Comparative and functional genomics of Toxoplasma and Hammondia hammondi

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 DESCRIPTION (provided by applicant): Toxoplasma gondii is an important opportunistic pathogen of humans where it can cause severe disease in the developing fetus, immunocompromised individuals, and in certain cases healthy adults. In contrast the nearest extant relative of T. gondii, Hammondia hammondi, does not infect humans and is highly avirulent in mice, and experimental infections are characterized by rapid conversion to "terminally differentiated" cyst stages. These in vivo virulence differences are recapitulated in vitro, where H. hammondi spontaneously converts into a terminally differentiated cyst stage. We have extensive published and unpublished preliminary data demonstrating that T. gondii and H. hammondi share >99% of their genes in nearly perfect synteny, and that the T. gondii and H. hammondi transcriptomes during development are similar but have key differences in the transcription of multiple candidate virulence effectors and transcription factors. In the work outlined in this 3 year R01 proposal we use the T. gondii/H. hammondi system to identify the gene regulatory mechanisms that drive T. gondii pathogenesis, with a particular focus on bradyzoite cyst stages that are the leading cause of severe toxoplasmosis in HIV/AIDS and transplant patients. In Aim 1 we use ultra-deep, strand-specific RNAseq at time points that encompass clear developmental transitions during in vitro growth to identify genes and genetic networks that regulated in a species-specific manner. In doing so we will identify 1) putative secreted virulence effectors and 2) transcription factors that are uniquely expressed in one species over the other. We will then determine their role in T. gondii biology using gene deletion and cross-species complementation experiments. Success of this Aim is facilitated by extensive published and preliminary data comparing the genomes, in vitro growth rates, transcription profiles, and host responses to T. gondii and H. hammondi. In Aim 2 we address the role of the host in species-specific growth phenotypes. To do this we will compare 1) infection dynamics of T. gondii and H. hammondi in vivo using parallel histological assays to identify differences in the cellular and humoral immune response and 2) the overall infectivity and virulence of H. hammondi in innate-immune deficient mouse strains that are highly susceptible to T. gondii infection. Through these studies we will identify new T. gondii secreted effectors and transcription factors that underlie the dramatic phenotypic differences between these species, and how these effectors might impact the host response to infection. These studies should also more broadly impact our understanding of how, on a gene- by-gene basis, virulent pathogens emerge from comparatively avirulent phenotypic backgrounds.