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 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 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 Viral Small-RNA Analysis of Bombyx mori Larval Midgut during Persistent and Pathogenic Cytoplasmic Polyhedrosis Virus Infection

2015 ◽  
Vol 89 (22) ◽  
pp. 11473-11486 ◽  
Author(s):  
Aris Zografidis ◽  
Filip Van Nieuwerburgh ◽  
Anna Kolliopoulou ◽  
Konstantinos Apostolou-Karampelis ◽  
Steven R. Head ◽  
...  
Keyword(s):  
Bombyx Mori ◽  
Small Rna ◽  
Rnai Pathway ◽  
Cold Spot ◽  
Antiviral Defense ◽  
Viral Dsrna ◽  
Cytoplasmic Polyhedrosis Virus ◽  
Double Stranded Rna ◽  
Content Type ◽  
Polyhedrosis Virus

ABSTRACTThe lepidopteran innate immune response against RNA viruses remains poorly understood, while in other insects several studies have highlighted an essential role for the exo-RNAi pathway in combating viral infection. Here, by using deep-sequencing technology for viral small-RNA (vsRNA) assessment, we provide evidence that exo-RNAi is operative in the silkwormBombyx moriagainst both persistent and pathogenic infection ofB. moricytoplasmic polyhedrosis virus (BmCPV) which is characterized by a segmented double-stranded RNA (dsRNA) genome. Further, we show that Dicer-2 predominantly targets viral dsRNA and produces 20-nucleotide (nt) vsRNAs, whereas an additional pathway is responsive to viral mRNA derived from segment 10. Importantly, vsRNA distributions, which define specific hot and cold spot profiles for each viral segment, to a considerable degree overlap between Dicer-2-related (19 to 21 nt) and Dicer-2-unrelated vsRNAs, suggesting a common origin for these profiles. We found a degenerate motif significantly enriched at the cut sites of vsRNAs of various lengths which link an unknown RNase to the origins of vsRNAs biogenesis and distribution. Accordingly, the indicated RNase activity may be an important early factor for the host's antiviral defense in Lepidoptera.IMPORTANCEThis work contributes to the elucidation of the lepidopteran antiviral response against infection of segmented double-stranded RNA (dsRNA) virus (CPV;Reoviridae) and highlights the importance of viral small-RNA (vsRNA) analysis for getting insights into host-pathogen interactions. Three vsRNA pathways are implicated in antiviral defense. For dsRNA, two pathways are proposed, either based on Dicer-2 cleavage to generate 20-nucleotide vsRNAs or based on the activity of an uncharacterized endo-RNase that cleaves the viral RNA substrate at a degenerate motif. The analysis also indicates the existence of a degradation pathway that targets the positive strand of segment 10.

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 Differential Contribution of RNA Interference Components in Response to Distinct Fusarium graminearum Virus Infections

2018 ◽  
Vol 92 (9) ◽  
pp. e01756-17 ◽  
Author(s):  
Jisuk Yu ◽  
Kyung-Mi Lee ◽  
Won Kyong Cho ◽  
Ju Yeon Park ◽  
Kook-Hyung Kim
Keyword(s):  
Rna Interference ◽  
Fusarium Graminearum ◽  
Small Rna ◽  
Defense Response ◽  
Rna Silencing ◽  
Cryphonectria Parasitica ◽  
Small Rna Sequencing ◽  
Sequencing Analysis ◽  
Antiviral Defense ◽  
Content Type

