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

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.

Entamoeba stage conversion: progress and new insights.

Entamoeba histolytica, an anaerobic protozoan, is an important global health problem. This parasite has a biphasic life cycle consisting of a dormant cyst stage which is environmentally resistant and transmits the infection, and the proliferative trophozoite stage which is motile and causes invasive disease. The stage conversion process remains poorly understood despite being central to amoebic biology. In this review, we will highlight recent progress in our understanding of Entamoeba stage conversion including dissecting transcriptome analysis in development, characterization of transcriptional networks, demonstration of epigenetic regulation, and role of small molecules that regulate Entamoeba development.

Identification of plicamycin, TG02, panobinostat, lestaurtinib, and GDC-0084 as promising compounds for the treatment of central nervous system infections caused by the free-living amebae Naegleria, Acanthamoeba and Balamuthia.

The free-living amebae Naegleria, Acanthamoeba, and Balamuthia cause rare but life-threatening infections. All three parasites can cause meningoencephalitis. Acanthamoeba can also cause chronic keratitis and both Balamuthia and Acanthamoeba can cause skin and systemic infections. There are minimal drug development pipelines for these pathogens despite a lack of available treatment regimens and high fatality rates. To identify anti-amebic drugs, we screened 159 compounds from a high-value repurposed library against trophozoites of the three amebae. Our efforts identified 38 compounds with activity against at least one ameba. Multiple drugs that bind the ATP-binding pocket of mTOR and PI3K are active, highlighting these compounds as important inhibitors of these parasites. Importantly, 24 active compounds have progressed at least to phase II clinical studies and overall 15 compounds were active against all three amebae. Based on central nervous system (CNS) penetration or exceptional potency against one amebic species, we identified sixteen priority compounds for the treatment of meningoencephalitis caused by these pathogens. The top five compounds are (i) plicamycin, active against all three free-living amebae and previously U.S. Food and Drug Administration (FDA) approved, (ii) TG02, active against all three amebae, (iii and iv) FDA-approved panobinostat and FDA orphan drug lestaurtinib, both highly potent against Naegleria, and (v) GDC-0084, a CNS penetrant mTOR inhibitor, active against at least two of the three amebae. These results set the stage for further investigation of these clinically advanced compounds for treatment of infections caused by the free-living amebae, including treatment of the highly fatal meningoencephalitis.

High-Throughput Screening of Entamoeba Identifies Compounds Which Target Both Life Cycle Stages and Which Are Effective Against Metronidazole Resistant Parasites.

Neglected tropical diseases, especially those caused by parasites, are significantly underserved by current drug development efforts, mostly due to the high costs and low economic returns. One method for lowering the costs of drug discovery and development for these diseases is to repurpose drugs developed for other indications. Here, we present the results of a screen of five repurposed drug libraries to identify potential new lead compounds to treat amebiasis, a disease that affects tens of millions of people and causes ~100,000 deaths annually. E. histolytica, the causative agent of amebiasis, has two major life cycle stages, the trophozoite and the cyst. The current primary treatment for amebiasis, nitroimidazole compounds, do not eliminate parasites from the colonic lumen, necessitating a multi-drug treatment regimen. We aimed to address this problem by screening against both life stages, with the aim of identifying a single drug that targets both. We successfully identified eleven compounds with activity against both cysts and trophozoites, as well as multiple compounds that killed trophozoites with improved efficacy over existing drugs. Two lead compounds (anisomycin and prodigiosin) were further characterized for activity against metronidazole (MNZ) resistant parasites and mature cysts. Anisomycin and prodigiosin were both able to kill MNZ resistant parasites while prodigiosin and its analog obatoclax were active against mature cysts. This work confirms the feasibility of identifying drugs that target both Entamoeba trophozoites and cysts, and is an important step toward developing improved treatment regimens for Entamoeba infection.

High Throughput Sequencing of Entamoeba 27nt Small RNA Population Reveals Role in Permanent Gene Silencing But No Effect on Regulating Gene Expression Changes during Stage Conversion, Oxidative, or Heat Shock Stress.

