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Identification of oligo-adenylated small RNAs in the parasite Entamoeba and a potential role for small RNA control. BMC genomics Zhang, H., Ehrenkaufer, G. M., Hall, N., Singh, U. 2020; 21 (1): 879
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 27nt 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 31nt sRNAs that results from the addition of a non-templated 3-4 adenosine nucleotides at the 3'-end of the 27nt 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 (27nt and 31nt) 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 27nt sRNAs with 5'-polyphosphate (5'-polyP) structure and share a largely overlapping sRNA repertoire. In addition, our data showed that a fraction of 31nt 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.

View details for DOI 10.1186/s12864-020-07275-6

View details for PubMedID 33297948
Development of a CRISPR/Cas9 system in Entamoeba histolytica: proof of concept. International journal for parasitology Kangussu-Marcolino, M. M., Morgado, P., Manna, D., Yee, H., Singh, U. 2020
Abstract

The protozoan parasite Entamoeba histolytica is an important human pathogen and a leading parasitic cause of death on a global scale. The lack of molecular tools for genome editing hinders the study of important biological functions of this parasite. Due to its versatility, the CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 system has been successfully used to induce site-specific genomic alterations, including in protozoan parasites. In this study, we optimized CRISPR-Cas9 for use as a genetic tool in E. histolytica. We chose a single plasmid approach containing both guide RNA (gRNA) and Cas9 nuclease expression cassettes. The amebic U6 promoter was used to drive the expression of the gRNA and its expression was confirmed by Northern blot analysis. Stable transfectant cell lines were obtained using a destabilizing domain of dihydrofolate reductase fused to myc-tagged Cas9 (ddCas9). With this system, we were able to induce ddCas9 expression 16 h following treatment with the small molecule ligand trimethoprim (TMP). Stable cell lines expressing ddCas9 and Luc-gRNA or non-specific (NS)-gRNA were transiently transfected with a plasmid containing a mutated luciferase gene (pDeadLuc) targeted by Luc-gRNA and another plasmid with a truncated luciferase gene (pDonorLuc) to restore luciferase expression and consequent activity. We observed that luminescence signal increased for the cell line expressing Luc-gRNA, suggesting that homologous recombination was facilitated by Cas9 activity. This evidence is supported by the presence of chimeric DNA detected by PCR and confirmed by sequencing of the resulting repaired DNA obtained by homologous recombination. We believe this represents the first report of a CRISPR/Cas9 system use in Entamoeba and provides evidence that this genome editing approach can be useful for genetic studies in this early branching eukaryote.

View details for DOI 10.1016/j.ijpara.2020.09.005

View details for PubMedID 33264648
Identification of anisomycin, prodigiosin and obatoclax as compounds with broad-spectrum anti-parasitic activity PLOS NEGLECTED TROPICAL DISEASES Ehrenkaufer, G., Li, P., Stebbins, E. E., Kangussu-Marcolino, M. M., Debnath, A., White, C., Moser, M. S., DeRisi, J., Gisselberg, J., Yeh, E., Wang, S. C., Company, A., Monti, L., Caffrey, C. R., Huston, C. D., Wang, B., Singh, U. 2020; 14 (3)
View details for DOI 10.1371/journal.pntd.0008150.r004

View details for Web of Science ID 000528655400050
Entamoeba stage conversion: progress and new insights. Current opinion in microbiology Manna, D., Ehrenkaufer, G. M., Lozano-Amado, D., Singh, U. 2020; 58: 62?68
Abstract

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.

View details for DOI 10.1016/j.mib.2020.09.005

View details for PubMedID 33032142
The NAD+ Responsive Transcription Factor ERM-BP Functions Downstream of Cellular Aggregation and Is an Early Regulator of Development and Heat Shock Response in Entamoeba. Frontiers in cellular and infection microbiology Manna, D., Lozano-Amado, D., Ehrenkaufer, G., Singh, U. 2020; 10: 363
Abstract

Entamoeba histolytica is a protozoan parasite and a major cause of dysentery and diarrheal disease in developing countries. Disease transmission from one host to another occurs via cysts which can survive in environmental extremes and are transmitted through contaminated food and water. Recent studies in our lab identified a novel transcription factor, Encystation Regulatory Motif- Binding Protein (ERM-BP), which is responsive to NAD+ and has an important role in encystation. The key residues important for ERM-BP function were demonstrated in vitro using recombinant protein. In this study we demonstrate the in vivo functional consequences of mutations in key domains and their impact on Entamoeba encystation. Our results show that mutations in the DNA binding domain (ERM-BP-DBM) and in the nicotinamidase domain (ERM-BP-C198A) lead to protein mis-localization in both trophozoites and cysts and significantly reduce encystation efficiency. Additionally, we showed that silencing of ERM-BP significantly decreased the size and number of multi-nucleated giant cells (MGC) that form during encystation, indicating that ERM-BP functions upstream of the cellular aggregation that precedes stage conversion. Dissection of epistatic interactions between ERM-BP and a second encystation-related transcription factor, NF-Y revealed that ERM-BP is upstream of NF-Y in controlling the developmental cascade and appears to be one of the earliest regulators of development identified to date in Entamoeba. We also demonstrated that ERM-BP is upregulated during heat stress in Entamoeba, another condition which increases intracellular NAD+ levels and that overexpression of ERM-BP makes E. histolytica and E. invadens parasites more resistant to heat stress. Overexpression of ERM-BP in E. histolytica also induced the formation of cyst-like quadrinucleated cells and formation of MGCs. Overall, our work has identified an important role of ERM-BP in Entamoeba stress response and links an NAD+-responsive transcription factor to both development and heat shock response. Characterization of stress and developmental cascades are important avenues to investigate for Entamoeba, an important human parasitic pathogen.

View details for DOI 10.3389/fcimb.2020.00363

View details for PubMedID 32766170

View details for PubMedCentralID PMC7379229
Interferon-gamma release assay for accurate detection of SARS-CoV-2 T cell response. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America Murugesan, K., Jagannathan, P., Pham, T. D., Pandey, S., Bonilla, H. F., Jacobson, K., Parsonnet, J., Andrews, J. R., Weiskopf, D., Sette, A., Pinsky, B. A., Singh, U., Banaei, N. 2020
Abstract

We investigated feasibility and accuracy of an interferon-gamma release assay (IGRA) for detection of T cell responses to SARS-CoV-2. Whole blood IGRA accurately distinguished between convalescents and uninfected healthy blood donors with a predominantly CD4+ T cell response. SARS-CoV-2 IGRA may serve as a useful diagnostic tool in managing the COVID-19 pandemic.

View details for DOI 10.1093/cid/ciaa1537

View details for PubMedID 33035306
Proinflammatory IgG Fc structures in patients with severe COVID-19 Nature Immunology Chakraborty, S., Gonzales, J., Edwards, K., Mallajosyulla, V., Buzzanco, A. S., Sherwood, R., Buffone, C., Kathale, N., Providenza, S., Xie, M. M., Andrews, J. R., Blish, C. A., Singh, U., Dugan, H., Wilson, P. C., Pham, T. D., Boyd, S. D., Nadeau, K. C., Pinsky, B. A., Zhang, S., Memoli, M. J., Taubenberger, J. K., Morales, T., Schapiro, J. M., Tan, G. S., et al 2020
View details for DOI 10.1038/s41590-020-00828-7
Characterization of extracellular vesicles from Entamoeba histolytica identifies roles in intercellular communication that regulates parasite growth and development. Infection and immunity Sharma, M., Morgado, P., Zhang, H., Ehrenkaufer, G., Manna, D., Singh, U. 2020
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, metabolism, and 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 27nt in size and represented a subset of the cellular antisense small RNA population that has previously been characterized in Entamoeba RNAi pathway proteins, including Argonaute were also present in amebic EVs. Interestingly, we found that the amebic EVs impact inter-cellular 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 reveals 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 amongst parasites and influence growth kinetics.

View details for DOI 10.1128/IAI.00349-20

View details for PubMedID 32719158
Identification of anisomycin, prodigiosin and obatoclax as compounds with broad-spectrum anti-parasitic activity. PLoS neglected tropical diseases Ehrenkaufer, G., Li, P., Stebbins, E. E., Kangussu-Marcolino, M. M., Debnath, A., White, C. V., Moser, M. S., DeRisi, J., Gisselberg, J., Yeh, E., Wang, S. C., Company, A. H., Monti, L., Caffrey, C. R., Huston, C. D., Wang, B., Singh, U. 2020; 14 (3): e0008150
Abstract

Parasitic infections are a major source of human suffering, mortality, and economic loss, but drug development for these diseases has been stymied by the significant expense involved in bringing a drug though clinical trials and to market. Identification of single compounds active against multiple parasitic pathogens could improve the economic incentives for drug development as well as simplifying treatment regimens. We recently performed a screen of repurposed compounds against the protozoan parasite Entamoeba histolytica, causative agent of amebic dysentery, and identified four compounds (anisomycin, prodigiosin, obatoclax and nithiamide) with low micromolar potency and drug-like properties. Here, we extend our investigation of these drugs. We assayed the speed of killing of E. histolytica trophozoites and found that all four have more rapid action than the current drug of choice, metronidazole. We further established a multi-institute collaboration to determine whether these compounds may have efficacy against other parasites and opportunistic pathogens. We found that anisomycin, prodigiosin and obatoclax all have broad-spectrum antiparasitic activity in vitro, including activity against schistosomes, T. brucei, and apicomplexan parasites. In several cases, the drugs were found to have significant improvements over existing drugs. For instance, both obatoclax and prodigiosin were more efficacious at inhibiting the juvenile form of Schistosoma than the current standard of care, praziquantel. Additionally, low micromolar potencies were observed against pathogenic free-living amebae (Naegleria fowleri, Balamuthia mandrillaris and Acanthamoeba castellanii), which cause CNS infection and for which there are currently no reliable treatments. These results, combined with the previous human use of three of these drugs (obatoclax, anisomycin and nithiamide), support the idea that these compounds could serve as the basis for the development of broad-spectrum anti-parasitic drugs.

View details for DOI 10.1371/journal.pntd.0008150

View details for PubMedID 32196500
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. International journal for parasitology. Drugs and drug resistance Kangussu-Marcolino, M. M., Ehrenkaufer, G. M., Chen, E., Debnath, A., Singh, U. 2019; 11: 80?94
Abstract

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.

View details for DOI 10.1016/j.ijpddr.2019.10.003

View details for PubMedID 31707263
Functional Characterization of Entamoeba histolytica Argonaute Proteins Reveals a Repetitive DR-Rich Motif Region That Controls Nuclear Localization. mSphere Zhang, H., Tran, V., Manna, D., Ehrenkaufer, G., Singh, U. 2019; 4 (5)
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.

View details for DOI 10.1128/mSphere.00580-19

View details for PubMedID 31619501
Drug treatment algorithms for water-borne parasitic pathogens Singh, U. AMER CHEMICAL SOC. 2019
View details for Web of Science ID 000525055501648
Nuclear Factor Y (NF-Y) Modulates Encystation in Entamoeba via Stage-Specific Expression of the NF-YB and NF-YC Subunits. mBio Manna, D., Singh, U. 2019; 10 (3)
Abstract

Nuclear factor Y (NF-Y) is a heterotrimeric transcription factor composed of three subunits, namely, NF-YA, NF-YB, and NF-YC, which are conserved throughout evolution. In higher eukaryotes, NF-Y plays important roles in several cellular processes (development, cell cycle regulation, apoptosis, and response to growth, stress, and DNA damage) by controlling gene expression through binding to a CCAAT promoter motif. We demonstrated that NF-Y subunits in the protist Entamoeba, while significantly divergent from those of higher eukaryotes, have well-conserved domains important for subunit interactions and DNA binding and that NF-YB and NF-YC are developmentally expressed during encystation. Electrophoretic mobility shift assays confirmed that the NF-Y protein(s) from Entamoeba cysts binds to a CCAAT motif. Consistent with a role as a transcription factor, the NF-Y proteins show nuclear localization during development. Additionally, we demonstrated that NF-YC localizes to the chromatoid body (an RNA processing center) during development, indicating that it may have a role in RNA processing. Finally, silencing of the NF-YC subunit resulted in reduced stability of the NF-Y complex and decreased encystation efficiency. We demonstrated that the NF-Y complex functions at a time point subsequent to the NAD+ flux and expression of the transcription factor encystation regulatory motif-binding protein, both of which are early regulators of Entamoeba development. Taken together, our results demonstrate that the NF-Y complex plays an important role in regulating encystation in Entamoeba and add to our understanding of the transcriptional networks and signals that control this essential developmental pathway in an important human pathogen.IMPORTANCE The human parasite Entamoeba histolytica is an important pathogen with significant global impact and is a leading cause of parasitic death in humans. Since only the cyst form can be transmitted, blocking encystation would prevent new infections, making the encystation pathway an attractive target for the development of new drugs. Identification of the genetic signals and transcriptional regulatory networks that control encystation would be an important advance in understanding the developmental cascade. We show that the Entamoeba NF-Y complex plays a crucial role in regulating the encystation process in Entamoeba.

