Bio

Clinical Focus


  • Infectious Disease
  • Infectious Diseases

Academic Appointments


Administrative Appointments


  • Division Chief, Infectious Diseases and Geographic Medicine (2010 - Present)
  • Fellowship Co-Director, Division of Infectious Diseases (2008 - 2013)

Honors & Awards


  • Member, ASCI (2010)

Professional Education


  • Residency:University of Virginia Health System (1995) VA
  • Fellowship:University of Virginia School of Medicine (1998) VA
  • Internship:University of Virginia Health System (1993) VA
  • Medical Education:Ohio State University (1992) OH
  • BS, Ohio State University, Biochemistry (1987)
  • MD, Ohio State University, Medicine (1992)

Community and International Work


  • Investigating E. histolytica genetic diversity, Bangladesh and Georgia

    Topic

    Investigating extent of genetic diversity among amebic strains

    Location

    International

    Ongoing Project

    Yes

    Opportunities for Student Involvement

    No

Research & Scholarship

Current Research and Scholarly Interests


Dr Singh studies the molecular basis of pathogenesis of a medically important parasite, Entamoeba histolytica. The work is aimed at understanding the virulence determinants that E. histolytica utilizes in causing invasive colonic and hepatic disease. Using a combination of genetic and genomic approaches we are identifying novel mechanisms that the parasite has developed for invading the human host. Additionally, we study the epidemiological trends of of amebic infection, with the goal of identifying a parasite molecular signature that correlates with invasive potential.

Teaching

2013-14 Courses


Publications

Journal Articles


  • 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

  • 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

  • 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

  • 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

  • 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)
  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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 Web of Science ID 000243660900001

    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

  • 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

  • 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

  • Genomic DNA microarrays for Entamoeba histolytica: Applications for use in expression profiling and strain genotyping EXPERIMENTAL PARASITOLOGY Macfarlane, R., Bhattacharya, D., Singh, U. 2005; 110 (3): 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

  • 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

  • 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

  • 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

  • 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 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

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