The human parasite Entamoeba histolytica has an active RNA interference (RNAi) pathway with an extensive repertoire of 27nt small RNAs that silence genes. However the role of this pathway in regulating amebic biology remains unknown. In this study, we address whether silencing via 27nt small RNAs may be a mechanism for controlling gene expression changes during conversion between the trophozoite and cyst stages of the parasite. We sequenced small RNA libraries generated from trophozoites, early cysts, mature cysts, and excysting cells and mapped them to the E. invadens genome. Our results show that, as in E. histolytica, small RNAs in E. invadens are largely ~27nt in length, have an unusual 5'-polyphosphate structure and mediate gene silencing. However, when comparing the libraries from each developmental time-point we found few changes in the composition of the small RNA populations. Furthermore, genes targeted by small RNAs were permanently silenced with no changes in transcript abundance during development. Thus, the E. invadens 27nt small RNA population does not mediate gene expression changes during development. In order to assess the generalizability of our observations, we examined whether small RNAs may be regulating gene expression changes during stress response in E. histolytica. Comparison of the 27nt small RNA populations from E. histolytica trophozoites from basal conditions, or after heat shock or exposure to oxidative stress showed few differences. Similar to data in E. invadens development, genes targeted by small RNAs were consistently silenced and did not change expression under tested stress conditions. Thus, the biological roles of the 27nt small RNA population in Entamoeba remain elusive. However, as the first characterization of the RNAi pathway in E. invadens these data serve as a useful resource for the study of Entamoeba development and open the door to the development of RNAi-based gene silencing tools in E. invadens.

Regulation of gene expression in the protozoan parasite Entamoeba invadens: identification of core promoter elements and promoters with stage-specific expression patterns.

Developmental switching between life-cycle stages is a common feature among many pathogenic organisms. Entamoeba histolytica is an important human pathogen and is a leading parasitic cause of death globally. During its life cycle, Entamoeba converts between cysts (essential for disease transmission) and trophozoites (responsible for tissue invasion). Despite being central to its biology, the triggers that are involved in the developmental pathways of this parasite are not well understood. In order to define the transcriptional network associated with stage conversion we used Entamoeba invadens which serves as a model system for Entamoeba developmental biology, and performed RNA sequencing at different developmental time points. In this study RNA-Seq data was utilised to define basal transcriptional control elements as well as to identify promoters which regulate stage-specific gene expression patterns. We discovered that the 5' and 3' untranslated regions of E. invadens genes are short, a median of 20 nucleotides (nt) and 26 nt respectively. Bioinformatics analysis of DNA sequences proximate to the start and stop codons identified two conserved motifs: (i) E. invadens Core Promoter Motif - GAAC-Like (EiCPM-GL) (GAACTACAAA), and (ii) E. invadens 3'-U-Rich Motif (Ei3'-URM) (TTTGTT) in the 5' and 3' flanking regions, respectively. Electrophoretic mobility shift assays demonstrated that both motifs specifically bind nuclear protein(s) from E. invadens trophozoites. Additionally, we identified select genes with stage-specific expression patterns and analysed the ability of each gene promoter to drive a luciferase reporter gene during the developmental cycle. This approach confirmed three trophozoite-specific, four encystation-specific and two excystation-specific promoters. This work lays the framework for use of stage-specific promoters to express proteins of interest in a particular life-cycle stage, adding to the molecular toolbox for genetic manipulation of E. invadens and allowing further dissection of factors controlling Entamoeba developmental biology.

The genome and transcriptome of the enteric parasite Entamoeba invadens, a model for encystation.

BACKGROUND: Several eukaryotic parasites form cysts that transmit infection. The process is found in diverse organisms such as Toxoplasma, Giardia, and nematodes. In Entamoeba histolytica this process cannot be induced in vitro, making it difficult to study. In Entamoeba invadens, stage conversion can be induced, but its utility as a model system to study developmental biology has been limited by a lack of genomic resources. We carried out genome and transcriptome sequencing of E. invadens to identify molecular processes involved in stage conversion. RESULTS: We report the sequencing and assembly of the E. invadens genome and use whole transcriptome sequencing to characterize changes in gene expression during encystation and excystation. The E. invadens genome is larger than that of E. histolytica, apparently largely due to expansion of intergenic regions; overall gene number and the machinery for gene regulation are conserved between the species. Over half the genes are regulated during the switch between morphological forms and a key signaling molecule, phospholipase D, appears to regulate encystation. We provide evidence for the occurrence of meiosis during encystation, suggesting that stage conversion may play a key role in recombination between strains. CONCLUSIONS: Our analysis demonstrates that a number of core processes are common to encystation between distantly related parasites, including meiosis, lipid signaling and RNA modification. These data provide a foundation for understanding the developmental cascade in the important human pathogen E. histolytica and highlight conserved processes more widely relevant in enteric pathogens.

Small RNA pyrosequencing in the protozoan parasite Entamoeba histolytica reveals strain-specific small RNAs that target virulence genes.

