Defining epistatic interactions among Coxiella burnetii effector proteins using a CRISPRi approach

项目概要/摘要 贝氏柯克斯体（Coxiella burnetii）是哺乳动物的专性胞内细菌病原体，并且是贝氏柯克斯体的病原体。 人畜共患病Q热。进入哺乳动物细胞后，C.伯内特虫被动地通过 在指导建立一个宽敞的，溶酶体衍生的细胞器之前， 允许细菌复制的含柯克斯体的空泡（CCV）。CCV的生物发生过程和 成熟是由大约130种效应蛋白的集体活性驱动的，这些效应蛋白被细胞膜转位， 感染时，细菌Dot/Icm型IVB分泌系统进入宿主细胞胞浆。尽管几年来 紧张的研究，细胞的目标，生化活动，以及相互之间的功能关系 对于大多数由C.伯内特氏菌在C. Burnetii，如靶基因缺失突变体的产生，已经证明是一个具有挑战性的，漫长的， 而且效率极低在这个项目中，我们建议开发一种可诱导和可逆的CRISPR。 干扰（CRISPRi）平台，以有效地沉默C。贝氏效应基因在细胞内表达 复制的在目标1中，我们将构建一个全面的CRISPRi菌株文库，靶向所有基因， 预测编码C。Burnetii Dot/Icm效应蛋白。这种有针对性的方法将使我们能够 评估单个效应蛋白对液泡生物发生和细胞内复制的贡献。在 目标2，我们将利用CRISPRi的强大优势，同时减少 在同一菌株中有多个基因。目标是开始梳理效应子之间的遗传相互作用。我们 将表达靶向一组17个效应基因的sgRNA的成对组合，这些效应基因先前已经被 被鉴定为对CCV生物发生和/或细胞内复制重要。这些目标将大大 增强了用于研究C.贝氏细胞内复制和 发病机制，并将开始阐明效应蛋白之间的上位关系，重要的是， 液泡龛的形成。

Project Summary/Abstract Coxiella burnetii is an obligate intracellular bacterial pathogen of mammals, and the causative agent of the zoonotic disease Q fever. Upon entry into mammalian cells, C. burnetii passively traffics through the endolysosomal pathway before directing the establishment of a spacious, lysosome-derived organelle called the Coxiella-containing vacuole (CCV) that allows bacterial replication. The process of CCV biogenesis and maturation is driven by the collective activity of the roughly 130 effector proteins that are translocated by the bacterial Dot/Icm type IVB secretion system into the host cell cytosol during infection. Despite several years of intense research, the cellular targets, biochemical activities, and functional relationships among each other remain unknown for most of the Dot/Icm effector proteins encoded by C. burnetii. Genetic manipulations in C. burnetii, such as the generation of targeted gene deletion mutants, have proven to be a challenging, lengthy, and highly inefficient undertaking. In this project we propose to develop an inducible and reversible CRISPR interference (CRISPRi) platform to efficiently silence C. burnetii effector gene expression during intracellular replication. In Aim 1, we will construct a comprehensive CRISPRi strain library targeting all genes that are predicted to encode C. burnetii Dot/Icm effector proteins, individually. This targeted approach will allow us to assess the contributions of individual effector proteins to vacuole biogenesis and intracellular replication. In Aim 2, we will leverage the powerful advantage of CRISPRi to simultaneously reduce the expression of multiple genes in the same strain. The goal is to begin teasing apart genetic interactions between effectors. We will express pairwise combinations of sgRNAs targeting a set of 17 effector genes that have previously been identified as important for CCV biogenesis and/or intracellular replication. Together, these aims will greatly enhance the toolkit available for studying virulence genes involved in C. burnetii intracellular replication and pathogenesis, and will start to shed light on epistatic relationships among effector proteins important for the formation of the vacuolar niche.