View details for DOI 10.1128/mBio.00737-19

View details for PubMedID 31213550
An NAD+-dependent novel transcription factor controls stage conversion in Entamoeba. eLife Manna, D., Lentz, C. S., Ehrenkaufer, G. M., Suresh, S., Bhat, A., Singh, U. 2018; 7
Abstract

Developmental switching between life-cycle stages is a common feature among parasitic pathogens to facilitate disease transmission and pathogenesis. The protozoan parasite Entamoeba switches between invasive trophozoites and dormant cysts, but the encystation process remains poorly understood despite being central to amoebic biology. We identify a transcription factor, Encystation Regulatory Motif-Binding Protein (ERM-BP), that regulates encystation. Down-regulation of ERM-BP decreases encystation efficiency resulting in abnormal cysts with defective cyst walls. We demonstrate that direct binding of NAD+ to ERM-BP affects ERM-BP conformation and facilitates its binding to promoter DNA. Additionally, cellular NAD+ levels increase during encystation and exogenous NAD+ enhances encystation consistent with the role of carbon source depletion in triggering Entamoeba encystation. Furthermore, ERM-BP catalyzes conversion of nicotinamide to nicotinic acid, which might have second messenger effects on stage conversion. Our findings link the metabolic cofactors nicotinamide and NAD+ to transcriptional regulation via ERM-BP and provide the first mechanistic insights into Entamoeba encystation.

View details for PubMedID 30375973
Supporting Research Career Development of Physician-Scientists JOURNAL OF INFECTIOUS DISEASES Singh, U. 2018; 218: S36?S39
View details for PubMedID 30124974
High-Throughput Screening of Entamoeba Identifies Compounds Which Target Both Life Cycle Stages and Which Are Effective Against Metronidazole Resistant Parasites FRONTIERS IN CELLULAR AND INFECTION MICROBIOLOGY Ehrenkaufer, G. M., Suresh, S., Solow-Cordero, D., Singh, U. 2018; 8
View details for DOI 10.3389/fcimb.2018.00276

View details for Web of Science ID 000441933600001
Policy Recommendations for Optimizing the Infectious Diseases Physician-Scientist Workforce. The Journal of infectious diseases Singh, U., Levy, J., Armstrong, W., Bedimo, R., Creech, C. B., Lautenbach, E., Popovich, K. J., Snowden, J., Vyas, J. M., Infectious Diseases Society of America, H. M. 2018; 218 (suppl_1): S49?S54
Abstract

The Infectious Diseases Society of America, HIV Medicine Association, and Pediatric Infectious Diseases Society are concerned by the continued decline in the number of infectious diseases trainees pursuing careers as physician-scientists and the attrition of junior and midcareer physician-scientists. The inability to replace the aging physician-scientist workforce will have a negative, long-lasting impact our biomedical research enterprise and its ability to drive the discovery of new treatments for important infectious diseases. We discuss policy recommendations for securing and optimizing the infectious diseases physician-scientist workforce in the areas of education, training, compensation, and mentorship, as well as ways to improve federal research funding, cross-sector collaboration, and workforce diversity.

View details for PubMedID 30124981
High-Throughput Screening of Entamoeba Identifies Compounds Which Target Both Life Cycle Stages and Which Are Effective Against Metronidazole Resistant Parasites. Frontiers in cellular and infection microbiology Ehrenkaufer, G. M., Suresh, S., Solow-Cordero, D., Singh, U. 2018; 8: 276
Abstract

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.

View details for PubMedID 30175074

View details for PubMedCentralID PMC6107840
Development of RNA Interference Trigger-Mediated Gene Silencing in Entamoeba invadens. Infection and immunity Suresh, S., Ehrenkaufer, G., Zhang, H., Singh, U. 2016; 84 (4): 964-975
Abstract

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.

View details for DOI 10.1128/IAI.01161-15

View details for PubMedID 26787723

View details for PubMedCentralID PMC4807475
Technical advances in trigger-induced RNA interference gene silencing in the parasite Entamoeba histolytica INTERNATIONAL JOURNAL FOR PARASITOLOGY Khalil, M. I., Foda, B. M., Suresh, S., Singh, U. 2016; 46 (3): 205-212
Abstract

Entamoeba histolytica has a robust endogenous RNA interference (RNAi) pathway. There are abundant 27 nucleotide (nt) anti-sense small RNAs (AS sRNAs) that target genes for silencing and the genome encodes many genes involved in the RNAi pathway such as Argonaute proteins. Importantly, an E. histolytica gene with numerous AS sRNAs can function as a "trigger" to induce silencing of a gene that is fused to the trigger. Thus, the amebic RNAi pathway regulates gene expression relevant to amebic biology and has additionally been harnessed as a tool for genetic manipulation. In this study we have further improved the trigger-induced gene silencing method. We demonstrate that rather than using the full-length gene, a short portion of the coding region fused to a trigger is sufficient to induce silencing; the first 537bp of the E. histolytica rhomboid gene (EhROM1) fused in-frame to the trigger was sufficient to silence EhROM1. We also demonstrated that the trigger method could silence two amebic genes concomitantly; fusion of the coding regions of EhROM1 and transcription factor, EhMyb, in-frame to a trigger gene resulted in both genes being silenced. Alternatively, two genes can be silenced sequentially: EhROM1-silenced parasites with no drug selection plasmid were transfected with trigger-EhMyb, resulting in parasites with both EhROM1 and EhMyb silenced. With all approaches tested, the trigger-mediated silencing was substantive and silencing was maintained despite loss of the G418 selectable marker. All gene silencing was associated with generation of AS sRNAs to the silenced gene. We tested the reversibility of the trigger system using inhibitors of histone modifications but found that the silencing was highly stable. This work represents a technical advance in the trigger gene silencing method in E. histolytica. Approaches that readily silence multiple genes add significantly to the genetic toolkit available to the ameba research community.

View details for DOI 10.1016/j.ijpara.2015.11.004

View details for Web of Science ID 000371951900006

View details for PubMedID 26747561

View details for PubMedCentralID PMC4767557
biology: RNA interference, drug discovery, and gut microbiome. F1000Research Morgado, P., Manna, D., Singh, U. 2016; 5: 2578-?
Abstract

In recent years, substantial progress has been made in understanding the molecular and cell biology of the human parasite Entamoeba histolytica, an important pathogen with significant global impact. This review outlines some recent advances in the Entamoeba field in the last five years, focusing on areas that have not recently been discussed in detail: (i) molecular mechanisms regulating parasite gene expression, (ii) new efforts at drug discovery using high-throughput drug screens, and (iii) the effect of gut microbiota on amoebiasis.

View details for PubMedID 27853522
Dimethylated H3K27 Is a Repressive Epigenetic Histone Mark in the Protist Entamoeba histolytica and Is Significantly Enriched in Genes Silenced via the RNAi Pathway JOURNAL OF BIOLOGICAL CHEMISTRY Foda, B. M., Singh, U. 2015; 290 (34): 21114-21130
Abstract

RNA interference (RNAi) is a fundamental biological process that plays a crucial role in regulation of gene expression in many organisms. Transcriptional gene silencing (TGS) is one of the important nuclear roles of RNAi. Our previous data show that Entamoeba histolytica has a robust RNAi pathway that links to TGS via Argonaute 2-2 (Ago2-2) associated 27-nucleotide small RNAs with 5'-polyphosphate termini. Here, we report the first repressive histone mark to be identified in E. histolytica, dimethylation of H3K27 (H3K27Me2), and demonstrate that it is enriched at genes that are silenced by RNAi-mediated TGS. An RNAi-silencing trigger can induce H3K27Me2 deposits at both episomal and chromosomal loci, mediating gene silencing. Our data support two phases of RNAi-mediated TGS: an active silencing phase where the RNAi trigger is present and both H3K27Me2 and Ago2-2 concurrently enrich at chromosomal loci; and an established silencing phase in which the RNAi trigger is removed, but gene silencing with H3K27Me2 enrichment persist independently of Ago2-2 deposition. Importantly, some genes display resistance to chromosomal silencing despite induction of functional small RNAs. In those situations, the RNAi-triggering plasmid that is maintained episomally gets partially silenced and has H3K27Me2 enrichment, but the chromosomal copy displays no repressive histone enrichment. Our data are consistent with a model in which H3K27Me2 is a repressive histone modification, which is strongly associated with transcriptional repression. This is the first example of an epigenetic histone modification that functions to mediate RNAi-mediated TGS in the deep-branching eukaryote E. histolytica.

View details for DOI 10.1074/jbc.M115.647263

View details for Web of Science ID 000360011800043

View details for PubMedCentralID PMC4543668
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 PLOS ONE Zhang, H., Ehrenkaufer, G. M., Manna, D., Hall, N., Singh, U. 2015; 10 (8)
Abstract

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.

View details for DOI 10.1371/journal.pone.0134481

View details for Web of Science ID 000359062300039

View details for PubMedID 26248204
A Single RNaseIII Domain Protein from Entamoeba histolytica Has dsRNA Cleavage Activity and Can Help Mediate RNAi Gene Silencing in a Heterologous System PLOS ONE Pompey, J. M., Foda, B., Singh, U. 2015; 10 (7)
Abstract

Dicer enzymes process double-stranded RNA (dsRNA) into small RNAs that target gene silencing through the RNA interference (RNAi) pathway. Dicer enzymes are complex, multi-domain RNaseIII proteins, however structural minimalism of this protein has recently emerged in parasitic and fungal systems. The most minimal Dicer, Saccharomyces castellii Dicer1, has a single RNaseIII domain and two double stranded RNA binding domains. In the protozoan parasite Entamoeba histolytica 27nt small RNAs are abundant and mediate silencing, yet no canonical Dicer enzyme has been identified. Although EhRNaseIII does not exhibit robust dsRNA cleavage in vitro, it can process dsRNA in the RNAi-negative background of Saccharomyces cerevisiae, and in conjunction with S. castellii Argonaute1 can partially reconstitute the RNAi pathway. Thus, although EhRNaseIII lacks the domain architecture of canonical or minimal Dicer enzymes, it has dsRNA processing activity that contributes to gene silencing via RNAi. Our data advance the understanding of small RNA biogenesis in Entamoeba as well as broaden the spectrum of non-canonical Dicer enzymes that contribute to the RNAi pathway.

