Effector diversification and Toxoplasma virulence

描述(申请人提供)：弓形虫是人类的一种专性细胞内寄生虫，可在免疫功能低下的患者和发育中的胎儿中致死。弓形虫的毒力是由蛋白质直接分泌到宿主细胞中驱动的。弓形虫分泌体庞大而复杂，大多数可能的分泌蛋白尚未被解释，它们在弓形虫发病机制中的作用尚不清楚。与弓形虫不同的是，哈蒙迪亚·哈蒙迪和犬新孢子虫不会感染人类，尽管这三个物种的基因含量、形态和生命周期特征都相同。该项目的目标是确定弓形虫独有的以前未知的毒力效应因子。为了实现这一目标，我们将把跨物种比较基因组学与功能基因组学和正向遗传学数据相结合，以确定候选效应基因座，然后利用分子遗传学和体内感染来测试候选基因。通过全基因组比较，我们已经确定了一个假定的分泌性蛋白的子集，这些蛋白由弓形虫中唯一扩展和多样化的基因座编码。这些弓形虫特异性扩展基因座(TSEL)是这项建议的重点，其重要性得到了广泛的初步数据的证实。具体地说，TSEL4(线粒体关联因子1；MAF1)在体内参与弓形虫的毒力，并负责宿主线粒体与含有寄生虫的液泡的结合(HMA)。在目标1中，我们将通过充分描述MAF1基因座的基因含量、多样性以及感染期间对HMA的影响来进一步研究这些观察结果。我们将使用靶向缺失和X射线结晶学来确定MAF1的功能结构域，并确定其生化功能。我们将通过与马蒂·布朗格(维多利亚大学)在结构研究方面的持续合作来促进这一目标。在目标2中，我们量化了MAF1的作用 寄主线粒体生物学的副对数多样化和HMA。同时，我们将确定相同的MAF1对弓形虫整个生命周期(包括孢子化卵囊)寄生虫毒力的影响。与Bennett Van Houten(匹兹堡大学癌症中心)在线粒体研究方面的合作以及JP Dubey(美国农业部)在卵囊生产方面的合作将促进这一目标的实现。最后，在目标3中，我们将描述在我们的比较基因组筛选中确定的另外6个TSEL。我们将确定每个TSEL基因座多样化的程度，并在寄生虫和宿主细胞中定位单个Paralog。我们还将删除每个TSEL，并确定其在体内对弓形虫致病的影响。意义：通过这些研究，我们将阐明弓形虫对宿主线粒体的操纵如何决定疾病的严重程度和结局。这可能揭示了线粒体驱动的新的先天免疫途径，因为线粒体在抵抗弓形虫等寄生虫方面的作用在很大程度上是未知的。这项工作还应该确定弓形虫毒力效应因子，这可能是人类毒力的关键决定因素。广泛地说，这项工作还可能为基因座扩展如何推动任何病原体毒力的快速变化提供新的见解。

DESCRIPTION (provided by applicant): Toxoplasma gondii is an obligate intracellular parasite of humans that can be lethal in immunocompromised patients and the developing fetus. Virulence in T. gondii is driven by secretion of proteins directly into the host cell. The T. gondi secretome is vast and complex, and the majority of the putative secretory proteins are unannotated and their role in T. gondii pathogenesis in unknown. In contrast to T. gondii, Hammondia hammondi and Neospora caninum do not infect humans despite shared gene content, morphology, and life cycle features across these three species. The goal of this project is to identify previously uncharacterized virulence effectors unique to T. gondii. To achieve this goal we will integrate cross-species comparative genomics with functional genomic and forward genetic data to identify candidate effector loci, and then test candidate genes using molecular genetics and infections in vivo. Through whole genome comparisons, we have identified a subset of putative secretory proteins encoded by loci that are uniquely expanded and diversified in T. gondii. These Toxoplasma-Specific Expanded Loci (TSELs) are the focus of this proposal and their importance is confirmed by extensive preliminary data. Specifically, TSEL4 (mitochondrial association factor 1; MAF1) contributes to T. gondii virulence in vivo and is responsible for host mitochondrial association (HMA) with the parasite-containing vacuole. In Aim 1, we will follow up these observations by fully characterizing the MAF1 locus in terms of gene content, diversity, and impact on HMA during infection. We will use targeted deletion and X-ray crystallography to identify the functional domains of MAF1 and to determine its biochemical function. This Aim will be facilitated by our ongoing collaboration with Marty Boulanger (University of Victoria) for structural studies. In Aim 2, we quantify the effect of MAF1 paralog diversification and HMA on host mitochondrial biology. In parallel, we will determine the impact of the same MAF1 paralogs on parasite virulence across the entire T. gondii life cycle (including sporulated oocysts). Collaborations with Bennett Van Houten (University of Pittsburgh Cancer Center) for mitochondrial studies and JP Dubey (USDA) for oocyst production will facilitate this Aim. Finally, in Aim 3 we will characterize 6 additional TSEL identified in our comparative genomic screen. We will determine the extent of locus diversification for each TSEL, and localize individual paralogs in the parasite and host cell. We will also delete each TSEL and determine its impact on T. gondii pathogenesis in vivo. SIGNIFICANCE: Through these studies, we will elucidate how T. gondii manipulation of host mitochondria determines disease severity and outcome. This may reveal novel mitochondrially-driven innate immune pathways since the role of mitochondria in resistance to parasites like T. gondii is largely unknown. This work should also identify T. gondii virulence effectors that may be key virulence determinants in humans. Broadly, this work may also provide new insight into how locus expansion can drive rapid changes in the virulence of any pathogen.