BACKGROUND: Small RNA mediated gene silencing is a well-conserved regulatory pathway. In the parasite Entamoeba histolytica an endogenous RNAi pathway exists, however, the depth and diversity of the small RNA population remains unknown. RESULTS: To characterize the small RNA population that associates with E. histolytica Argonaute-2 (EhAGO2-2), we immunoprecipitated small RNAs that associate with it and performed one full pyrosequencing run. Data analysis revealed new features of the 27nt small RNAs including the 5'-G predominance, distinct small RNA distribution patterns on protein coding genes, small RNAs mapping to both introns and exon-exon junctions, and small RNA targeted genes that are clustered particularly in sections of genome duplication. Characterization of genomic loci to which both sense and antisense small RNAs mapped showed that both sets of small RNAs have 5'-polyphosphate termini; strand-specific RT-PCR detected transcripts in both directions at these loci suggesting that both transcripts may serve as template for small RNA generation. In order to determine whether small RNA abundance patterns account for strain-specific gene expression profiles of E. histolytica virulent and non-virulent strains, we sequenced small RNAs from a non-virulent strain and found that small RNAs mapped to genes in a manner consistent with their regulation of strain-specific virulence genes. CONCLUSIONS: We provided a full spectrum analysis for E. histolytica AGO2-2 associated 27nt small RNAs. Additionally, comparative analysis of small RNA populations from virulent and non-virulent amebic strains indicates that small RNA populations may regulate virulence genes.

Transient and stable transfection in the protozoan parasite Entamoeba invadens.

Entamoeba histolytica is an important human pathogen and a major health problem worldwide. Many aspects of parasite biology can be studied with the exception of stage conversion, which cannot be reproduced adequately in E. histolytica. The reptile parasite Entamoeba invadens is a vital model system for studying stage conversion since it can be induced to undergo both encystation and excystation with high efficiency in vitro. However, functional studies using E. invadens have been limited by the lack of genetic tools in this species. Here, we report a new method for both transient and stable transfection of E. invadens. These new tools will greatly enhance research into Entamoeba development.

A developmentally regulated Myb domain protein regulates expression of a subset of stage-specific genes in Entamoeba histolytica.

Conversion between a cyst and trophozoite stage is essential to disease transmission and pathogenesis in the parasitic protist Entamoeba histolytica. A transcriptomic analysis of E. histolytica cysts and trophozoites has recently been accomplished, but the molecular basis of the regulation of encystation is not known. We have now identified a developmentally regulated Myb protein (belonging to the SHAQKY family of Myb proteins), which controls expression of a subset of amoebic stage-specific genes. Overexpression of the nuclear localized Myb protein resulted in a transcriptome that overlapped significantly with the expression profile of amoebic cysts. Analysis of promoters from genes regulated by the Myb protein identified a CCCCCC promoter motif to which amoebic nuclear protein(s) bind in a sequence-specific manner. Chromatin immunoprecipitation demonstrated that the E. histolytica Myb protein binds to promoters of genes which contain the CCCCCC motif and which are regulated by the Myb protein. This work is the first identification of a transcription factor, which regulates expression of a subset of stage-specific genes in E. histolytica. Identification of transcriptional regulatory networks that control developmental pathways will provide novel insights into the biology of this important human pathogen.

Recent insights into Entamoeba development: identification of transcriptional networks associated with stage conversion.

Entamoeba histolytica is an important human pathogen and a leading parasitic cause of death globally. The parasite life cycle alternates between the trophozoite form, which is motile and causes invasive disease and the cyst stage, which is environmentally resistant and transmits infection. Understanding the triggers that initiate stage conversion is an important yet understudied area of investigation. Recent progress in dissecting the transcriptional networks that regulate E. histolytica development is outlined in this paper.

Entamoeba histolytica modulates a complex repertoire of novel genes in response to oxidative and nitrosative stresses: implications for amebic pathogenesis.

Upon host infection, the protozoan parasite Entamoeba histolytica is confronted with reactive oxygen and nitrogen species and must survive these stresses in order to cause invasive disease. We analysed the parasite's response to oxidative and nitrosative stresses, probing the transcriptional changes of trophozoites of a pathogenic strain after a 60 min exposure to H2O2 (1 mM) or a NO donor (dipropylenetriamine-NONOate, 200 microM), using whole-genome DNA microarrays. Genes encoding reactive oxygen and nitrogen species detoxification enzymes had high transcriptional levels under basal conditions and upon exposure to both stresses. On a whole-genome level, there was significant modulation of gene expression by H2O2 (286 genes regulated) and dipropylenetriamine-NONOate (1036 genes regulated) with a significant overlap of genes modulated under both conditions (164 genes). A number of transcriptionally regulated genes were in signalling/regulatory and repair/metabolic pathways. However, the majority of genes with altered transcription encode unknown proteins, suggesting as yet unraveled response pathways in E. histolytica. Trophozoites of a non-pathogenic E. histolytica strain had a significantly muted transcriptional response to H2O2 compared with the pathogenic strain, hinting that differential response to oxidative stress may be one factor that contributes to the pathogenic potential of E. histolytica.