View details for DOI 10.1371/journal.pone.0133740

View details for Web of Science ID 000358838400054

View details for PubMedID 26230096

View details for PubMedCentralID PMC4521922
Entamoeba histolytica rhomboid protease 1 has a role in migration and motility as validated by two independent genetic approaches EXPERIMENTAL PARASITOLOGY Rastew, E., Morf, L., Singh, U. 2015; 154: 33-42
Abstract

Rhomboid proteins represent a recently discovered family of intramembrane proteases present in a broad range of organisms and with increasing links to human diseases. The enteric parasite Entamoeba histolytica has evolved multiple mechanisms to adapt to the human host environment and establish infection. Our recent studies identified EhROM1 as a functional E. histolytica rhomboid protease with roles in adhesion to and phagocytosis of host cells. Since those studies were performed in a non-virulent strain, roles in parasite virulence could not be assessed. We focused this study on the comparison and validation of two genetic manipulation techniques: overexpression of a dominant-negative catalytic mutant of EhROM1 and knock down of EhROM1 using a RNAi-based silencing approach followed by functional studies of phenotypic analyses in virulent parasites. Both the EhROM1 catalytic mutant and parasites with EhROM1 downregulation were reduced in cytotoxicity, hemolytic activity, and directional and non-directional transwell migration. Importantly, the role for EhROM1 in cell migration mimics similar roles for rhomboid proteases from mammalian and apicomplexan systems. However, the EhROM1 catalytic mutant and EhROM1 downregulation parasites had different phenotypes for erythrophagocytosis, while complement resistance was not affected in either strain. In summary, in this study we genetically manipulated E. histolytica rhomboid protease EhROM1 by two different approaches and identified similarly attenuated phenotypes by both approaches, suggesting a novel role for EhROM1 in amebic motility.

View details for DOI 10.1016/j.exppara.2015.04.004

View details for Web of Science ID 000355640500006

View details for PubMedID 25889553

View details for PubMedCentralID PMC4444385
Dimethylated H3K27 Is a Repressive Epigenetic Histone Mark in the Protist Entamoeba histolytica and Is Significantly Enriched in Genes Silenced via the RNAi Pathway. The Journal of biological chemistry Foda, B. M., Singh, U. 2015; 290 (34): 21114?30
Abstract

RNA interference (RNAi) is a fundamental biological process that plays a crucial role in regulation of gene expression in many organisms. Transcriptional gene silencing (TGS) is one of the important nuclear roles of RNAi. Our previous data show that Entamoeba histolytica has a robust RNAi pathway that links to TGS via Argonaute 2-2 (Ago2-2) associated 27-nucleotide small RNAs with 5'-polyphosphate termini. Here, we report the first repressive histone mark to be identified in E. histolytica, dimethylation of H3K27 (H3K27Me2), and demonstrate that it is enriched at genes that are silenced by RNAi-mediated TGS. An RNAi-silencing trigger can induce H3K27Me2 deposits at both episomal and chromosomal loci, mediating gene silencing. Our data support two phases of RNAi-mediated TGS: an active silencing phase where the RNAi trigger is present and both H3K27Me2 and Ago2-2 concurrently enrich at chromosomal loci; and an established silencing phase in which the RNAi trigger is removed, but gene silencing with H3K27Me2 enrichment persist independently of Ago2-2 deposition. Importantly, some genes display resistance to chromosomal silencing despite induction of functional small RNAs. In those situations, the RNAi-triggering plasmid that is maintained episomally gets partially silenced and has H3K27Me2 enrichment, but the chromosomal copy displays no repressive histone enrichment. Our data are consistent with a model in which H3K27Me2 is a repressive histone modification, which is strongly associated with transcriptional repression. This is the first example of an epigenetic histone modification that functions to mediate RNAi-mediated TGS in the deep-branching eukaryote E. histolytica.

View details for PubMedID 26149683

View details for PubMedCentralID PMC4543668
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. PloS one Zhang, H., Ehrenkaufer, G. M., Manna, D., Hall, N., Singh, U. 2015; 10 (8)
Abstract

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.

View details for DOI 10.1371/journal.pone.0134481

View details for PubMedID 26248204
Regulation of gene expression in the protozoan parasite Entamoeba invadens: identification of core promoter elements and promoters with stage-specific expression patterns INTERNATIONAL JOURNAL FOR PARASITOLOGY Manna, D., Ehrenkaufer, G. M., Singh, U. 2014; 44 (11): 837-845
Abstract

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.

View details for DOI 10.1016/j.ijpara.2014.06.008

View details for Web of Science ID 000343361100009
Regulation of gene expression in the protozoan parasite Entamoeba invadens: identification of core promoter elements and promoters with stage-specific expression patterns. International journal for parasitology Manna, D., Ehrenkaufer, G. M., Singh, U. 2014; 44 (11): 837-845
Abstract

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.

View details for DOI 10.1016/j.ijpara.2014.06.008

View details for PubMedID 25075445
RNAi Pathway Genes Are Resistant to Small RNA Mediated Gene Silencing in the Protozoan Parasite Entamoeba histolytica PLOS ONE Pompey, J. M., Morf, L., Singh, U. 2014; 9 (9)
Abstract

The RNA interference pathway in the protist Entamoeba histolytica plays important roles in permanent gene silencing as well as in the regulation of virulence determinants. Recently, a novel RNA interference (RNAi)-based silencing technique was developed in this parasite that uses a gene endogenously silenced by small RNAs as a "trigger" to induce silencing of other genes that are fused to it. Fusion to a trigger gene induces the production of gene-specific antisense small RNAs, resulting in robust and permanent silencing of the cognate gene. This approach has silenced multiple genes including those involved in virulence and transcriptional regulation. We now demonstrate that all tested genes of the amebic RNAi pathway are unable to be silenced using the trigger approach, including Argonaute genes (Ago2-1, Ago2-2, and Ago2-3), RNaseIII, and RNA-dependent RNA polymerase (RdRP). In all situations (except for RdRP), fusion to a trigger successfully induces production of gene-specific antisense small RNAs to the cognate gene. These small RNAs are capable of silencing a target gene in trans, indicating that they are functional; despite this, however, they cannot silence the RNAi pathway genes. Interestingly, when a trigger is fused to RdRP, small RNA induction to RdRP does not occur, a unique phenotype hinting that either RdRP is highly resistant to being a target of small RNAs or that small RNA generation may be controlled by RdRP. The inability of the small RNA pathway to silence RNAi genes in E. histolytica, despite the generation of functional small RNAs to these loci suggest that epigenetic factors may protect certain genomic loci and thus determine susceptibility to small RNA mediated silencing.

View details for DOI 10.1371/journal.pone.0106477

View details for Web of Science ID 000341304700029

View details for PubMedCentralID PMC4157801
Destabilization domain approach adapted for regulated protein expression in the protozoan parasite Entamoeba histolytica. International journal for parasitology Liu, Y., Singh, U. 2014; 44 (10): 729-735
Abstract

A plethora of information has been gained by sequencing the genome of the human parasite Entamoeba histolytica, however a lack of robust genetic tools hampers experimental elucidation of gene functions. We adapted the destabilization domain approach for modulation of protein levels in E. histolytica using the destabilization domains of FK506 binding protein (ddFKBP) and dihydrofolate reductase (ddDHFR), respectively. In our studies, the ddFKBP appears to be more tightly regulated than ddDHFR, with minimal detectable protein in trophozoites in the absence of the stabilizing compound. The on- and off-rate kinetics for ddFKBP were rapid, with stabilization and degradation within 3h of addition or removal of stabilizing compound, respectively. The kinetics for ddDHFR was different, with rapid stabilization (within 3h of stabilizing compound being added) but much slower degradation (protein not destabilized until 24h after compound removal). Furthermore, we demonstrated that for the ddFKBP, the standard stabilizing compound Shield-1 could be effectively replaced by two cheaper alternatives (rapamycin and FK506), indicating that the more cost-effective alternatives are viable options for use with E. histolytica. Thus, the destabilization domain approach represents a powerful method to study protein functions in E. histolytica and adds to the catalog of genetic tools that could be used to study this important human pathogen.

View details for DOI 10.1016/j.ijpara.2014.05.002

View details for PubMedID 24929134

View details for PubMedCentralID PMC4138259
RNAi pathway genes are resistant to small RNA mediated gene silencing in the protozoan parasite Entamoeba histolytica. PloS one Pompey, J. M., Morf, L., Singh, U. 2014; 9 (9)
Abstract

The RNA interference pathway in the protist Entamoeba histolytica plays important roles in permanent gene silencing as well as in the regulation of virulence determinants. Recently, a novel RNA interference (RNAi)-based silencing technique was developed in this parasite that uses a gene endogenously silenced by small RNAs as a "trigger" to induce silencing of other genes that are fused to it. Fusion to a trigger gene induces the production of gene-specific antisense small RNAs, resulting in robust and permanent silencing of the cognate gene. This approach has silenced multiple genes including those involved in virulence and transcriptional regulation. We now demonstrate that all tested genes of the amebic RNAi pathway are unable to be silenced using the trigger approach, including Argonaute genes (Ago2-1, Ago2-2, and Ago2-3), RNaseIII, and RNA-dependent RNA polymerase (RdRP). In all situations (except for RdRP), fusion to a trigger successfully induces production of gene-specific antisense small RNAs to the cognate gene. These small RNAs are capable of silencing a target gene in trans, indicating that they are functional; despite this, however, they cannot silence the RNAi pathway genes. Interestingly, when a trigger is fused to RdRP, small RNA induction to RdRP does not occur, a unique phenotype hinting that either RdRP is highly resistant to being a target of small RNAs or that small RNA generation may be controlled by RdRP. The inability of the small RNA pathway to silence RNAi genes in E. histolytica, despite the generation of functional small RNAs to these loci suggest that epigenetic factors may protect certain genomic loci and thus determine susceptibility to small RNA mediated silencing.

View details for DOI 10.1371/journal.pone.0106477

View details for PubMedID 25198343
Robust gene silencing mediated by antisense small RNAs in the pathogenic protist Entamoeba histolytica. Nucleic acids research Morf, L., Pearson, R. J., Wang, A. S., Singh, U. 2013; 41 (20): 9424-9437
Abstract

RNA interference uses small RNAs (sRNA), which target genes for sequence-specific silencing. The parasite Entamoeba histolytica contains an abundant repertoire of 27 nt antisense (AS) sRNA with 5'-polyphosphate termini, but their roles in regulating gene expression have not been well established. We demonstrate that a gene-coding region to which large numbers of AS sRNAs map can serve as a 'trigger' and silence the gene fused to it. Silencing is mediated by generation of AS sRNAs with 5'-polyphosphate termini that have sequence specificity to the fused gene. The mechanism of silencing is independent of the placement of the trigger relative to the silenced gene but is dependent on the sRNA concentration to the trigger. Silencing requires transcription of the trigger-gene fusion and is maintained despite loss of the trigger plasmid. We used this approach to silence multiple amebic genes, including an E. histolytica Myb gene, which is upregulated during oxidative stress response. Silencing of the EhMyb gene decreased parasite viability under oxidative stress conditions. Thus, we have developed a new tool for genetic manipulation in E. histolytica with many advantages over currently available technologies. Additionally, these data shed mechanistic insights into a eukaryotic RNA interference pathway with many novel aspects.

View details for DOI 10.1093/nar/gkt717

View details for PubMedID 23935116

View details for PubMedCentralID PMC3814356
Regulation of H2O2 Stress-responsive Genes through a Novel Transcription Factor in the Protozoan Pathogen Entamoeba histolytica JOURNAL OF BIOLOGICAL CHEMISTRY Pearson, R. J., Morf, L., Singh, U. 2013; 288 (6): 4462-4474
Abstract

Outcome of infection depends upon complex interactions between the invading pathogen and the host. As part of the host's innate immune response, the release of reactive oxygen and nitrogen species by phagocytes represents a major obstacle to the establishment of infection. The ability of the human parasite Entamoeba histolytica to survive reactive oxygen and nitrogen species is central to its pathogenic potential and contributes to disease outcome. In order to define the transcriptional network associated with oxidative stress, we utilized the MEME and MAST programs to analyze the promoter regions of 57 amoebic genes that had increased expression specifically in response to H(2)O(2) exposure. We functionally characterized an H(2)O(2)-regulatory motif (HRM) ((1)AAACCTCAATGAAGA(15)), which was enriched in these promoters and specifically bound amoebic nuclear protein(s). Assays with promoter-luciferase fusions established the importance of key residues and that the HRM motif directly impacted the ability of H(2)O(2)-responsive promoters to drive gene expression. DNA affinity chromatography and mass spectrometry identified EHI_108720 as an HRM DNA-binding protein. Overexpression and down-regulation of EHI_108720 demonstrated the specificity of EHI_108720 protein binding to the HRM, and overexpression increased basal expression from an H(2)O(2)-responsive wild-type promoter but not from its mutant counterpart. Thus, EHI_108720, or HRM-binding protein, represents a new stress-responsive transcription factor in E. histolytica that controls a transcriptional regulatory network associated with oxidative stress. Overexpression of EHI_108720 increased parasite virulence. Insight into how E. histolytica responds to oxidative stress increases our understanding of how this important human pathogen establishes invasive disease.

