAgo2-2, and EhAgo2-3) all bind 27-nt small RNAs and have distinct subcellular localizations, which change in response to stress conditions. The EhAgos bind small RNA populations via their PAZ domains. An unusual repetitive DR-rich motif region is identified in EhAgo2-2 that functions as a nuclear localization signal. Our results show for the first time an active nuclear transport process of the EhAgo2-2 RNA-induced silencing complex (RISC) in this parasite. These data add to the novel observations that can be made when studies of the RNAi pathway are extended to nonmodel systems.

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A Genomewide Overexpression Screen Identifies Genes Involved in the Phosphatidylinositol 3-Kinase Pathway in the Human Protozoan Parasite Entamoeba histolytica

Eukaryotic Cell ◽

10.1128/ec.00329-13 ◽

2014 ◽

Vol 13 (3) ◽

pp. 401-411 ◽

Cited By ~ 15

Author(s):

Amrita B. Koushik ◽

Brenda H. Welter ◽

Michelle L. Rock ◽

Lesly A. Temesvari

Keyword(s):

Entamoeba Histolytica ◽

Mutant Cell ◽

Lethal Dose ◽

Protozoan Parasite ◽

Pi3k Pathway ◽

Forward Genetics ◽

Pi3k Inhibitor ◽

Phosphatidylinositol 3 Kinase ◽

Content Type ◽

Phosphatidylinositol 3

ABSTRACTEntamoeba histolyticais a protozoan parasite that causes amoebic dysentery and liver abscess.E. histolyticarelies on motility, phagocytosis, host cell adhesion, and proteolysis of extracellular matrix for virulence. In eukaryotic cells, these processes are mediated in part by phosphatidylinositol 3-kinase (PI3K) signaling. Thus, PI3K may be critical for virulence. We utilized a functional genomics approach to identify genes whose products may operate in the PI3K pathway inE. histolytica. We treated a population of trophozoites that were overexpressing genes from a cDNA library with a near-lethal dose of the PI3K inhibitor wortmannin. This screen was based on the rationale that survivors would be overexpressing gene products that directly or indirectly function in the PI3K pathway. We sequenced the overexpressed genes in survivors and identified a cDNA encoding a Rap GTPase, a protein previously shown to participate in the PI3K pathway. This supports the validity of our approach. Genes encoding a coactosin-like protein, EhCoactosin, and a serine-richE. histolyticaprotein (SREHP) were also identified. Cells overexpressing EhCoactosin or SREHP were also less sensitive to a second PI3K inhibitor, LY294002. This corroborates the link between these proteins and PI3K. Finally, a mutant cell line with an increased level of phosphatidylinositol (3,4,5)-triphosphate, the product of PI3K activity, exhibited increased expression of SREHP and EhCoactosin. This further supports the functional connection between these proteins and PI3K inE. histolytica. To our knowledge, this is the first forward-genetics screen adapted to reveal genes participating in a signal transduction pathway in this pathogen.

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Localization of Phosphatidylinositol 4,5-Bisphosphate to Lipid Rafts and Uroids in the Human Protozoan Parasite Entamoeba histolytica

Infection and Immunity ◽

10.1128/iai.00040-13 ◽

2013 ◽

Vol 81 (6) ◽

pp. 2145-2155 ◽

Cited By ~ 16

Author(s):

Amrita B. Koushik ◽

Rhonda R. Powell ◽

Lesly A. Temesvari

Keyword(s):