Small RNAs with 5'-polyphosphate termini associate with a Piwi-related protein and regulate gene expression in the single-celled eukaryote Entamoeba histolytica.

Small interfering RNAs regulate gene expression in diverse biological processes, including heterochromatin formation and DNA elimination, developmental regulation, and cell differentiation. In the single-celled eukaryote Entamoeba histolytica, we have identified a population of small RNAs of 27 nt size that (i) have 5'-polyphosphate termini, (ii) map antisense to genes, and (iii) associate with an E. histolytica Piwi-related protein. Whole genome microarray expression analysis revealed that essentially all genes to which antisense small RNAs map were not expressed under trophozoite conditions, the parasite stage from which the small RNAs were cloned. However, a number of these genes were expressed in other E. histolytica strains with an inverse correlation between small RNA and gene expression level, suggesting that these small RNAs mediate silencing of the cognate gene. Overall, our results demonstrate that E. histolytica has an abundant 27 nt small RNA population, with features similar to secondary siRNAs from C. elegans, and which appear to regulate gene expression. These data indicate that a silencing pathway mediated by 5'-polyphosphate siRNAs extends to single-celled eukaryotic organisms.

Transcriptional regulatory networks in Entamoeba histolytica.

Expression profiling with microarray technology has revolutionized exploration of transcriptional regulatory networks on a genome-wide scale. This approach has been successfully applied to the study of Entamoeba histolytica, which causes dysentery and liver abscesses and is a leading parasitic cause of death globally. A variety of microarray platforms have been developed for this system including those generated from genomic DNA, long oligonucleotides, and short oligonucleotides. Using these tools researchers have identified parasite genes whose transcript abundance is differentially regulated during stress, host invasion, and stage conversion. Additionally, novel virulence factors have been identified by identifying genes that are highly expressed in virulent but with low expression in non-virulent Entamoeba strains. All combined, these studies have provided new data on molecular aspects of amebic biology, pathogenic potential and stage conversion and provide investigators with the first insights into potential novel drug targets against amebic disease.

Trichostatin A effects on gene expression in the protozoan parasite Entamoeba histolytica.

BACKGROUND: Histone modification regulates chromatin structure and influences gene expression associated with diverse biological functions including cellular differentiation, cancer, maintenance of genome architecture, and pathogen virulence. In Entamoeba, a deep-branching eukaryote, short chain fatty acids (SCFA) affect histone acetylation and parasite development. Additionally, a number of active histone modifying enzymes have been identified in the parasite genome. However, the overall extent of gene regulation tied to histone acetylation is not known. RESULTS: In order to identify the genome-wide effects of histone acetylation in regulating E. histolytica gene expression, we used whole-genome expression profiling of parasites treated with SCFA and Trichostatin A (TSA). Despite significant changes in histone acetylation patterns, exposure of parasites to SCFA resulted in minimal transcriptional changes (11 out of 9,435 genes transcriptionally regulated). In contrast, exposure to TSA, a more specific inhibitor of histone deacetylases, significantly affected transcription of 163 genes (122 genes upregulated and 41 genes downregulated). Genes modulated by TSA were not regulated by treatment with 5-Azacytidine, an inhibitor of DNA-methyltransferase, indicating that in E. histolytica the crosstalk between DNA methylation and histone modification is not substantial. However, the set of genes regulated by TSA overlapped substantially with genes regulated during parasite development: 73/122 genes upregulated by TSA exposure were upregulated in E. histolytica cysts (p-value = 6 x 10(-53)) and 15/41 genes downregulated by TSA exposure were downregulated in E. histolytica cysts (p-value = 3 x 10(-7)). CONCLUSION: This work represents the first genome-wide analysis of histone acetylation and its effects on gene expression in E. histolytica. The data indicate that SCFAs, despite their ability to influence histone acetylation, have minimal effects on gene transcription in cultured parasites. In contrast, the effect of TSA on E. histolytica gene expression is more substantial and includes genes involved in the encystation pathway. These observations will allow further dissection of the effects of histone acetylation and the genetic pathways regulating stage conversion in this pathogenic parasite.