View details for DOI 10.1074/jbc.M112.423467

View details for Web of Science ID 000314845000072

View details for PubMedID 23250742

View details for PubMedCentralID PMC3567695
Small RNA pyrosequencing in the protozoan parasite Entamoeba histolytica reveals strain-specific small RNAs that target virulence genes BMC GENOMICS Zhang, H., Ehrenkaufer, G. M., Hall, N., Singh, U. 2013; 14
Abstract

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.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.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.

View details for DOI 10.1186/1471-2164-14-53

View details for Web of Science ID 000316679600001

View details for PubMedID 23347563

View details for PubMedCentralID PMC3610107
Distinct Distal Gut Microbiome Diversity and Composition in Healthy Children from Bangladesh and the United States PLOS ONE Lin, A., Bik, E. M., Costello, E. K., Dethlefsen, L., Haque, R., Relman, D. A., Singh, U. 2013; 8 (1)
Abstract

Our current understanding of the composition and stability of the human distal gut microbiota is based largely on studies of infants and adults living in developed countries. In contrast, little is known about the gut microbiota and its variation over time in older children and adolescents, especially in developing countries.We compared the diversity, composition, and temporal stability of the fecal microbiota of healthy children, ages 9 to 14 years, living in an urban slum in Bangladesh with that of children of the same age range in an upper-middle class suburban community in the United States. We analyzed >8,000 near full-length 16S rRNA gene sequences and over 845,000 pyrosequencing reads of the 16S rRNA V1-V3 region. The distal gut of Bangladeshi children harbored significantly greater bacterial diversity than that of U.S. children, including novel lineages from several bacterial phyla. Bangladeshi and U.S. children had distinct fecal bacterial community membership and structure; the microbiota of Bangladeshi children was enriched in Prevotella, Butyrivibrio, and Oscillospira and depleted in Bacteroides relative to U.S. children (although similar to Bangladeshi adults). Furthermore, community membership and structure in Bangladeshi children was significantly less stable month-to-month than U.S. children.Together, these results suggest that differing environmental or genetic factors may shape the microbiota of healthy children in the two countries. Further investigation is necessary to understand the mechanisms and factors that underlie these differences, and to incorporate these findings into new strategies for the prevention and treatment of childhood and adolescent diseases.

View details for DOI 10.1371/journal.pone.0053838

View details for Web of Science ID 000314019100034

View details for PubMedID 23349750

View details for PubMedCentralID PMC3551965
The genome and transcriptome of the enteric parasite Entamoeba invadens, a model for encystation. Genome biology Ehrenkaufer, G. M., Weedall, G. D., Williams, D., Lorenzi, H. A., Caler, E., Hall, N., Singh, U. 2013; 14 (7): R77
Abstract

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.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.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.

View details for DOI 10.1186/gb-2013-14-7-r77

View details for PubMedID 23889909
The genome and transcriptome of the enteric parasite Entamoeba invadens, a model for encystation GENOME BIOLOGY Ehrenkaufer, G. M., Weedall, G. D., Williams, D., Lorenzi, H. A., Caler, E., Hall, N., Singh, U. 2013; 14 (7)
Abstract

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.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.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.

View details for DOI 10.1186/gb-2013-14-7-r77

View details for Web of Science ID 000328194900009
Distinct distal gut microbiome diversity and composition in healthy children from Bangladesh and the United States. PloS one Lin, A., Bik, E. M., Costello, E. K., Dethlefsen, L., Haque, R., Relman, D. A., Singh, U. 2013; 8 (1)
Abstract

Our current understanding of the composition and stability of the human distal gut microbiota is based largely on studies of infants and adults living in developed countries. In contrast, little is known about the gut microbiota and its variation over time in older children and adolescents, especially in developing countries.We compared the diversity, composition, and temporal stability of the fecal microbiota of healthy children, ages 9 to 14 years, living in an urban slum in Bangladesh with that of children of the same age range in an upper-middle class suburban community in the United States. We analyzed >8,000 near full-length 16S rRNA gene sequences and over 845,000 pyrosequencing reads of the 16S rRNA V1-V3 region. The distal gut of Bangladeshi children harbored significantly greater bacterial diversity than that of U.S. children, including novel lineages from several bacterial phyla. Bangladeshi and U.S. children had distinct fecal bacterial community membership and structure; the microbiota of Bangladeshi children was enriched in Prevotella, Butyrivibrio, and Oscillospira and depleted in Bacteroides relative to U.S. children (although similar to Bangladeshi adults). Furthermore, community membership and structure in Bangladeshi children was significantly less stable month-to-month than U.S. children.Together, these results suggest that differing environmental or genetic factors may shape the microbiota of healthy children in the two countries. Further investigation is necessary to understand the mechanisms and factors that underlie these differences, and to incorporate these findings into new strategies for the prevention and treatment of childhood and adolescent diseases.

View details for DOI 10.1371/journal.pone.0053838

View details for PubMedID 23349750

View details for PubMedCentralID PMC3551965
Oxidative stress resistance genes contribute to the pathogenic potential of the anaerobic protozoan parasite, Entamoeba histolytica INTERNATIONAL JOURNAL FOR PARASITOLOGY Rastew, E., Vicente, J. B., Singh, U. 2012; 42 (11): 1007-1015
Abstract

The protozoan parasite, Entamoeba histolytica, invades the host colon causing significant tissue destruction and inflammation. Upon host infection, the parasite is confronted with reactive oxygen and nitrogen species (ROS/RNS) that cause large-scale changes in gene expression profiles, which likely support the parasite's adaptation to the host environment. We have previously identified oxidative and nitrosative stress responsive genes using whole-genome expression profiling. Functional studies on two such genes are now reported and demonstrate that they have roles in parasite virulence. EHI_056680 encodes a small hypothetical protein named E. histolytica stress-induced adhesion factor (EhSIAF); EHI_188210 encodes a putative phospholipid transporting P-type ATPase/flippase (EhPTPA). Over-expression of each protein in E. histolytica trophozoites enhanced parasite survival in response to oxidative stress. Exposure to oxidative and nitrosative stress did not affect the localization of EhSIAF or EhPTPA but markedly increased EhPTPA protein levels. Interestingly, over-expression of each gene resulted in parasites with increased adherence to healthy mammalian cells, but increased adherence to apoptotic cells was noted only in EhSIAF over-expressing parasites. However, despite having increased adherence to both healthy and apoptotic host cells, EhSIAF-over-expressing parasites were reduced in their ability to destroy mammalian cell monolayers, raising the intriguing possibility that EhSIAF over-expression caused signaling defects or resulted in a dominant negative phenotype. Over-expression of EhSIAF and EhPTPA also resulted in decreased motility in a transwell motility assay. Thus, we have confirmed that two genes that are upregulated by ROS confer increased resistance to oxidative stress and have identified an unexpected role of EhSIAF and EhPTPA in host cell adherence and a role of EhSIAF in parasite virulence. Our data imply that stress response genes may play multi-factorial roles in amoebic pathogenesis.

View details for DOI 10.1016/j.ijpara.2012.08.006

View details for Web of Science ID 000311331200006

View details for PubMedID 23009748

View details for PubMedCentralID PMC3483436
A Detoxifying Oxygen Reductase in the Anaerobic Protozoan Entamoeba histolytica EUKARYOTIC CELL Vicente, J. B., Vy Tran, V., Pinto, L., Teixeira, M., Singh, U. 2012; 11 (9): 1112-1118
Abstract

We report the characterization of a bacterial-type oxygen reductase abundant in the cytoplasm of the anaerobic protozoan parasite Entamoeba histolytica. Upon host infection, E. histolytica is confronted with various oxygen tensions in the host intestine, as well as increased reactive oxygen and nitrogen species at the site of local tissue inflammation. Resistance to oxygen-derived stress thus plays an important role in the pathogenic potential of E. histolytica. The genome of E. histolytica has four genes that encode flavodiiron proteins, which are bacterial-type oxygen or nitric oxide reductases and were likely acquired by lateral gene transfer from prokaryotes. The EhFdp1 gene has higher expression in virulent than in nonvirulent Entamoeba strains and species, hinting that the response to oxidative stress may be one correlate of virulence potential. We demonstrate that EhFdp1 is abundantly expressed in the cytoplasm of E. histolytica and that the protein levels are markedly increased (up to ~5-fold) upon oxygen exposure. Additionally, we produced fully functional recombinant EhFdp1 and demonstrated that this enzyme is a specific and robust oxygen reductase but has poor nitric oxide reductase activity. This observation represents a new mechanism of oxygen resistance in the anaerobic protozoan pathogen E. histolytica.

View details for DOI 10.1128/EC.00149-12

View details for Web of Science ID 000308446200004

View details for PubMedID 22798391

View details for PubMedCentralID PMC3445978
Entamoeba histolytica: a snapshot of current research and methods for genetic analysis CURRENT OPINION IN MICROBIOLOGY Morf, L., Singh, U. 2012; 15 (4): 469-475
Abstract

Entamoeba histolytica represents one of the leading causes of parasitic death worldwide. Although identified as the causative agent of amebiasis since 1875, the molecular mechanisms by which the parasite causes disease are still not fully understood. Studying Entamoeba reveals insights into a eukaryotic cell that differs in many ways from better-studied model organisms. Thus, much can be learned from this protozoan parasite on evolution, cell biology, and RNA biology. In this review we discuss selected research highlights in Entamoeba research and focus on the development of molecular biological techniques to study this pathogen. We end by highlighting some of the many questions that remain to be answered in order to fully understand this important human pathogen.

View details for DOI 10.1016/j.mib.2012.04.011

View details for Web of Science ID 000308622300012

View details for PubMedID 22664276

View details for PubMedCentralID PMC3424301
Transient and stable transfection in the protozoan parasite Entamoeba invadens MOLECULAR AND BIOCHEMICAL PARASITOLOGY Ehrenkaufer, G. M., Singh, U. 2012; 184 (1): 59-62
Abstract

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.

View details for DOI 10.1016/j.molbiopara.2012.04.007

View details for Web of Science ID 000305112200011

View details for PubMedID 22561071

View details for PubMedCentralID PMC3358517
Nucleus-localized Antisense Small RNAs with 5 '-Polyphosphate Termini Regulate Long Term Transcriptional Gene Silencing in Entamoeba histolytica G3 Strain JOURNAL OF BIOLOGICAL CHEMISTRY Zhang, H., Alramini, H., Vy Tran, V., Singh, U. 2011; 286 (52): 44467-44479
Abstract

In the deep-branching eukaryotic parasite Entamoeba histolytica, transcriptional gene silencing (TGS) of the Amoebapore A gene (ap-a) in the G3 strain has been reported with subsequent development of this parasite strain for gene silencing. However, the mechanisms underlying this gene silencing approach are poorly understood. Here we report that antisense small RNAs (sRNAs) specific to the silenced ap-a gene can be identified in G3 parasites. Furthermore, when additional genes are silenced in the G3 strain, antisense sRNAs to the newly silenced genes can also be detected. Characterization of these sRNAs demonstrates that they are ~27 nucleotides in size, have 5'-polyphosphate termini, and persist even after removal of the silencing plasmid. Immunofluorescence analysis (IFA) and fluorescence in situ hybridization (FISH) show that both the Argonaute protein EhAGO2-2 and antisense sRNAs to the silenced genes are localized to the parasite nucleus. Furthermore, ?-EhAGO2-2 immunoprecipitation confirmed the direct association of the antisense sRNAs with EhAGO2-2. Finally, chromatin immunoprecipitation (ChIP) assays demonstrate that the loci of the silenced genes are enriched for histone H3 and EhAGO2-2, indicating that both chromatin modification and the RNA-induced transcriptional silencing complex are involved in permanent gene silencing in G3 parasites. In conclusion, our data demonstrate that G3-based gene silencing in E. histolytica is mediated by an siRNA pathway, which utilizes antisense 5'-polyphosphate sRNAs. To our knowledge, this is the first study to show that 5'- polyphosphate antisense sRNAs can mediate TGS, and it is the first example of RNAi-mediated TGS in protozoan parasites.

