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 (~95% of all annotated amebic genes) and compared the expression profile of E. histolytica HM-1:IMSS parasites with those treated with 23 μM 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

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

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

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/TAAACCCT) 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, AACTATTTAAACATC/TC 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

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

Trichostatin A effects on gene expression in the protozoan parasite <it>Entamoeba histolytica</it>

Abstract 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 × 10-53) and 15/41 genes downregulated by TSA exposure were downregulated in E. histolytica cysts (p-value = 3 × 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.</p

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 × 10(-53)) and 15/41 genes downregulated by TSA exposure were downregulated in E. histolytica cysts (p-value = 3 × 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

An amebic nuclear protein(s) binds in a sequence-specific manner to M9 under heat shock conditions, but not under standard culture conditions

<p><b>Copyright information:</b></p><p>Taken from "Identification of putative transcriptional regulatory networks in using Bayesian inference"</p><p></p><p>Nucleic Acids Research 2007;35(7):2141-2152.</p><p>Published online 13 Mar 2007</p><p>PMCID:PMC1874630.</p><p>© 2007 The Author(s)</p> () EMSAs with the M9 oligonucleotide (TGAACTTATAAACATC) were performed using nuclear extracts from trophozoites under standard culture conditions. No significant binding proteins were identified. Lanes: (1) nuclear extract plus labeled oligonucleotide without competitor, (2) 2-fold excess specific competitor, (3) 10-fold excess specific competitor, (4) 2-fold excess competitor with M9A mutated, (5) 10-fold excess competitor with M9A mutated, (6) 2-fold excess with M9B mutated and (7) 10-fold excess competitor with M9B mutated. () EMSAs with the M9 oligonucleotide were performed using nuclear extract from trophozoites subjected to heat shock. Several bands were found in the lane with nuclear extract alone, all of which were competed using the specific oligonucleotide competitor, but two of which were not competed with mutated oligonucleotides. Lanes: (1) nuclear extract plus labeled oligonucleotide without competitor, (2) 2-fold excess specific competitor, (3) 10-fold excess specific competitor, (4) 2-fold excess competitor with M9A mutated, (5) 10-fold excess competitor with M9A mutated, (6) 2-fold excess with M9B mutated and (7) 10-fold excess competitor with M9B mutated. Arrows indicate complexes we believe to be specific. Top and bottom panels were separated to allow clearer visualization of the different bands