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

点击翻译按钮获取中文摘要

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.