View details for DOI 10.1074/jbc.M111.278184

View details for Web of Science ID 000298645500019

View details for PubMedID 22049083

View details for PubMedCentralID PMC3247957
Antiparasitic Therapy MAYO CLINIC PROCEEDINGS Kappagoda, S., Singh, U., Blackburn, B. G. 2011; 86 (6): 561-583
Abstract

Parasitic diseases affect more than 2 billion people globally and cause substantial morbidity and mortality, particularly among the world's poorest people. This overview focuses on the treatment of the major protozoan and helminth infections in humans. Recent developments in antiparasitic therapy include the expansion of artemisinin-based therapies for malaria, new drugs for soil-transmitted helminths and intestinal protozoa, expansion of the indications for antiparasitic drug treatment in patients with Chagas disease, and the use of combination therapy for leishmaniasis and human African trypanosomiasis.

View details for DOI 10.4065/mcp.2011.0203

View details for Web of Science ID 000291288400012

View details for PubMedID 21628620

View details for PubMedCentralID PMC3104918
RNA interference in Entamoeba histolytica: implications for parasite biology and gene silencing FUTURE MICROBIOLOGY Zhang, H., Pompey, J. M., Singh, U. 2011; 6 (1): 103-117
Abstract

Entamoeba histolytica is a major health threat to people in developing countries, where it causes invasive diarrhea and liver abscesses. The study of this important human pathogen has been hindered by a lack of tools for genetic manipulation. Recently, a number of genetic approaches based on variations of the RNAi method have been successfully developed and cloning of endogenous small-interfering RNAs from E. histolytica revealed an abundant population of small RNAs with an unusual 5�-polyphosphate structure. However, little is known about the implications of these findings to amebic biology or the mechanisms of gene silencing in this organism. In this article we review the literature relevant to RNAi in E. histolytica, discuss its implications for advances in gene silencing in this organism and outline potential future directions towards understanding the repertoire of RNAi and its impact on the biology of this deep-branching eukaryotic parasite.

View details for DOI 10.2217/FMB.10.154

View details for Web of Science ID 000286686500013

View details for PubMedID 21162639

View details for PubMedCentralID PMC3038252
Approaches to characterizing Entamoeba histolytica transcriptional regulation CELLULAR MICROBIOLOGY Pearson, R. J., Singh, U. 2010; 12 (12): 1681-1690
Abstract

Entamoeba histolytica causes an estimated 100,000 deaths per year and is one of the leading causes of death among parasitic infections. Studies using E. histolytica-specific polymerase chain reaction identified that 13.8% of adults in a rural Mexican community and 11.2% of adults in central Vietnam are asymptomatically colonized. Such high incidents of asymptomatic infection suggest that only a minority of infections proceed to invasive disease. Understanding the mechanisms that underpin variable disease outcome will be critical in developing therapeutic strategies. In recent years there have been a plethora of gene expression profiling data documenting the transcriptome differences between virulent and non-virulent strains of E. histolytica as well as changes induced by external environmental changes or stimuli. While these studies have successfully identified co-regulated genes and potential virulence factors, there is still little known about the transcriptional mechanisms that induce the changes observed in this non-model organism. In this review, we have looked at how molecular technological advances have shaped our understanding of transcriptional regulation in amoeba and what we may expect from the application of powerful new techniques.

View details for DOI 10.1111/j.1462-5822.2010.01524.x

View details for Web of Science ID 000284318600001

View details for PubMedID 20812994
Downregulation of an Entamoeba histolytica Rhomboid Protease Reveals Roles in Regulating Parasite Adhesion and Phagocytosis EUKARYOTIC CELL Baxt, L. A., Rastew, E., Bracha, R., Mirelman, D., Singh, U. 2010; 9 (8): 1283-1293
Abstract

Entamoeba histolytica is a deep-branching eukaryotic pathogen. Rhomboid proteases are intramembrane serine proteases, which cleave transmembrane proteins in, or in close proximity to, their transmembrane domain. We have previously shown that E. histolytica contains a single functional rhomboid protease (EhROM1) and has unique substrate specificity. EhROM1 is present on the trophozoite surface and relocalizes to internal vesicles during erythrophagocytosis and to the base of the cap during surface receptor capping. In order to further examine the biological function of EhROM1 we downregulated EhROM1 expression by >95% by utilizing the epigenetic silencing mechanism of the G3 parasite strain. Despite the observation that EhROM1 relocalized to the cap during surface receptor capping, EhROM1 knockdown [ROM(KD)] parasites had no gross changes in cap formation or complement resistance. However, ROM(KD) parasites demonstrated decreased host cell adhesion, a result recapitulated by treatment of wild-type parasites with DCI, a serine protease inhibitor with activity against rhomboid proteases. The reduced adhesion phenotype of ROM(KD) parasites was noted exclusively with healthy cells, and not with apoptotic cells. Additionally, ROM(KD) parasites had decreased phagocytic ability with reduced ingestion of healthy cells, apoptotic cells, and rice starch. Decreased phagocytic ability is thus independent of the reduced adhesion phenotype, since phagocytosis of apoptotic cells was reduced despite normal adhesion levels. The defect in host cell adhesion was not explained by altered expression or localization of the heavy subunit of the Gal/GalNAc surface lectin. These results suggest no significant role of EhROM1 in complement resistance but unexpected roles in parasite adhesion and phagocytosis.

View details for DOI 10.1128/EC.00015-10

View details for Web of Science ID 000280577300014

View details for PubMedID 20581296

View details for PubMedCentralID PMC2918930
Entamoeba histolytica Genomic Analyses ANAEROBIC PARASITIC PROTOZOA: GENOMICS AND MOLECULAR BIOLOGY Singh, U., Ehrenkaufer, G. M., Clark, C. G., Johnson, P. J., Adam, R. D. 2010: 157?73
View details for Web of Science ID 000277629500006
A developmentally regulated Myb domain protein regulates expression of a subset of stage-specific genes in Entamoeba histolytica CELLULAR MICROBIOLOGY Ehrenkaufer, G. M., Hackney, J. A., Singh, U. 2009; 11 (6): 898-910
Abstract

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.

View details for DOI 10.1111/j.1462-5822.2009.01300.x

View details for Web of Science ID 000265883500005

View details for PubMedID 19239479

View details for PubMedCentralID PMC3424735
A Pseudouridine Synthase Homologue Is Critical to Cellular Differentiation in Toxoplasma gondii EUKARYOTIC CELL Anderson, M. Z., Brewer, J., Singh, U., Boothroyd, J. C. 2009; 8 (3): 398-409
Abstract

Toxoplasma gondii is a haploid protozoan parasite infecting about one in seven people in the United States. Key to the worldwide prevalence of T. gondii is its ability to establish a lifelong, chronic infection by evading the immune system, and central to this is the developmental switch between the two asexual forms, tachyzoites and bradyzoites. A library of mutants defective in tachyzoite-to-bradyzoite differentiation (Tbd(-)) was created through insertional mutagenesis. This library contains mutants that, compared to the wild type, are between 20% and 74% as efficient at stage conversion. Two mutants, TBD5 and TBD8, with disruptions in a gene encoding a putative pseudouridine synthase, PUS1, were identified. The disruption in TBD8 is in the 5' end of the PUS1 gene and appears to produce a null allele with a 50% defect in differentiation. This is about the same switch efficiency as obtained with an engineered pus1 deletion mutant (Deltapus1). The insertion in TBD5 is within the PUS1 coding region, and this appears to result in a more extreme phenotype of only approximately 10% switch efficiency. Complementation of TBD8 with the genomic PUS1 allele restored wild-type differentiation efficiency. Infection of mice with pus1 mutant strains results in increased mortality during the acute phase and higher cyst burdens during the chronic infection, demonstrating an aberrant differentiation phenotype in vivo due to PUS1 disruption. Our results suggest a surprising and important role for RNA modification in this biological process.

View details for DOI 10.1128/EC.00329-08

View details for Web of Science ID 000263935200014

View details for PubMedID 19124578

View details for PubMedCentralID PMC2653242
Short hairpin RNA-mediated knockdown of protein expression in Entamoeba histolytica BMC MICROBIOLOGY Linford, A. S., Moreno, H., Good, K. R., Zhang, H., Singh, U., Petri, W. A. 2009; 9
Abstract

Entamoeba histolytica is an intestinal protozoan parasite of humans. The genome has been sequenced, but the study of individual gene products has been hampered by the lack of the ability to generate gene knockouts. We chose to test the use of RNA interference to knock down gene expression in Entamoeba histolytica.An episomal vector-based system, using the E. histolytica U6 promoter to drive expression of 29-basepair short hairpin RNAs, was developed to target protein-encoding genes in E. histolytica. The short hairpin RNAs successfully knocked down protein levels of all three unrelated genes tested with this system: Igl, the intermediate subunit of the galactose- and N-acetyl-D-galactosamine-inhibitable lectin; the transcription factor URE3-BP; and the membrane binding protein EhC2A. Igl levels were reduced by 72%, URE3-BP by 89%, and EhC2A by 97%.Use of the U6 promoter to drive expression of 29-basepair short hairpin RNAs is effective at knocking down protein expression for unrelated genes in Entamoeba histolytica, providing a useful tool for the study of this parasite.

View details for DOI 10.1186/1471-2180-9-38

View details for Web of Science ID 000264160800001

View details for PubMedID 19222852

View details for PubMedCentralID PMC2652455
Entamoeba histolytica modulates a complex repertoire of novel genes in response to oxidative and nitrosative stresses: implications for amebic pathogenesis CELLULAR MICROBIOLOGY Vicente, J. B., Ehrenkaufer, G. M., Saraiva, L. M., Teixeira, M., Singh, U. 2009; 11 (1): 51-69
Abstract

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.

View details for DOI 10.1111/j.1462-5822.2008.01236.x

View details for Web of Science ID 000262150100005

View details for PubMedID 18778413

View details for PubMedCentralID PMC3418052
Recent insights into Entamoeba development: Identification of transcriptional networks associated with stage conversion INTERNATIONAL JOURNAL FOR PARASITOLOGY Singh, U., Ehrenkaufer, G. M. 2009; 39 (1): 41-47
Abstract

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.

View details for DOI 10.1016/j.ijpara.2008.09.004

View details for Web of Science ID 000262735000004

View details for PubMedID 18938171

View details for PubMedCentralID PMC2648836
Small RNAs with 5 '- Polyphosphate Termini Associate with a Piwi-Related Protein and Regulate Gene Expression in the Single-Celled Eukaryote Entamoeba histolytica PLOS PATHOGENS Zhang, H., Ehrenkaufer, G. M., Pompey, J. M., Hackney, J. A., Singh, U. 2008; 4 (11)
Abstract

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.

View details for DOI 10.1371/journal.ppat.1000219

View details for Web of Science ID 000261481200023

View details for PubMedID 19043551

View details for PubMedCentralID PMC2582682
Transcriptional Regulatory Networks in Entamoeba histolytica CURRENT DRUG TARGETS Ehrenkaufer, G. M., Singh, U. 2008; 9 (11): 931-937
Abstract

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.

