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

mSphere ◽  
2019 ◽  
Vol 4 (5) ◽  
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.

scholarly journals Functional Characterization of the Nuclear Localization Signal for a Suppressor of Posttranscriptional Gene Silencing

2003 ◽  
Vol 77 (12) ◽  
pp. 7026-7033 ◽  
Author(s):  
Xiangli Dong ◽  
Rene van Wezel ◽  
John Stanley ◽  
Yiguo Hong
Keyword(s):  
Amino Acid ◽  
Gene Silencing ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Fluorescent Protein ◽  
Functional Characterization ◽  
Potato Virus X ◽  
Posttranscriptional Gene Silencing ◽  
Arginine Residues ◽  
Localization Signal

ABSTRACT The nucleus-localized C2 protein of Tomato yellow leaf curl virus-China (TYLCV-C) is an active suppressor of posttranscriptional gene silencing (PTGS). Consistently, infection with TYLCV-C resulted in PTGS arrest in plants. The C2 protein possesses a functional, arginine-rich nuclear localization signal within the basic amino acid-rich region 17KVQHRIAKKTTRRRR31. When expressed from potato virus X, C2-RRRR31DVGG (in which the four consecutive arginine residues 28RRRR31 were replaced with DVGG) that had been tagged with a green fluorescent protein (GFP) failed to transport GFP into nuclei and was dysfunctional in inducing necrosis and suppressing PTGS in plants. Amino acid substitution mutants C2-K17D-GFP, C2-HR21DV-GFP, and C2-KK25DI-GFP localized to nuclei and produced necrosis, but only C2-K17D-GFP suppressed PTGS. The N-terminal portions C21-31 and C217-31 fused in frame to GFP were capable of targeting GFP to nuclei, but neither caused necrosis nor affected PTGS. Our data establish that nuclear localization is likely required for C2 protein to function in C2-mediated induction of necrosis and suppression of PTGS, which may follow diverse pathways in plants. Possible mechanisms of how the C2 protein involves these biological functions are discussed.

scholarly journals Characterization of Extracellular Vesicles from Entamoeba histolytica Identifies Roles in Intercellular Communication That Regulates Parasite Growth and Development

2020 ◽  
Vol 88 (10) ◽  
Author(s):  
Manu Sharma ◽  
Pedro Morgado ◽  
Hanbang Zhang ◽  
Gretchen Ehrenkaufer ◽  
Dipak Manna ◽  
...  
Keyword(s):  
Entamoeba Histolytica ◽  
Extracellular Vesicles ◽  
Small Rna ◽  
Intercellular Communication ◽  
Cell Communication ◽  
Protozoan Parasite ◽  
Cytoskeletal Proteins ◽  
Rnai Pathway ◽  
Sequencing Analysis ◽  
Content Type

ABSTRACT Extracellular vesicles (EVs) secreted by eukaryotic and prokaryotic cells to transport lipids, proteins, and nucleic acids to the external environment have important roles in cell-cell communication through cargo transfer. We identified and characterized EVs from Entamoeba histolytica, a protozoan parasite and a human pathogen. Conditioned medium from amebic parasites contained particles consistent with the expected size and morphology of EVs. Mass spectrometry was used to characterize the EV proteome and showed that it was enriched in common exosome marker proteins, including proteins associated with vesicle formation, cell signaling, and metabolism, as well as cytoskeletal proteins. Additionally, the EVs were found to selectively package small RNAs (sRNA), which were protected within the vesicles against RNase treatment. Sequencing analysis of the sRNA contained in EVs revealed that the majority were 27 nucleotides (nt) in size and represented a subset of the cellular antisense small RNA population that has previously been characterized in Entamoeba. RNA interference (RNAi) pathway proteins, including Argonaute, were also present in amebic EVs. Interestingly, we found that the amebic EVs impacted intercellular communication between parasites and altered encystation efficiency. EVs isolated from encysting parasites promoted encystation in other parasites, whereas EVs from metabolically active trophozoites impeded encystation. Overall, the data reveal that Entamoeba secrete EVs that are similar in size and shape to previously characterized exosomes from other organisms and that these EVs contain a defined protein and small RNA cargo and have roles in intercellular communication among parasites and influence growth kinetics.

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

2010 ◽  
Vol 9 (6) ◽  
pp. 926-933 ◽  
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.