Lipid Rafts ◽

Entamoeba Histolytica ◽

Protozoan Parasite ◽

Invasive Infection ◽

Cellular Functions ◽

Intestinal Protozoan ◽

Content Type ◽

Downstream Effector ◽

Key Factor ◽

Actomyosin Cytoskeleton

ABSTRACTEntamoeba histolyticais an intestinal protozoan parasite and is the causative agent of amoebiasis. During invasive infection, highly motile amoebae destroy the colonic epithelium, enter the blood circulation, and disseminate to other organs such as liver, causing liver abscess. Motility is a key factor inE. histolyticapathogenesis, and this process relies on a dynamic actomyosin cytoskeleton. In other systems, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is known to regulate a wide variety of cellular functions, including signal transduction, actin remodeling, and cell motility. Little is known about the role of PI(4,5)P2inE. histolyticapathogenicity. In this study, we demonstrate that PI(4,5)P2is localized to cholesterol-rich microdomains, lipid rafts, and the actin-rich fractions of theE. histolyticamembrane. Microscopy revealed that the trailing edge of polarized trophozoites, uroids, are highly enriched in lipid rafts and their constituent lipid, PI(4,5)P2. Polarization and enrichment of uroids and rafts with PI(4,5)P2were enhanced upon treatment ofE. histolyticacells with cholesterol. Exposure to cholesterol also increased intracellular calcium, which is a downstream effector of PI(4,5)P2, with a concomitant increase in motility. Together, our data suggest that inE. histolytica, PI(4,5)P2may signal from lipid rafts and cholesterol may play a role in triggering PI(4,5)P2-mediated signaling to enhance the motility of this pathogen.

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Overexpression of Specific Cysteine Peptidases Confers Pathogenicity to a Nonpathogenic Entamoeba histolytica Clone

mBio ◽

10.1128/mbio.00072-13 ◽

2013 ◽

Vol 4 (2) ◽

Cited By ~ 27

Author(s):

Jenny Matthiesen ◽

Ann-Katrein Bär ◽

Anne-Kathrin Bartels ◽

Dennis Marien ◽

Susann Ofori ◽

...

Keyword(s):

Entamoeba Histolytica ◽

Liver Abscess ◽

Clinical Symptoms ◽

Axenic Culture ◽

Protozoan Parasite ◽

Culture Conditions ◽

Amoebic Liver Abscess ◽

Content Type ◽

Pathogenicity Factors ◽

Cysteine Peptidases

ABSTRACTCysteine peptidases (CPs) ofEntamoeba histolyticaare considered to be important pathogenicity factors. Previous studies have found that under standard axenic culture conditions, only four (ehcp-a1,ehcp-a2,ehcp-a5, andehcp-a7) out of 35 papain-likeehcpgenes present in theE. histolyticagenome are expressed at high levels. Little is known about the expression of CPs inE. histolyticaduring amoebic liver abscess (ALA) formation. In the current study, a quantitative real-time PCR assay was developed to determine the expression of the variousehcpgenes during ALA formation in animal models. Increased expression of fourehcpgenes (ehcp-a3,-a4,-a10, and-c13) was detected in the gerbil and mouse models. Increased expression of another threeehcpgenes (ehcp-a5,-a6, and-a7) was detected in the mouse model only, and two otherehcpgenes (ehcp-b8and-b9) showed increased expression in the gerbil model only. Trophozoites of the nonpathogenicE. histolyticaHM-1:IMSS clone A1, which was unable to induce ALAs, were transfected with vectors enabling overexpression of those CPs that are expressed at high levels under culture conditions or during ALA formation. Interestingly, overexpression ofehcp-b8,-b9, and-c13restored the pathogenic phenotype of the nonpathogenic clone A1 whereas overexpression of various other peptidase genes had no effect on the pathogenicity of this clone.IMPORTANCEEntamoeba histolyticais a widespread and clinically important protozoan parasite. It normally exists in the human intestine without causing clinical symptoms but can invade the intestinal mucosa, which causes serious intestinal (amoebic colitis) and extraintestinal (amoebic liver abscess [ALA]) diseases. The identification of factors responsible for the invasion of the parasite and disease formation is a major topic in the field. Here, we investigate the roles of different papain-like cysteine peptidases (CPs) as pathogenicity factors. We show that the expression of some of the peptidases that are normally expressed at low levels increases during ALA formation. Furthermore, nonpathogenic amoebae can be transformed to pathogenic amoebae, simply by specific overexpression of some of these CPs. Our findings reinforce the importance of CPs as pathogenicity factors ofE. histolytica.