ABSTRACT The mechanisms of RNA interference (RNAi) as a defense response against viruses remain unclear in many plant-pathogenic fungi. In this study, we used reverse genetics and virus-derived small RNA profiling to investigate the contributions of RNAi components to the antiviral response against Fusarium graminearum viruses 1 to 3 (FgV1, -2, and -3). Real-time reverse transcription-quantitative PCR (qRT-PCR) indicated that infection of Fusarium graminearum by FgV1, -2, or -3 differentially induces the gene expression of RNAi components in F. graminearum. Transcripts of the DICER-2 and AGO-1 genes of F. graminearum (FgDICER-2 and FgAGO-1) accumulated at lower levels following FgV1 infection than following FgV2 or FgV3 infection. We constructed gene disruption and overexpression mutants for each of the Argonaute and dicer genes and for two RNA-dependent RNA polymerase (RdRP) genes and generated virus-infected strains of each mutant. Interestingly, mycelial growth was significantly faster for the FgV1-infected FgAGO-1 overexpression mutant than for the FgV1-infected wild type, while neither FgV2 nor FgV3 infection altered the colony morphology of the gene deletion and overexpression mutants. FgV1 RNA accumulation was significantly decreased in the FgAGO-1 overexpression mutant. Furthermore, the levels of induction of FgAGO-1, FgDICER-2, and some of the FgRdRP genes caused by FgV2 and FgV3 infection were similar to those caused by hairpin RNA-induced gene silencing. Using small RNA sequencing analysis, we documented different patterns of virus-derived small interfering RNA (vsiRNA) production in strains infected with FgV1, -2, and -3. Our results suggest that the Argonaute protein encoded by FgAGO-1 is required for RNAi in F. graminearum, that FgAGO-1 induction differs in response to FgV1, -2, and -3, and that FgAGO-1 might contribute to the accumulation of vsiRNAs in FgV1-infected F. graminearum. IMPORTANCE To increase our understanding of how RNAi components in Fusarium graminearum react to mycovirus infections, we characterized the role(s) of RNAi components involved in the antiviral defense response against Fusarium graminearum viruses (FgVs). We observed differences in the levels of induction of RNA silencing-related genes, including FgDICER-2 and FgAGO-1, in response to infection by three different FgVs. FgAGO-1 can efficiently induce a robust RNAi response against FgV1 infection, but FgDICER genes might be relatively redundant to FgAGO-1 with respect to antiviral defense. However, the contribution of this gene in the response to the other FgV infections might be small. Compared to previous studies of Cryphonectria parasitica, which showed dicer-like protein 2 and Argonaute-like protein 2 to be important in antiviral RNA silencing, our results showed that F. graminearum developed a more complex and robust RNA silencing system against mycoviruses and that FgDICER-1 and FgDICER-2 and FgAGO-1 and FgAGO-2 had redundant roles in antiviral RNA silencing.

scholarly journals Identification of oligo-adenylated small RNAs in the parasite Entamoeba and a potential role for small RNA control

BMC Genomics ◽  
2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Hanbang Zhang ◽  
Gretchen M. Ehrenkaufer ◽  
Neil Hall ◽  
Upinder Singh
Keyword(s):  
Small Rna ◽  
Cellular Localization ◽  
Potential Role ◽  
Target Genes ◽  
Protozoan Parasite ◽  
Rnai Pathway ◽  
Regulation Mechanism ◽  
Major Step ◽  
Entamoeba Invadens ◽  
Related Parasite

Abstract Background The RNA interference (RNAi) pathway is a gene regulation mechanism that utilizes small RNA (sRNA) and Argonaute (Ago) proteins to silence target genes. Our previous work identified a functional RNAi pathway in the protozoan parasite Entamoeba histolytica, including abundant 27 nt antisense sRNA populations which associate with EhAgo2–2 protein. However, there is lack of understanding about the sRNAs that are bound to two other EhAgos (EhAgo2–1 and 2–3), and the mechanism of sRNA regulation itself is unclear in this parasite. Therefore, identification of the entire pool of sRNA species and their sub-populations that associate with each individual EhAgo protein would be a major step forward. Results In the present study, we sequenced sRNA libraries from both total RNAs and EhAgo bound RNAs. We identified a new population of 31 nt sRNAs that results from the addition of a non-templated 3–4 adenosine nucleotides at the 3′-end of the 27 nt sRNAs, indicating a non-templated RNA-tailing event in the parasite. The relative abundance of these two sRNA populations is linked to the efficacy of gene silencing for the target gene when parasites are transfected with an RNAi-trigger construct, indicating that non-templated sRNA-tailing likely play a role in sRNA regulation in this parasite. We found that both sRNA populations (27 nt and 31 nt) are present in the related parasite Entamoeba invadens, and are unchanged during the development. In sequencing the sRNAs associating with the three EhAgo proteins, we observed that despite distinct cellular localization, all three EhAgo sRNA libraries contain 27 nt sRNAs with 5′-polyphosphate (5′-polyP) structure and share a largely overlapping sRNA repertoire. In addition, our data showed that a fraction of 31 nt sRNAs associate with EhAgo2–2 but not with its mutant protein (C-terminal deletion), nor other two EhAgos, indicating a specific EhAgo site may be required for sRNA modification process in the parasite. Conclusion We identified a new population of sRNA with non-templated oligo-adenylation modification, which is the first such observation amongst single celled protozoan parasites. Our sRNA sequencing libraries provide the first comprehensive sRNA dataset for all three Entamoeba Ago proteins, which can serve as a useful database for the amoeba community.

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