Integrated Bioinformatic and Targeted Deletion Analyses of the SRS Gene Superfamily Identify SRS29C as a Negative Regulator of Toxoplasma Virulence

Program in Molecular Structure and Function, Hospital for Sick Children, Toronto, Ontario, Canada.
mBio (Impact Factor: 6.79). 10/2012; 3(6). DOI: 10.1128/mBio.00321-12
Source: PubMed


The Toxoplasma gondii SRS gene superfamily is structurally related to SRS29B (formerly SAG1), a surface adhesin that binds host cells and stimulates host immunity. Comparative genomic analyses of three Toxoplasma strains identified 182 SRS genes distributed across 14 chromosomes at 57 genomic loci. Eight distinct SRS subfamilies were resolved. A core 69 functional gene orthologs were identified, and strain-specific expansions and pseudogenization were common. Gene expression profiling demonstrated differential expression of SRS genes in a developmental-stage- and strain-specific fashion and identified nine SRS genes as priority targets for gene deletion among the tissue-encysting coccidia. A Δsag1 sag2A mutant was significantly attenuated in murine acute virulence and showed upregulated SRS29C (formerly SRS2) expression. Transgenic overexpression of SRS29C in the virulent RH parent was similarly attenuated. Together, these findings reveal SRS29C to be an important regulator of acute virulence in mice and demonstrate the power of integrated genomic analysis to guide experimental investigations.

Parasitic species employ large gene families to subvert host immunity to enable pathogen colonization and cause disease. Toxoplasma gondii contains a large surface coat gene superfamily that encodes adhesins and virulence factors that facilitate infection in susceptible hosts. We generated an integrated bioinformatic resource to predict which genes from within this 182-gene superfamily of adhesin-encoding genes play an essential role in the host-pathogen interaction. Targeted gene deletion experiments with predicted candidate surface antigens identified SRS29C as an important negative regulator of acute virulence in murine models of Toxoplasma infection. Our integrated computational and experimental approach provides a comprehensive framework, or road map, for the assembly and discovery of additional key pathogenesis genes contained within other large surface coat gene superfamilies from a broad array of eukaryotic pathogens.