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Biochemical and Kinetic Characterization of the Recombinant ADP-Forming Acetyl Coenzyme A Synthetase from the Amitochondriate Protozoan Entamoeba histolytica

Eukaryotic Cell ◽

10.1128/ec.00192-14 ◽

2014 ◽

Vol 13 (12) ◽

pp. 1530-1537 ◽

Cited By ~ 6

Author(s):

Cheryl P. Jones ◽

Cheryl Ingram-Smith

Keyword(s):

Entamoeba Histolytica ◽

Coenzyme A ◽

Protozoan Parasite ◽

Acetyl Coa ◽

Lower Efficiency ◽

Acetyl Coenzyme A ◽

Content Type ◽

Atp Production ◽

Acetyl Coenzyme ◽

Atp Generation

ABSTRACTEntamoeba histolytica, an amitochondriate protozoan parasite that relies on glycolysis as a key pathway for ATP generation, has developed a unique extended PPi-dependent glycolytic pathway in which ADP-forming acetyl-coenzyme A (CoA) synthetase (ACD; acetate:CoA ligase [ADP-forming]; EC 6.2.1.13) converts acetyl-CoA to acetate to produce additional ATP and recycle CoA. We characterized the recombinantE. histolyticaACD and found that the enzyme is bidirectional, allowing it to potentially play a role in ATP production or in utilization of acetate. In the acetate-forming direction, acetyl-CoA was the preferred substrate and propionyl-CoA was used with lower efficiency. In the acetyl-CoA-forming direction, acetate was the preferred substrate, with a lower efficiency observed with propionate. The enzyme can utilize both ADP/ATP and GDP/GTP in the respective directions of the reaction. ATP and PPiwere found to inhibit the acetate-forming direction of the reaction, with 50% inhibitory concentrations of 0.81 ± 0.17 mM (mean ± standard deviation) and 0.75 ± 0.20 mM, respectively, which are both in the range of their physiological concentrations. ATP and PPidisplayed mixed inhibition versus each of the three substrates, acetyl-CoA, ADP, and phosphate. This is the first example of regulation of ACD enzymatic activity, and possible roles for this regulation are discussed.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1423718112 ◽

2015 ◽

Vol 112 (22) ◽

pp. E2884-E2890 ◽

Cited By ~ 33

Author(s):

Fumika Mi-ichi ◽

Tomofumi Miyamoto ◽

Shouko Takao ◽

Ghulam Jeelani ◽

Tetsuo Hashimoto ◽

...

Keyword(s):

Culture Media ◽

Cyst Formation ◽

Close Relative ◽

Dependent Manner ◽

Gene Encoding ◽

Major Function ◽

Activation Pathway ◽

Sulfate Activation ◽

Cholesteryl Sulfate

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.

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Cysteine Protease-Binding Protein Family 6 Mediates the Trafficking of Amylases to Phagosomes in the Enteric Protozoan Entamoeba histolytica

Infection and Immunity ◽

10.1128/iai.00915-12 ◽

2013 ◽

Vol 81 (5) ◽

pp. 1820-1829 ◽

Cited By ~ 16

Author(s):

Atsushi Furukawa ◽

Kumiko Nakada-Tsukui ◽

Tomoyoshi Nozaki

Keyword(s):

Entamoeba Histolytica ◽

Cysteine Protease ◽

Binding Protein ◽

Proteome Analysis ◽

Protozoan Parasite ◽

Cysteine Proteases ◽

Protein Family ◽

Intestinal Protozoan ◽

Α Subunit ◽

Content Type

ABSTRACTPhagocytosis plays a pivotal role in nutrient acquisition and evasion from the host defense systems inEntamoeba histolytica, the intestinal protozoan parasite that causes amoebiasis. We previously reported thatE. histolyticapossesses a unique class of a hydrolase receptor family, designated the cysteine protease-binding protein family (CPBF), that is involved in trafficking of hydrolases to lysosomes and phagosomes, and we have also reported that CPBF1 and CPBF8 bind to cysteine proteases or β-hexosaminidase α-subunit and lysozymes, respectively. In this study, we showed by immunoprecipitation that CPBF6, one of the most highly expressed CPBF proteins, specifically binds to α-amylase and γ-amylase. We also found that CPBF6 is localized in lysosomes, based on immunofluorescence imaging. Immunoblot and proteome analyses of the isolated phagosomes showed that CPBF6 mediates transport of amylases to phagosomes. We also demonstrated that the carboxyl-terminal cytosolic region of CPBF6 is engaged in the regulation of the trafficking of CPBF6 to phagosomes. Our proteome analysis of phagosomes also revealed new potential phagosomal proteins.

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