Identification of putative transcriptional regulatory networks in Entamoeba histolytica using Bayesian inference.

Few transcriptional regulatory networks have been described in non-model organisms. In Entamoeba histolytica seminal aspects of pathogenesis are transcriptionally controlled, however, little is known about transcriptional regulatory networks that effect gene expression in this parasite. We used expression data from two microarray experiments, cis-regulatory motif elucidation, and a naïve Bayesian classifier to identify genome-wide transcriptional regulatory patterns in E. histolytica. Our algorithm identified promoter motifs that accurately predicted the gene expression level of 68% of genes under trophozoite conditions. We identified a promoter motif ((A)/(T)AAACCCT) associated with high gene expression, which is highly enriched in promoters of ribosomal protein genes and tRNA synthetases. Additionally, we identified three promoter motifs (GAATGATG, AACTATTTAAACAT(C)/(T)C and TGAACTTATAAACATC) associated with low gene expression. The promoters of a large gene family were highly enriched for these motifs, and in these genes the presence of >/=2 motifs predicted low baseline gene expression and transcriptional activation by heat shock. We demonstrate that amebic nuclear protein(s) bind specifically to four of the motifs identified herein. Our analysis suggests that transcriptional regulatory networks can be identified using limited expression data. Thus, this approach is applicable to the multitude of systems for which microarray and genome sequence data are emerging.

Growth of the protozoan parasite Entamoeba histolytica in 5-azacytidine has limited effects on parasite gene expression.

BACKGROUND: In higher eukaryotes DNA methylation regulates important biological functions including silencing of gene expression and protection from adverse effects of retrotransposons. In the protozoan parasite Entamoeba histolytica, a DNA methyltransferase has been identified and treatment with 5-azacytidine (5-AzaC), a potent inhibitor of DNA methyltransferase, has been reported to attenuate parasite virulence. However, the overall extent of DNA methylation and its subsequent effects on global gene expression in this parasite are currently unknown. RESULTS: In order to identify the genome-wide effects of DNA methylation in E. histolytica, we used a short oligonucleotide microarray representing 9,435 genes (approximately 95% of all annotated amebic genes) and compared the expression profile of E. histolytica HM-1:IMSS parasites with those treated with 23 microM 5-AzaC for up to one week. Overall, 2.1% of genes tested were transcriptionally modulated under these conditions. 68 genes were upregulated and 131 genes down regulated (2-fold change; p-value < 0.05). Sodium-bisulfite treatment and sequencing of genes indicated that there were at least two subsets of genes with genomic DNA methylation in E. histolytica: (i) genes that were endogenously silenced by genomic DNA methylation and for which 5-AzaC treatment induced transcriptional de-repression, and (ii) genes that have genomic DNA methylation, but which were not endogenously silenced by the methylation. We identified among the genes down regulated by 5-AzaC treatment a cysteine proteinase (2.m00545) and lysozyme (52.m00148) both of which have known roles in amebic pathogenesis. Decreased expression of these genes in the 5-AzaC treated E. histolytica may account in part for the parasites reduced cytolytic abilities. CONCLUSION: This work represents the first genome-wide analysis of DNA-methylation in Entamoeba histolytica and indicates that DNA methylation has relatively limited effects on gene expression in this parasite.

Identification of developmentally regulated genes in Entamoeba histolytica: insights into mechanisms of stage conversion in a protozoan parasite.

Developmental switching between life-cycle stages is a common feature among many pathogenic organisms. The protozoan parasite Entamoeba histolytica converts between cysts (essential for disease transmission) and trophozoites (responsible for tissue invasion). Identification of genes involved in the developmental pathway has been severely hindered by the inability to generate E. histolytica cysts in vitro. Using parasite strains derived from recent human infections and whole-genome transcriptional profiling, we determined that 1439 genes (approximately 15% of annotated genes) were potentially developmentally regulated. Genes enriched in cysts (672 in total) included cysteine proteinases and transmembrane protein kinases, which may be involved in signal transduction. Genes enriched in trophozoites (767 in total) included genes typically thought of as important in tissue invasion by trophozoites, including the Gal/GalNAc lectin light subunit and cysteine protease 1. Putative regulators of differentiation including possible G-protein coupled receptors, signal transduction proteins and transcription factors were identified. A number of E. histolytica stage-specific genes were also developmentally regulated in the reptilian parasite E. invadens, indicating that they likely have conserved functions in Entamoeba development. These advances lay the groundwork for dissection of the molecular signals that initiate stage conversion and development of novel diagnostic and therapeutic measures targeting E. histolytica cysts.