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Available from: Jon P Boyle, Oct 04, 2015
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    • "Toxoplasma contains several distinct, coccidian-specific multicopy gene families throughout its genome, including those that encode the SRS, ROPK, and SUSA proteins [51-53]. Recently, a bioinformatics study showed that 60 out of the 144 ME49 SRS genes (42%) are located in subtelomeric sites [54]. However, none of these genes are near TgTAS-like regions in the current genome assembly. "
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    ABSTRACT: Chromosome ends are composed of telomeric repeats and subtelomeric regions, which are patchworks of genes interspersed with repeated elements. Although chromosome ends display similar arrangements in different species, their sequences are highly divergent. In addition, these regions display a particular nucleosomal composition and bind specific factors, therefore producing a special kind of heterochromatin. Using data from currently available draft genomes we have characterized these putative Telomeric Associated Sequences in Toxoplasma gondii. An all-vs-all pairwise comparison of T. gondii assembled chromosomes revealed the presence of conserved regions of ~ 30 Kb located near the ends of 9 of the 14 chromosomes of the genome of the ME49 strain. Sequence similarity among these regions is ~ 70%, and they are also highly conserved in the GT1 and VEG strains. However, they are unique to Toxoplasma with no detectable similarity in other Apicomplexan parasites. The internal structure of these sequences consists of 3 repetitive regions separated by high-complexity sequences without annotated genes, except for a gene from the Toxoplasma Specific Family. ChIP-qPCR experiments showed that nucleosomes associated to these sequences are enriched in histone H4 monomethylated at K20 (H4K20me1), and the histone variant H2A.X, suggesting that they are silenced sequences (heterochromatin). A detailed characterization of the base composition of these sequences, led us to identify a strong long-range compositional bias, which was similar to that observed in other genomic silenced fragments such as those containing centromeric sequences, and was negatively correlated to gene density. We identified and characterized a region present in most Toxoplasma assembled chromosomes. Based on their location, sequence features, and nucleosomal markers we propose that these might be part of subtelomeric regions of T. gondii. The identified regions display a unique trinucleotide compositional bias, which is shared (despite the lack of any detectable sequence similarity) with other silenced sequences, such as those making up the chromosome centromeres. We also identified other genomic regions with this compositional bias (but no detectable sequence similarity) that might be functionally similar.
    BMC Genomics 01/2014; 15(1):21. DOI:10.1186/1471-2164-15-21 · 3.99 Impact Factor
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    • "Compared to Type II and Type III, Type I strains display relatively high growth rates and are acutely virulent in mice (LD 100 ¼1 parasite) (Howe and Sibley, 1995). Recent work has identified that murine virulence is highly dependent on the expression level of virulence factors, such as ROP18, GRA15, and SRS29C (Melo et al, 2011; Wasmuth et al, 2012), proteins that target host immune signalling pathways. At the same time, due to its importance in providing energy and the basic building blocks required for growth, metabolic potential is increasingly being viewed as a critical element governing a pathogen's virulence potential, as well as its ability to survive in infected hosts (McKinney et al, 2000; Olszewski et al, 2009; Willger et al, 2009; Ensminger et al, 2012). "
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    ABSTRACT: Increasingly, metabolic potential is proving to be a critical determinant governing a pathogen's virulence as well as its capacity to expand its host range. To understand the potential contribution of metabolism to strain-specific infectivity differences, we present a constraint-based metabolic model of the opportunistic parasite, Toxoplasma gondii. Dominated by three clonal strains (Type I, II, and III demonstrating distinct virulence profiles), T. gondii exhibits a remarkably broad host range. Integrating functional genomic data, our model (which we term as iCS382) reveals that observed strain-specific differences in growth rates are driven by altered capacities for energy production. We further predict strain-specific differences in drug susceptibilities and validate one of these predictions in a drug-based assay, with a Type I strain demonstrating resistance to inhibitors that are effective against a Type II strain. We propose that these observed differences reflect an evolutionary strategy that allows the parasite to extend its host range, as well as result in a subsequent partitioning into discrete strains that display altered virulence profiles across different hosts, different organs, and even cell types.
    Molecular Systems Biology 11/2013; 9(1):708. DOI:10.1038/msb.2013.62 · 10.87 Impact Factor
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    • "SRS29A was also 1.7 fold more highly expressed in RH-ERP extracellular tachyzoites compared to RH-ERP intracellular tachyzoites (Additional file 1: Table S1). A recent study showed that RH-ERP overexpressing SRS29C was significantly attenuated in mouse virulence compared to parental RH-ERP strain, though this overexpressing strain did not have any significant differences from the parental strain with regards to invasion, attachment or growth in vitro[38]. "
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    ABSTRACT: Toxoplasma gondii has a largely clonal population in North America and Europe, with types I, II and III clonal lineages accounting for the majority of strains isolated from patients. RH, a particular type I strain, is most frequently used to characterize Toxoplasma biology. However, compared to other type I strains, RH has unique characteristics such as faster growth, increased extracellular survival rate and inability to form orally infectious cysts. Thus, to identify candidate genes that could account for these parasite phenotypic differences, we determined genetic differences and differential parasite gene expression between RH and another type I strain, GT1. Moreover, as differences in host cell modulation could affect Toxoplasma replication in the host, we determined differentially modulated host processes among the type I strains through host transcriptional profiling. Through whole genome sequencing, we identified 1,394 single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) between RH and GT1. These SNPs/indels together with parasite gene expression differences between RH and GT1 were used to identify candidate genes that could account for type I phenotypic differences. A polymorphism in dense granule protein, GRA2, determined RH and GT1 differences in the evasion of the interferon gamma response. In addition, host transcriptional profiling identified that genes regulated by NF-[latin small letter kra]B, such as interleukin (IL)-12p40, were differentially modulated by the different type I strains. We subsequently showed that this difference in NF-[latin small letter kra]B activation was due to polymorphisms in GRA15. Furthermore, we observed that RH, but not other type I strains, recruited phosphorylated I[latin small letter kra]Balpha (a component of the NF-[latin small letter kra]B complex) to the parasitophorous vacuole membrane and this recruitment of p- I[latin small letter kra]Balpha was partially dependent on GRA2. We identified candidate parasite genes that could be responsible for phenotypic variation among the type I strains through comparative genomics and transcriptomics. We also identified differentially modulated host pathways among the type I strains, and these can serve as a guideline for future studies in examining the phenotypic differences among type I strains.
    BMC Genomics 07/2013; 14(1):467. DOI:10.1186/1471-2164-14-467 · 3.99 Impact Factor
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