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

2016 ◽  
Vol 84 (4) ◽  
pp. 1045-1053 ◽  
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.

scholarly journals RISC in Entamoeba histolytica: Identification of a Protein-Protein Interaction Network for the RNA Interference Pathway in a Deep-Branching Eukaryote

mBio ◽  
2021 ◽  
Author(s):  
Hanbang Zhang ◽  
Juliana Veira ◽  
Sarah Twitty Bauer ◽  
Christopher Yip ◽  
Upinder Singh
Keyword(s):  
Developing Countries ◽  
Rna Interference ◽  
Entamoeba Histolytica ◽  
Molecular Basis ◽  
Interaction Network ◽  
Rnai Pathway ◽  
Content Type ◽  
Protein Protein Interaction ◽  
Endogenous Genes ◽  
Protein Protein Interaction Network

Entamoeba histolytica is a leading parasitic cause of death in developing countries, and our efforts are focused on defining the molecular basis of RNA interference (RNAi) gene regulation in this parasite. The Entamoeba RNAi pathway effectively silences a subset of endogenous genes and has also been harnessed as a gene silencing tool to study gene function in this organism.

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

mBio ◽  
2014 ◽  
Vol 5 (6) ◽  
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.

scholarly journals Development of RNA Interference Trigger-Mediated Gene Silencing in Entamoeba invadens

2016 ◽  
Vol 84 (4) ◽  
pp. 964-975 ◽  
Author(s):  
Susmitha Suresh ◽  
Gretchen Ehrenkaufer ◽  
Hanbang Zhang ◽  
Upinder Singh
Keyword(s):  
Oxidative Stress ◽  
Rna Interference ◽  
Gene Silencing ◽  
Pathogen Transmission ◽  
Rnai Pathway ◽  
Protease Gene ◽  
Coding Region ◽  
Content Type ◽  
Myb Gene ◽  
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 invadensis the model system to studyEntamoebadevelopmental biology, as high-grade regulated encystation and excystation are readily achievable. However, the lack of gene-silencing tools inE. invadenshas limited the molecular studies that can be performed. Using the endogenous RNA interference (RNAi) pathway inEntamoeba, we developed an RNAi-based trigger gene-silencing approach inE. 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 twoE. invadensgenes: 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 inE. invadens. Dissection of the molecular basis ofEntamoebastage conversion is now possible, representing an important technical advance for the system.

scholarly journals Functional Characterization of an Interferon Gamma Receptor-Like Protein on Entamoeba histolytica

2019 ◽  
Vol 87 (11) ◽  
Author(s):  
Julieta Pulido-Ortega ◽  
Patricia Talamás-Rohana ◽  
Martín Humberto Muñoz-Ortega ◽  
Liseth Rubí Aldaba-Muruato ◽  
Sandra Luz Martínez-Hernández ◽  
...  
Keyword(s):  
Entamoeba Histolytica ◽  
Interferon Gamma ◽  
Functional Characterization ◽  
Protein Sequencing ◽  
Content Type ◽  
Human Red Blood Cells ◽  
Gamma Receptor ◽  
Cyclooxygenase 1 ◽  
Human Proteins ◽  
Ifn Γ

ABSTRACT Entamoeba histolytica is an anaerobic parasitic protozoan and the causative agent of amoebiasis. E. histolytica expresses proteins that are structurally homologous to human proteins and uses them as virulence factors. We have previously shown that E. histolytica binds exogenous interferon gamma (IFN-γ) on its surface, and in this study, we explored whether exogenous IFN-γ could modulate parasite virulence. We identified an IFN-γ receptor-like protein on the surface of E. histolytica trophozoites by using anti-IFN-γ receptor 1 (IFN-γR1) antibody and performing immunofluorescence, Western blot, protein sequencing, and in silico analyses. Coupling of human IFN-γ to the IFN-γ receptor-like protein on live E. histolytica trophozoites significantly upregulated the expression of E. histolytica cysteine protease A1 (EhCP-A1), EhCP-A2, EhCP-A4, EhCP-A5, amebapore A (APA), cyclooxygenase 1 (Cox-1), Gal-lectin (Hgl), and peroxiredoxin (Prx) in a time-dependent fashion. IFN-γ signaling via the IFN-γ receptor-like protein enhanced E. histolytica’s erythrophagocytosis of human red blood cells, which was abrogated by the STAT1 inhibitor fludarabine. Exogenous IFN-γ enhanced chemotaxis of E. histolytica, its killing of Caco-2 colonic and Hep G2 liver cells, and amebic liver abscess formation in hamsters. These results demonstrate that E. histolytica expresses a surface IFN-γ receptor-like protein that is functional and may play a role in disease pathogenesis and/or immune evasion.

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