Identification of novel Toxoplasma genes involved in host-parasite interactions

 描述(申请人提供)：复合体顶体是人类最常见的寄生虫之一，通过疟疾、弓形虫病和隐孢子虫病等疾病导致大量死亡和发病率。作为专性的细胞内寄生虫，它们与宿主建立了亲密的相互作用。弓形虫是这种适应的一个极端例子，能够在任何温血宿主的几乎每种细胞类型中复制。将需要基因组规模的分析来系统地探索宿主-寄生虫相互作用的遗传学。我们提出了使用CRISPR/Cas9来靶向寄生虫中所有预测的蛋白质编码基因的初步证据。这一进展使得能够快速和经济有效地评估每个寄生虫基因在不同条件下对生长的相对贡献。使用这个平台，我们可以在遗传多样性的宿主中识别对寄生虫生存至关重要的基因。在来自不同品系的小鼠细胞系中观察到的可变寄生虫生长，表明弓形虫和小鼠之间的共同进化是显而易见的。因此，我们建议测量宿主变异对成纤维细胞最佳生长所需的寄生虫基因的影响。为了最大限度地利用可用于下游应用的资源，我们将重点研究两个重组近交系(RI)的创始菌株，合作杂交(CC)和BXD家族，这两个家族总共占实验室小鼠遗传多样性的90%以上。这项研究的第一个目标是将我们的CRISPR/Cas9介导的全基因组分析扩展到来自九个近交系小鼠品系的细胞系。这一目标将定义两组寄生虫基因：一组对不同宿主之间的寄生虫很重要，另一组只对某些小鼠品系的生长有贡献。第二个目标将利用CRISPR/CAS9、自动显微镜和图像分析的效率，检查与破坏特定寄生虫基因相关的表型。在我们前期工作的基础上，我们选择了14个基因进行研究，这些基因对人成纤维细胞的适合性有很大的贡献，在顶端复合体中保守，并被预测可以分泌。对这些重要寄生虫因子的研究将扩大我们对宿主-寄生虫相互作用的理解，并建立高通量寄生虫基因表型的方法。这些方法将对研究第一个目的中确定的其他相关基因的表型有价值。这项拟议的工作将是第一次在自然寄主变异的背景下对顶端复合体基因组进行系统分析。定义广泛或特定影响感染的基因类别将有助于理解寄生虫的细胞学基础。

 DESCRIPTION (provided by applicant): Apicomplexans are among the most common parasites of humans, accounting for significant mortality and morbidity through diseases like malaria, toxoplasmosis and cryptosporidiosis. As obligate intracellular parasites, they establish intimate interactions with their hosts. Toxoplasma gondii is an extreme example of this adaptation, able to replicate within nearly every cell type in any warm-blooded host. Genome-scale analyses will be needed to systematically explore the genetics of host-parasite interactions. We present preliminary evidence for the use of CRISPR/Cas9 to target all predicted protein-coding genes in the parasite. This advance enables rapid and cost-effective assessment of the relative contribution of each parasite gene to growth under different conditions. Using this platform we can identify genes important for parasite survival in genetically diverse hosts. Coevolution between T. gondii and mice is evident in the variable parasite growth observed in cell lines derived from different mouse strains. We therefore propose to measure the effect of host variation on the parasite genes required for optimal growth in fibroblasts. To maximize the resources available to downstream applications we will focus our study on the founder strains for two recombinant inbred (RI) families, the Collaborative Cross (CC) and the BXD family, which together account for more than 90% of the genetic diversity in laboratory mice. The first aim of this study will extend our CRISPR/Cas9-mediated genome-wide analysis to cell lines derived from the nine inbred mouse strains. This aim will define two sets of parasite genes: those important for parasitism across different hosts, and those that only contribute to growth in certain mouse strains. The second aim will examine the phenotypes associated with disrupting specific parasite genes, leveraging the efficiency of CRISPR/Cas9, automated microscopy, and image analysis. Based on our preliminary work, we have selected 14 genes to study, which strongly contribute to fitness in human fibroblasts, are conserved among apicomplexans, and are predicted to be secreted. Study of these important parasite factors will extend our understanding of host-parasite interactions, and establish the methods for high-throughput phenotyping of parasite genes. These methods will be valuable to investigate the phenotypes of other relevant genes identified in the first aim. The proposed work will be the first systematic analysis of the apicomplexan genome in the context of natural host variation. Defining the categories of genes that broadly or specifically affect infection will helpus understand the cellular basis of parasitism.