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

刚地弓形虫几乎可以在任何温血动物的任何有核细胞中感染和生长。虽然弓形虫主要被认为是一种机会性寄生虫，通常受到宿主免疫系统的有效控制，但也存在导致人类和牲畜患病的毒性菌株。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。最近的研究发现，弓形虫向宿主细胞分泌的因子超过200种，其中绝大多数具有未知的功能。我的目的是确定所有分泌毒力因子重要的在体内生长的小鼠在四种遗传不同的寄生虫菌株。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，因为这些被认为是弓形虫感染期间最相关的免疫细胞群。先前的毒力因子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，包括体外系统中的弓形虫。Treeck实验室最近对该方案进行了优化，以便对任何潜在的寄生虫遗传背景进行靶向筛选，并在体内对这些菌株进行感染。除了确定分泌的毒力因子外，我将选择其中的两个，用细胞生物学、生化和免疫学的方法详细研究它们的功能。总之，这些实验将揭示小鼠宿主的全部毒力因子，以及寄生虫在巨噬细胞中生存所必需的毒力因子。使用具有不同遗传背景的寄生虫将有助于更好地全面了解弓形虫的毒力。通过对候选病毒的详细调查，我将了解弓形虫如何干扰小鼠免疫反应的新特征，同时也揭示了这种流行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.