Upinder Singh

The genome of the protist parasite Entamoeba histolytica

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. histolyticas 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.

Toxoplasma gondii Asexual Development: Identification of Developmentally Regulated Genes and Distinct Patterns of Gene Expression

ABSTRACT Asexual development in Toxoplasma gondii is a vital aspect of the parasites 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 ∼4,400 Toxoplasma cDNAs, representing a minimum of ∼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.

Distinct Distal Gut Microbiome Diversity and Composition in Healthy Children from Bangladesh and the United States

Background 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. Methodology/Principal Findings 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. Conclusions/Significance 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.

Structure and Content of the Entamoeba histolytica Genome

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.

Genetic analysis of tachyzoite to bradyzoite differentiation mutants in Toxoplasma gondii reveals a hierarchy of gene induction

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.

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

Developmental switching between life‐cycle stages is a common feature among many pathogenic organisms. The protozoan parasite Entamoeba histolytica converts between cysts (essential for disease transmission) and trophozoites (responsible for tissue invasion). Identification of genes involved in the developmental pathway has been severely hindered by the inability to generate E. histolytica cysts in vitro. Using parasite strains derived from recent human infections and whole‐genome transcriptional profiling, we determined that 1439 genes (∼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.

Identification of Differentially Expressed Genes in Virulent and Nonvirulent Entamoeba Species: Potential Implications for Amebic Pathogenesis

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.

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

Upon host infection, the protozoan parasite Entamoeba histolytica is confronted with reactive oxygen and nitrogen species and must survive these stresses in order to cause invasive disease. We analysed the parasites 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 μM), 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.

An Entamoeba histolytica rhomboid protease with atypical specificity cleaves a surface lectin involved in phagocytosis and immune evasion

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.

Identification and characterization of differentiation mutants in the protozoan parasite Toxoplasma gondii

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.