View details for Web of Science ID 000261495900003

View details for PubMedID 18991605
New insights into Entamoeba histolytica pathogenesis CURRENT OPINION IN INFECTIOUS DISEASES Baxt, L. A., Singh, U. 2008; 21 (5): 489-494
Abstract

Entamoeba histolytica is an important global pathogen and a leading cause of parasitic death worldwide. This article summarizes significant research findings over the last year.Efforts have focused primarily on identification of novel virulence determinants in E. histolytica, transcriptional profiling during tissue invasion and stage conversion, and characterization of basic cell biological processes. Additionally, new techniques for gene silencing have been identified.A comprehensive examination of the parasite lifestyle on a whole genome level has been undertaken, allowing identification of new virulence genes and signaling pathways and processes relevant to amebic biology.

View details for DOI 10.1097/QCO.0b013e32830ce75f

View details for Web of Science ID 000259078900007

View details for PubMedID 18725798

View details for PubMedCentralID PMC2688559
An Entamoeba histolytica rhomboid protease with atypical specificity cleaves a surface lectin involved in phagocytosis and immune evasion GENES & DEVELOPMENT Baxt, L. A., Baker, R. P., Singh, U., Urban, S. 2008; 22 (12): 1636-1646
Abstract

Rhomboid proteases are membrane-embedded enzymes conserved in all kingdoms of life, but their cellular functions across evolution are largely unknown. Prior work has uncovered a role for rhomboid enzymes in host cell invasion by malaria and related intracellular parasites, but this is unlikely to be a widespread function, even in pathogens, since rhomboid proteases are also conserved in unrelated protozoa that maintain an extracellular existence. We examined rhomboid function in Entamoeba histolytica, an extracellular, parasitic ameba that is second only to malaria in medical burden globally. Despite its large genome, E. histolytica encodes only one rhomboid (EhROM1) with residues necessary for protease activity. EhROM1 displayed atypical substrate specificity, being able to cleave Plasmodium adhesins but not the canonical substrate Drosophila Spitz. We searched for substrates encoded in the ameba genome and found EhROM1 was able to cleave a cell surface lectin specifically. In E. histolytica trophozoites, EhROM1 changed localization to vesicles during phagocytosis and to the posterior cap structure during surface receptor shedding for immune evasion, in both cases colocalizing with lectins. Collectively these results implicate rhomboid proteases for the first time in immune evasion and suggest that a common function of rhomboid enzymes in widely divergent protozoan pathogens is to break down adhesion proteins.

View details for DOI 10.1101/gad.1667708

View details for Web of Science ID 000256797300009

View details for PubMedID 18559479

View details for PubMedCentralID PMC2428061
Loss of dsRNA-based gene silencing in Entamoeba histolytica: Implications for approaches to genetic analysis EXPERIMENTAL PARASITOLOGY MacFarlane, R. C., Singh, U. 2008; 119 (2): 296-300
Abstract

The ability to regulate gene expression in the protozoan parasite Entamoeba histolytica is critical in determining gene function. We previously published that expression of dsRNA specific to E. histolytica serine threonine isoleucine rich protein (EhSTIRP) resulted in reduction of gene expression [MacFarlane, R.C., Singh, U., 2007. Identification of an Entamoeba histolytica serine, threonine, isoleucine, rich protein with roles in adhesion and cytotoxicity. Eukaryotic Cell 6, 2139-2146]. However, after approximately one year of continuous drug selection, the expression of EhSTIRP reverted to wild-type levels. We confirmed that the parasites (i) contained the appropriate dsRNA plasmid, (ii) were not contaminated with other plasmids, (iii) the drug selectable marker was functional, and (iv) sequenced the dsRNA portion of the construct. This work suggests that in E. histolytica long term cultivation of parasites expressing dsRNA can lead to the loss of dsRNA based silencing through the selection of "RNAi" negative parasites. Thus, users of the dsRNA silencing approach should proceed with caution and regularly confirm gene down regulation. The development and use of constructs for inducible expression of dsRNA may help alleviate this potential problem.

View details for DOI 10.1016/j.exppara.2008.02.001

View details for Web of Science ID 000256607700016

View details for PubMedID 18346737

View details for PubMedCentralID PMC2426738
Identification of an Entamoeba histolytica serine-, threonine-, and isoleucine-rich protein with roles in adhesion and cytotoxicity EUKARYOTIC CELL MacFarlane, R. C., Singh, U. 2007; 6 (11): 2139-2146
Abstract

Entamoeba histolytica is a leading cause of parasitic death globally. However, the molecular framework regulating pathogenesis is poorly understood. We have previously used expression profiling to identify Entamoeba genes whose expressions were strictly associated with virulent strains (R. C. MacFarlane and U. Singh, Infect. Immun. 74:340-351, 2006). One gene, which we have named EhSTIRP (Entamoeba histolytica serine-, threonine-, and isoleucine-rich protein), was exclusively expressed in virulent but not in nonvirulent Entamoeba strains. EhSTIRP is predicted to be a transmembrane protein and is encoded by a multigene family. In order to characterize its function in amebic biology, we used a double-stranded RNA-based approach and were able to selectively down-regulate expression of this gene family. Upon EhSTIRP down-regulation, we were able to ascribe cytotoxic and adhesive properties to the protein family using lactate dehydrogenase release and Chinese hamster ovary cell adhesion assays. EhSTIRP thus likely represents a novel determinant of virulence in Entamoeba histolytica. This work validates the fact that genes expressed exclusively in virulent strains may represent virulence determinants and highlights the need for further functional analyses of other genes with similar expression profiles.

View details for DOI 10.1128/EC.00174-07

View details for Web of Science ID 000251410200021

View details for PubMedID 17827347

View details for PubMedCentralID PMC2168410
Trichostatin A effects on gene expression in the protozoan parasite Entamoeba histolytica BMC GENOMICS Ehrenkaufer, G. M., Eichinger, D. J., Singh, U. 2007; 8
Abstract

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.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)).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.

View details for DOI 10.1186/1471-2164-8-216

View details for Web of Science ID 000248701500001

View details for PubMedID 17612405

View details for PubMedCentralID PMC1940012
Identification of developmentally regulated genes in Entamoeba histolytica: insights into mechanisms of stage conversion in a protozoan parasite CELLULAR MICROBIOLOGY Ehrenkaufer, G. M., Haque, R., Hackney, J. A., Eichinger, D. J., Singh, U. 2007; 9 (6): 1426-1444
Abstract

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.

View details for DOI 10.1111/j.1462-5822.2006.00882.x

View details for Web of Science ID 000246577400006

View details for PubMedID 17250591
Functional characterization of spliceosomal introns and identification of U2, U4, and U5 snRNAs in the deep-branching eukaryote Entamoeba histolytica EUKARYOTIC CELL Davis, C. A., Brown, M. P., Singh, U. 2007; 6 (6): 940-948
Abstract

Pre-mRNA splicing is essential to ensure accurate expression of many genes in eukaryotic organisms. In Entamoeba histolytica, a deep-branching eukaryote, approximately 30% of the annotated genes are predicted to contain introns; however, the accuracy of these predictions has not been tested. In this study, we mined an expressed sequence tag (EST) library representing 7% of amoebic genes and found evidence supporting splicing of 60% of the testable intron predictions, the majority of which contain a GUUUGU 5' splice site and a UAG 3' splice site. Additionally, we identified several splice site misannotations, evidence for the existence of 30 novel introns in previously annotated genes, and identified novel genes through uncovering their spliced ESTs. Finally, we provided molecular evidence for the E. histolytica U2, U4, and U5 snRNAs. These data lay the foundation for further dissection of the role of RNA processing in E. histolytica gene expression.

View details for DOI 10.1128/EC.00059-07

View details for Web of Science ID 000247439700006

View details for PubMedID 17468393

View details for PubMedCentralID PMC1951529
Identification of putative transcriptional regulatory networks in Entamoeba histolytica using Bayesian inference NUCLEIC ACIDS RESEARCH Hackney, J. A., Ehrenkaufer, G. M., Singh, U. 2007; 35 (7): 2141-2152
Abstract

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 na�ve 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.

View details for DOI 10.1093/nar/gkm028

View details for Web of Science ID 000246294700013

View details for PubMedID 17355990

View details for PubMedCentralID PMC1874630
Growth of the protozoan parasite Entamoeba histolytica in 5-azacytidine has limited effects on parasite gene expression BMC GENOMICS Ali, I. K., Ehrenkaufer, G. M., Hackney, J. A., Singh, U. 2007; 8
Abstract

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.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.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.

View details for DOI 10.1186/1471-2164-8-7

View details for PubMedID 17207281
Structure and content of the Entamoeba histolytica genome ADVANCES IN PARASITOLOGY, VOL 65 Clark, C. G., Alsmark, U. C., Tazreiter, M., Saito-Nakano, Y., Ali, V., Marion, S., Weber, C., Mukherjee, C., Bruchhaus, I., Tannich, E., Leippe, M., Sicheritz-Ponten, T., Foster, P. G., Samuelson, J., NOEL, C. J., Hirt, R. P., Embley, T. M., Gilchrist, C. A., Mann, B. J., Singh, U., Ackers, J. P., Bhattacharya, S., Bhattacharya, A., Lohia, A., Guillen, N., Duchene, M., Nozaki, T., Hall, N. 2007; 65: 51-190
Abstract

The intestinal parasite Entamoeba histolytica is one of the first protists for which a draft genome sequence has been published. Although the genome is still incomplete, it is unlikely that many genes are missing from the list of those already identified. In this chapter we summarise the features of the genome as they are currently understood and provide previously unpublished analyses of many of the genes.

View details for DOI 10.1016/S0065-308X(07)65002-7

View details for Web of Science ID 000252405100002

View details for PubMedID 18063096
Impact of intestinal colonization and invasion on the Entamoeba histolytica transcriptome MOLECULAR AND BIOCHEMICAL PARASITOLOGY Gilchrist, C. A., Houpt, E., Trapaidze, N., Fei, Z., Crasta, O., Asgharpour, A., Evans, C., Martino-Catt, S., Baba, D. J., Stroup, S., Hamano, S., Ehrenkaufer, G., Okada, M., Singh, U., Nozaki, T., Mann, B. J., Petri, W. A. 2006; 147 (2): 163-176
Abstract

A genome-wide transcriptional analysis of Entamoeba histolytica was performed on trophozoites isolated from the colon of six infected mice and from in vitro culture. An Affymetrix platform gene expression array was designed for this analysis that included probe sets for 9435 open reading frames (ORFs) and 9066 5' and 3' flanking regions. Transcripts were detected for > 80% of all ORFs. A total of 523 transcripts (5.2% of all E. histolytica genes) were significantly changed in amebae isolated from the intestine on Days 1 and 29 after infection: 326 and 109 solely on Days 1 and 29, and 88 on both days. Quantitative real-time reverse transcriptase PCR confirmed these changes in 11/12 genes tested using mRNA isolated from an additional six mice. Adaptation to the intestinal environment was accompanied by increases in a subset of cell signaling genes including transmembrane kinases, ras and rho family GTPases, and calcium binding proteins. Significant decreases in mRNA abundance for genes involved in glycolysis and concomitant increases in lipases were consistent with a change in energy metabolism. Defense against bacteria present in the intestine (but lacking from in vitro culture) was suggested by alterations in mRNA levels of genes similar to the AIG1 plant antibacterial proteins. Decreases in oxygen detoxification pathways were observed as expected in the anaerobic colonic lumen. Of the known virulence factors the most remarkable changes were a 20-35-fold increase in a cysteine proteinase four-like gene, and a 2-3-fold decrease in two members of the Gal/GalNAc lectin light subunit family. Control of the observed changes in mRNA abundance in the intestine might potentially rest with four related proteins with DNA binding domains that were down-regulated 6-16-fold in the intestinal environment. In conclusion, the first genome-wide analysis of the transcriptome of E. histolytica demonstrated that the vast majority of genes are transcribed in trophozoites, and that in the host intestine trophozoites altered the expression of mRNAs for genes implicated in metabolism, oxygen defense, cell signaling, virulence, antibacterial activity, and DNA binding.

