Comparative and functional genomics of Toxoplasma and Hammondia hammondi

 描述（由申请方提供）：弓形虫是一种重要的人类机会致病菌，可导致发育中的胎儿、免疫功能低下的个体和某些情况下的健康成人发生严重疾病。与此相反，T.弓形虫（Hammondiahamondi）不感染人类，并且在小鼠中是高度无毒的，并且实验性感染的特征在于快速转化为“终末分化的”包囊阶段。这些体内毒力差异在体外也得到了重现，其中H。Hammondi自发地转化为终末分化的囊肿阶段。我们有大量的已发表和未发表的初步数据表明，T。gondii和H. hammondi和T. gondii和H.发育期间的Hammondi转录组是相似的，但在多种候选毒力效应子和转录因子的转录中具有关键差异。在本3年R 01提案中概述的工作中，我们使用T。gondii/H. hammondi系统来识别驱动T.弓形虫病的发病机制，特别关注缓殖子包囊阶段，这是艾滋病毒/艾滋病和移植患者严重弓形虫病的主要原因。在目标1中，我们在体外生长过程中包含明确发育转变的时间点使用超深链特异性RNAseq，以鉴定以物种特异性方式调控的基因和遗传网络。在此过程中，我们将鉴定1）推定的分泌型毒力效应子和2）在一个物种中相对于另一个物种独特表达的转录因子。然后我们将确定它们在T中的作用。利用基因缺失和跨物种互补实验进行弓形虫生物学研究。这一目标的成功是由广泛的出版和初步的数据比较基因组，在体外生长速率，转录谱，和宿主对T。gondii和H.哈蒙迪在目标2中，我们讨论了宿主在物种特异性生长表型中的作用。为了做到这一点，我们将比较1）T。gondii和H.使用平行的组织学测定法在体内进行Hammondi，以鉴定 细胞和体液免疫反应; 2）H. hammondi在对T.弓形虫感染通过这些研究，我们将确定新的T。弓形虫分泌的效应子和转录因子是这些物种之间显著表型差异的基础，以及这些效应子如何影响宿主对感染的反应。这些研究也应该更广泛地影响我们的理解，在一个基因接一个基因的基础上，有毒的病原体是如何从相对无毒的表型背景中出现的。

 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 We have extensive published and unpublished preliminary data demonstrating that T. gondii and H. hammondi share 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.