Identification and characterisation of novel Toxoplasma gondii virulence factors using tailored CRISPR screens in vivo and in macrophages

弓形虫可以感染任何温血动物的任何有核细胞并在其中生长。而T.弓形虫主要被认为是一种机会性寄生虫，并且通常被宿主免疫系统有效地控制，存在引起人类和牲畜疾病的毒性菌株。寄生虫和宿主之间的相互作用是毒力因子和宿主免疫反应的良好平衡。最近的研究揭示了200多种T.弓形虫进入宿主细胞，其中绝大多数具有未知的功能。我的目的是确定所有分泌的毒力因子在体内生长的小鼠在四个遗传不同的寄生虫株。作为一种补充方法，我将确定巨噬细胞中寄生虫生存所必需的分泌因子，因为这些被认为是T细胞中最相关的免疫细胞群。弓形虫感染先前关于毒力因子的研究需要敲除单个寄生虫基因的小鼠感染，而CRISPR-Cas9技术被用于许多模式生物的高通量遗传筛选，包括T.体外系统中的弓形虫。Treeck实验室最近优化了该方案，以进行具有潜在任何寄生虫遗传背景的靶向筛选，并在体内进行这些菌株的感染。除了鉴定分泌的毒力因子外，我将选择其中的两个，并使用细胞生物学、生物化学和免疫学方法详细研究它们的功能。总之，这些实验将揭示小鼠宿主中毒力因子的全部库以及巨噬细胞中寄生虫存活所必需的毒力因子。使用不同遗传背景的寄生虫将导致对T.弓形虫毒力通过对候选人的详细调查，我将了解T。弓形虫干扰小鼠的免疫反应，也揭示了这种流行的病原体及其宿主的共同进化。

Toxoplasma gondii can infect and grow in virtually any nucleated cell of any warm-blooded animal. While T. gondii is mainly regarded as an opportunistic parasite and is often efficiently controlled by the host immune system, virulent strains exist which cause disease in humans and livestock. The interplay between the parasite and the host is a fine balance of virulence factors and host immune response. Recent studies have revealed over 200 putative secreted factors by T. gondii into the host cell, of which the vast majority have an unknown function. I aim to identify all secreted virulence factors important for in vivo growth in mice in four genetically different parasite strains. As a complementary approach I will identify the secreted factors essential for parasite survival in macrophages, since these are regarded as the most relevant immune cell population during T. gondii infection. Previous studies on virulence factors required murine infections with individual parasite genes knocked out whereas the CRISPR-Cas9 technique was adopted in high throughput genetic screenings in many model organisms, including T. gondii in in vitro systems. The Treeck laboratory has recently optimized this protocol to perform targeted screens with potentially any parasite genetic background and to perform infections with those strains in vivo. Apart from identifying secreted virulence factors, I will select two of them and investigate their function in detail using cell biological, biochemical and immunological methods. Together these experiments will uncover the full repertoire of virulence factors in murine hosts as well as those necessary for parasite survival in macrophages. The use of parasites with different genetic backgrounds will lead to a better overall understanding of T. gondii virulence. By investigating candidates in detail, I will learn novel features of how T. gondii interferes with the mouse immune response, also shedding some light on the co-evolution of this prevalent pathogen and its host.