View details for DOI 10.1016/j.molbiopara.2006.02.007

View details for Web of Science ID 000237525600003

View details for PubMedID 16569449
Identification of differentially expressed genes in virulent and nonvirulent Entamoeba species: Potential implications for amebic pathogenesis INFECTION AND IMMUNITY MacFarlane, R. C., Singh, U. 2006; 74 (1): 340-351
Abstract

Entamoeba histolytica is a protozoan parasite that causes colitis and liver abscesses. Several Entamoeba species and strains with differing levels of virulence have been identified. E. histolytica HM-1:IMSS is a virulent strain, E. histolytica Rahman is a nonvirulent strain, and Entamoeba dispar is a nonvirulent species. We used an E. histolytica DNA microarray consisting of 2,110 genes to assess the transcriptional differences between these species/strains with the goal of identifying genes whose expression correlated with a virulence phenotype. We found 415 genes expressed at lower levels in E. dispar and 32 genes with lower expression in E. histolytica Rahman than in E. histolytica HM-1:IMSS. Overall, 29 genes had decreased expression in both the nonvirulent species/strains than the virulent E. histolytica HM-1:IMSS. Interestingly, a number of genes with potential roles in stress response and virulence had decreased expression in either one or both nonvirulent Entamoeba species/strains. These included genes encoding Fe hydrogenase (9.m00419), peroxiredoxin (176.m00112), type A flavoprotein (6.m00467), lysozyme (6.m00454), sphingomyelinase C (29.m00231), and a hypothetical protein with homology to both a Plasmodium sporozoite threonine-asparagine-rich protein (STARP) and a streptococcal hemagglutinin (238.m00054). The function of these genes in Entamoeba and their specific roles in parasite virulence need to be determined. We also found that a number of the non-long-terminal-repeat retrotransposons (EhLINEs and EhSINEs), which have been shown to modulate gene expression and genomic evolution, had lower expression in the nonvirulent species/strains than in E. histolytica HM-1:IMSS. Our results, identifying expression profiles and patterns indicative of a virulence phenotype, may be useful in characterizing the transcriptional framework of virulence.

View details for DOI 10.1128/IAI.74.1.340-351.2006

View details for Web of Science ID 000234276400037

View details for PubMedID 16368989

View details for PubMedCentralID PMC1346599
Disruption of a locus encoding a nucleolar zinc finger protein decreases tachyzoite-to-bradyzoite differentiation in Toxoplasma gondii INFECTION AND IMMUNITY Vanchinathan, P., Brewer, J. L., Harb, O. S., Boothroyd, J. C., Singh, U. 2005; 73 (10): 6680-6688
Abstract

During its life cycle in intermediate hosts, Toxoplasma gondii exists in two interconverting developmental stages: tachyzoites and bradyzoites. This interconversion is essential for the survival and pathogenicity of the parasite, but little is known about the genetic mechanisms that control this process. We have previously generated tachyzoite-to-bradyzoite differentiation (Tbd(-)) mutants using chemical mutagenesis and a green fluorescent protein-based selection strategy. The genetic loci responsible for the Tbd(-) phenotype, however, could not be identified. We have now used an insertional mutagenesis strategy to generate two differentiation mutants: TBD-5 and TBD-6 that switch to bradyzoites at 10 and 50% of wild-type levels, respectively. In TBD-6 there is a single insertion of the mutagenesis vector 164 bp upstream of the transcription start site of a gene encoding a zinc finger protein (ZFP1). Disruption of this locus in wild-type parasites reproduces the decreased stage conversion phenotype. ZFP1 is targeted to the parasite nucleolus by CCHC motifs and significantly altered expression levels are toxic to the parasites. This represents the first identification of a gene necessary for efficient conversion of tachyzoites to bradyzoites.

View details for DOI 10.1128/IAI.73.10.6680-6688.2005

View details for Web of Science ID 000232087600055

View details for PubMedID 16177345

View details for PubMedCentralID PMC1230886
Coding and noncoding genomic regions of Entamoeba histolytica have significantly different rates of sequence polymorphisms: Implications for epidemiological studies JOURNAL OF CLINICAL MICROBIOLOGY Bhattacharya, D., Haque, R., Singh, U. 2005; 43 (9): 4815-4819
Abstract

To evaluate genetic variability among Entamoeba histolytica strains, we sequenced 9,077 bp from each of 14 isolates. The polymorphism rates from coding and noncoding regions were significantly different (0.07% and 0.37%, respectively), indicating that these regions are subject to different selection pressures. Additionally, single nucleotide polymorphisms (SNPs) potentially associated with specific clinical outcomes were identified.

View details for DOI 10.1128/JCM.43.9.4815-4819.2005

View details for Web of Science ID 000232020400078

View details for PubMedID 16145147

View details for PubMedCentralID PMC1234137
Genomic DNA microarrays for Entamoeba histolytica: Applications for use in expression profiling and strain genotyping 2nd EMBO Workshop on Pathogenesis and Amoebiasis Macfarlane, R., Bhattacharya, D., Singh, U. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2005: 196?202
Abstract

The parasite Entamoeba histolytica is a causative agent of dysentery and liver abscesses. Found predominantly in developing countries, this parasitic infection is responsible for significant morbidity and mortality. We have developed a genomic DNA microarray for E. histolytica. The array composed of 11,328 clones contains >2000 unique genes and was utilized for expression profiling and comparative genomic hybridizations of Entamoeba strains. We present a synopsis of our results to date and potential future applications of microarray technology for the study of Entamoeba biology.

View details for DOI 10.1016/j.exppara.2005.03.006

View details for Web of Science ID 000230067400007

View details for PubMedID 15955312
Transcriptional profiling of Entamoeba histolytica trophozoites INTERNATIONAL JOURNAL FOR PARASITOLOGY MacFarlane, R. C., Shah, P. H., Singh, U. 2005; 35 (5): 533-542
Abstract

We have developed an Entamoeba histolytica genomic DNA microarray and used it to develop a transcriptional profile of 1,971 E. histolytica (HM-1:IMSS) genes. The arrays accurately detected message abundance and 31-47% of amebic genes were expressed under standard tissue culture conditions (levels detectable by Northern blot analysis or RT-PCR respectively). Genes expressed at high levels ( approximately 2% of total) included actin (8.m00351), and ribosomal genes (20.m00312). Moderately expressed genes ( approximately 14% of total) included cysteine proteinase (191.m00117), profilin (156.m00098), and an Argonaute family member (11.m00378). Genes with low-level expression ( approximately 15% of total) included Ariel1 (160.m00087). Genes with very low expression ( approximately 16% of total) and those not expressed ( approximately 52% of total) included encystation-specific genes such as Jacob cyst wall glycoprotein (33.m00261), chitin synthase (3.m00544), and chitinase (22.m00311). Transcriptional modulation could be detected using the arrays with 17% of genes upregulated at least two-fold in response to heat shock. These included heat shock proteins (119.m00119 and 279.m00091), cyst wall glycoprotein Jacob (33.m00261), and ubiquitin-associated proteins (16.m00343; 195.m00092). Using Caco-2 cells to model the host-parasite interaction, we verified that host cell killing was dependent on live ameba. However, surprisingly these events did not appear to induce major transcriptional changes in the parasites.

View details for DOI 10.1016/j.ijpara.2005.02.006

View details for Web of Science ID 000228765900007

View details for PubMedID 15826645
Comparative genomic hybridizations of Entamoeba strains reveal unique genetic fingerprints that correlate with virulence EUKARYOTIC CELL Shah, P. H., MacFarlane, R. C., Bhattacharya, D., Matese, J. C., Demeter, J., Stroup, S. E., Singh, U. 2005; 4 (3): 504-515
Abstract

Variable phenotypes have been identified for Entamoeba species. Entamoeba histolytica is invasive and causes colitis and liver abscesses but only in approximately 10% of infected individuals; 90% remain asymptomatically colonized. Entamoeba dispar, a closely related species, is avirulent. To determine the extent of genetic diversity among Entamoeba isolates and potential genotype-phenotype correlations, we have developed an E. histolytica genomic DNA microarray and used it to genotype strains of E. histolytica and E. dispar. On the basis of the identification of divergent genetic loci, all strains had unique genetic fingerprints. Comparison of divergent genetic regions allowed us to distinguish between E. histolytica and E. dispar, identify novel genetic regions usable for strain and species typing, and identify a number of genes restricted to virulent strains. Among the four E. histolytica strains, a strain with attenuated virulence was the most divergent and phylogenetically distinct strain, raising the intriguing possibility that genetic subtypes of E. histolytica may be partially responsible for the observed variability in clinical outcomes. This microarray-based genotyping assay can readily be applied to the study of E. histolytica clinical isolates to determine genetic diversity and potential genotypic-phenotypic associations.

View details for DOI 10.1128/EC.4.3.504-515.2005

View details for Web of Science ID 000227781300002

View details for PubMedID 15755913

View details for PubMedCentralID PMC1087797
The genome of the protist parasite Entamoeba histolytica NATURE Loftus, B., Anderson, I., Davies, R., Alsmark, U. C., Samuelson, J., Amedeo, P., Roncaglia, P., Berriman, M., Hirt, R. P., Mann, B. J., Nozaki, T., Suh, B., Pop, M., Duchene, M., Ackers, J., Tannich, E., Leippe, M., Hofer, M., Bruchhaus, I., Willhoeft, U., Bhattacharya, A., Chillingworth, T., Churcher, C., Hance, Z., Harris, B., Harris, D., Jagels, K., Moule, S., Mungall, K., Ormond, D., Squares, R., Whitehead, S., Quail, M. A., Rabbinowitsch, E., Norbertczak, H., Price, C., Wang, Z., Guillen, N., Gilchrist, C., Stroup, S. E., Bhattacharya, S., Lohia, A., Foster, P. G., Sicheritz-Ponten, T., Weber, C., Singh, U., Mukherjee, C., El-Sayed, N. M., Petri, W. A., Clark, C. G., Embley, T. M., Barrell, B., Fraser, C. M., Hall, N. 2005; 433 (7028): 865-868
Abstract

Entamoeba histolytica is an intestinal parasite and the causative agent of amoebiasis, which is a significant source of morbidity and mortality in developing countries. Here we present the genome of E. histolytica, which reveals a variety of metabolic adaptations shared with two other amitochondrial protist pathogens: Giardia lamblia and Trichomonas vaginalis. These adaptations include reduction or elimination of most mitochondrial metabolic pathways and the use of oxidative stress enzymes generally associated with anaerobic prokaryotes. Phylogenomic analysis identifies evidence for lateral gene transfer of bacterial genes into the E. histolytica genome, the effects of which centre on expanding aspects of E. histolytica's metabolic repertoire. The presence of these genes and the potential for novel metabolic pathways in E. histolytica may allow for the development of new chemotherapeutic agents. The genome encodes a large number of novel receptor kinases and contains expansions of a variety of gene families, including those associated with virulence. Additional genome features include an abundance of tandemly repeated transfer-RNA-containing arrays, which may have a structural function in the genome. Analysis of the genome provides new insights into the workings and genome evolution of a major human pathogen.

View details for DOI 10.1038/nature03291

View details for Web of Science ID 000227174600042

View details for PubMedID 15729342
DNA content analysis on microarrays. Methods in molecular biology (Clifton, N.J.) Singh, U., Shah, P. H., MacFarlane, R. C. 2004; 270: 237-248
Abstract

The genome sequencing of protozoan parasites has facilitated the development of powerful postgenomics tools such as DNA microarrays and revolutionized the study of parasite biology. Large-scale genomic comparisons are useful in identifying the extent of genomic variability among related strains and isolates. Identification of deletions between geographically diverse clinical isolates is important in understanding parasite biology and the "fitness" of a given strain in dissemination. Additionally, the development of reliable diagnostic tests or identification of potential vaccine candidates is predicated on the large-scale conservation of the candidate genes. Parasites with variable virulence phenotypes (vaccine strain vs virulent strain) can also be studied for their genomic variability and provide further insights into the potential role of genotypic variability and its relationship to virulence. This chapter outlines the utilization of DNA microarrays to study genomic content.

View details for PubMedID 15153631
DNA microarrays in parasitology: strengths and limitations TRENDS IN PARASITOLOGY Boothroyd, J. C., Blader, I., Cleary, M., Singh, U. 2003; 19 (10): 470-476
Abstract

Genome sequencing efforts have provided a wealth of new biological information that promises to have a major impact on our understanding of parasites. Microarrays provide one of the major high-throughput platforms by which this information can be exploited in the laboratory. Many excellent reviews and technique articles have recently been published on applying microarrays to organisms for which fully annotated genomes are at hand. However, many parasitologists work on organisms whose genomes have been only partially sequenced and where little, if any, annotation is available. The focus of this review is on how to use and apply microarrays to these situations.

View details for DOI 10.1016/j.pt.2003.08.002

View details for Web of Science ID 000186058400012

View details for PubMedID 14519585
Investigating amoebic pathogenesis using Entamoeba histolytica DNA microarrays JOURNAL OF BIOSCIENCES Singh, U., Shah, P. 2002; 27 (6): 595-601
View details for Web of Science ID 000179604900008
Toxoplasma gondii asexual development: Identification of developmentally regulated genes and distinct patterns of gene expression EUKARYOTIC CELL Cleary, M. D., Singh, U., Blader, I. J., Brewer, J. L., Boothroyd, J. C. 2002; 1 (3): 329-340
Abstract

Asexual development in Toxoplasma gondii is a vital aspect of the parasite's life cycle, allowing transmission and avoidance of the host immune response. Differentiation of rapidly dividing tachyzoites into slowly growing, encysted bradyzoites involves significant changes in both physiology and morphology. We generated microarrays of approximately 4,400 Toxoplasma cDNAs, representing a minimum of approximately 600 genes (based on partial sequencing), and used these microarrays to study changes in transcript levels during tachyzoite-to-bradyzoite differentiation. This approach has allowed us to (i) determine expression profiles of previously described developmentally regulated genes, (ii) identify novel developmentally regulated genes, and (iii) identify distinct classes of genes based on the timing and magnitude of changes in transcript levels. Whereas microarray analysis typically involves comparisons of mRNA levels at different time points, we have developed a method to measure relative transcript abundance between genes at a given time point. This method was used to determine transcript levels in parasites prior to differentiation and to further classify bradyzoite-induced genes, thus allowing a more comprehensive view of changes in gene expression than is provided by standard expression profiles. Newly identified developmentally regulated genes include putative surface proteins (a SAG1-related protein, SRS9, and a mucin-domain containing protein), regulatory and metabolic enzymes (methionine aminopeptidase, oligopeptidase, aminotransferase, and glucose-6-phosphate dehydrogenase homologues), and a subset of genes encoding secretory organelle proteins (MIC1, ROP1, ROP2, ROP4, GRA1, GRA5, and GRA8). This analysis permits the first in-depth look at changes in gene expression during development of this complex protozoan parasite.

View details for DOI 10.1128/EC.1.3.329-340.2002

View details for Web of Science ID 000178729300002

View details for PubMedID 12455982

View details for PubMedCentralID PMC118016
Genetic analysis of tachyzoite to bradyzoite differentiation mutants in Toxoplasma gondii reveals a hierarchy of gene induction MOLECULAR MICROBIOLOGY Singh, U., Brewer, J. L., Boothroyd, J. C. 2002; 44 (3): 721-733
Abstract

Developmental switching in Toxoplasma gondii, from the virulent tachyzoite to the relatively quiescent bradyzoite stage, is responsible for disease propagation and reactivation. We have generated tachyzoite to bradyzoite differentiation (Tbd-) mutants in T. gondii and used these in combination with a cDNA microarray to identify developmental pathways in bradyzoite formation. Four independently generated Tbd- mutants were analysed and had defects in bradyzoite development in response to multiple bradyzoite-inducing conditions, a stable phenotype after in vivo passages and a markedly reduced brain cyst burden in a murine model of chronic infection. Transcriptional profiles of mutant and wild-type parasites, growing under bradyzoite conditions, revealed a hierarchy of developmentally regulated genes, including many bradyzoite-induced genes whose transcripts were reduced in all mutants. A set of non-developmentally regulated genes whose transcripts were less abundant in Tbd- mutants were also identified. These may represent genes that mediate downstream effects and/or whose expression is dependent on the same transcription factors as the bradyzoite-induced set. Using these data, we have generated a model of transcription regulation during bradyzoite development in T. gondii. Our approach shows the utility of this system as a model to study developmental biology in single-celled eukaryotes including protozoa and fungi.

View details for Web of Science ID 000175466900011

View details for PubMedID 11994153
Identification and characterization of differentiation mutants in the protozoan parasite Toxoplasma gondii MOLECULAR MICROBIOLOGY Matrajt, M., Donald, R. G., Singh, U., Roos, D. S. 2002; 44 (3): 735?47
Abstract

Two forms of the protozoan parasite Toxoplasma gondii are associated with intermediate hosts such as humans: rapidly growing tachyzoites are responsible for acute illness, whereas slowly dividing encysted bradyzoites can remain latent within the tissues for the life of the host. In order to identify genetic factors associated with parasite differentiation, we have used a strong bradyzoite-specific promoter (identified by promoter trapping) to drive the expression of T. gondii hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) in stable transgenic parasites, providing a stage-specific positive/negative selectable marker. Insertional mutagenesis has been carried out on this parental line, followed by bradyzoite induction in vitro and selection in 6-thioxanthine to identify misregulation mutants. Two different mutants fail to induce the HXGPRT gene efficiently during bradyzoite differentiation. These mutants are also defective in other aspects of differentiation: they replicate well under bradyzoite growth conditions, lysing the host cell monolayer as effectively as tachyzoites. Expression of the major bradyzoite antigen BAG1 is reduced, and staining with Dolichos biflorus lectin shows reduced cyst wall formation. Microarray hybridizations show that these mutants behave more like tachyzoites at a global level, even under bradyzoite differentiation conditions.

View details for DOI 10.1046/j.1365-2958.2002.02904.x

View details for Web of Science ID 000175466900012

View details for PubMedID 11994154
Context-dependent roles of the Entamoeba histolytica core promoter element GAAC in transcriptional activation and protein complex assembly MOLECULAR AND BIOCHEMICAL PARASITOLOGY Singh, U., Gilchrist, C. A., Schaenman, J. M., Rogers, J. B., Hockensmith, J. W., Mann, B. J., Petri, W. A. 2002; 120 (1): 107-116
Abstract

Transcriptional control of the hgl5 gene of Entamoeba histolytica is mediated through an unusual core promoter composed of TATA, GAAC and Initiator elements. In the hgl5 promoter the GAAC element (AATGAACT) determines the site and rate of transcription initiation. Here we tested the role of the GAAC element in transcription activation from upstream regulatory elements (UREs) in the hgl5 promoter. We also examined the function of the GAAC element in the ferredoxin (fdx) promoter and characterized the protein binding to the GAAC element. Electrophoretic mobility shift assays (EMSA) demonstrated that the GAAC region is necessary for higher-order nuclear protein complex assembly. The function of the GAAC element in transcription activation mediated by UREs revealed that mutation of the GAAC element did not affect transcription activation mediated by the hgl5 URE4 but abrogated activation by the hgl5 URE3. We compared the role of the GAAC elements in the hgl5 and fdx promoters. Competitive gel shift assays were consistent with the same nuclear protein binding to the GAAC elements in both genes. Mutation of the GAAC element in the fdx gene decreased reporter gene expression, however, in contrast to hgl5 gene, had no effect on the site of transcription initiation. These results support a role for the GAAC element in assembly of nuclear proteins at the core promoter and in transcription activation mediated by URE3. The differing effect on transcription initiation in the hgl5 and fdx genes upon mutation of the GAAC element suggests a context-dependence of the GAAC-binding protein in gene expression.

View details for Web of Science ID 000174312300010

View details for PubMedID 11849710
Diagnosis and management of amebiasis CLINICAL INFECTIOUS DISEASES Petri, W. A., Singh, U. 1999; 29 (5): 1117-1125
View details for Web of Science ID 000083512600001

View details for PubMedID 10524950
Diagnosis and management of amebiasis. Clinical Infectious Diseases. Singh, U., Petri, Jr, WA 1999: 1117-25
The novel core promoter element GAAC in the hgl5 gene of Entamoeba histolytica is able to direct a transcription start site independent of TATA or initiator regions JOURNAL OF BIOLOGICAL CHEMISTRY Singh, U., Rogers, J. B. 1998; 273 (34): 21663-21668
Abstract

Entamoeba histolytica, an enteric protozoa, is the third leading parasitic cause of death worldwide. Investigation of the transcriptional machinery of this eukaryotic pathogen has revealed an unusual core promoter structure that consists of nonconsensus TATA and initiator regions and a novel third conserved core promoter sequence, the GAAC element. Mutation of this region in the hgl5 promoter decreases reporter gene expression and alters the transcription start site. Using positional analysis of this element, we have now demonstrated that it is able to direct a new transcription start site, 2-7 bases downstream of itself, independent of TATA and Inr regions. The GAAC region was also shown to control the rate of transcription via nuclear run on analysis and an amebic nuclear protein was demonstrated to specifically interact with this sequence. This is the first description in the eukaryotic literature of a third conserved core promoter element, distinct from TATA or initiator regions, that is able to direct a transcription start site. We have formulated two models for the role of the GAAC region: (i) the GAAC-binding protein is a part of the TFIID complex and (ii) the GAAC-binding protein functions to "tether" TATA-binding protein to the TATA box.

View details for Web of Science ID 000075492600034

View details for PubMedID 9705300
The novel core promoter element GAAC in the hgl5 gene of Entamoeba histolytica is able to direct a transcription start site independent of TATA or Inr regions. Journal of Biological Chemistry. Singh U, Rogers J. 1998: 21663-21668
Transcription initiation is controlled by three core promoter elements in the hgl5 gene of the protozoan parasite Entamoeba histolytica PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Singh, U., Rogers, J. B., Mann, B. J., Petri, W. A. 1997; 94 (16): 8812-8817
Abstract

Entamoeba histolytica is a single cell eukaryote that is the etiologic agent of amoebic colitis. Core promoter elements of E. histolytica protein encoding genes include a TATA-like sequence (GTATTTAAAG/C) at -30, a novel element designated GAAC (GAACT) that has a variable location between TATA and the site of transcription initiation, and a putative initiator (Inr) element (AAAAATTCA) overlying the site of transcription initiation. The presence of three separate conserved sequences in a eukaryotic core promoter is unprecedented and prompted examination of their roles in regulating transcription initiation. Alterations of all three regions in the hgl5 gene decreased reporter gene activity with the greatest effect seen by mutation of the GAAC element. Positional analysis of the TATA box demonstrated that transcription initiated consistently 30-31 bases downstream of the TATA region. Mutation of either the TATA or GAAC elements resulted in the appearance of new transcription start sites upstream of +1 in the promoter of the hgl5 gene. Mutation of the Inr element resulted in no change in the site of transcription initiation; however, in the presence of a mutated TATA and GAAC regions, the Inr element controlled the site of transcription initiation. We conclude that all three elements play a role in determining the site of transcription initiation. The variable position of the GAAC element relative to the site of transcription initiation, and the multiple transcription initiations that resulted from its mutation, indicate that the GAAC element has an important and apparently novel role in transcriptional control in E. histolytica.

View details for Web of Science ID A1997XQ12400093

View details for PubMedID 9238060
Three conserved cis-acting sequences in the core promoter control gene expression in the protozoan parasite Entamoeba histolytica. Archives of medical research Singh, U., Purdy, J., Mann, B. J., Petri, W. A. 1997; 28: 41-42
View details for PubMedID 9033006

Upinder Singh

Professor of Medicine (Infectious Diseases & Geographic Medicine) and of Microbiology and Immunology

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