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

项目摘要/摘要 伯氏柯克斯体是哺乳动物的一种专性细胞内细菌病原体，是引起猪传染性支气管炎的病原体。 人畜共患病Q热。在进入哺乳动物细胞后，伯氏梭菌被动地通过 在引导建立一个宽敞的、由溶酶体衍生的细胞器之前， 柯克斯氏菌空泡(CCV)，允许细菌复制。CCV的生物发生过程和 成熟是由大约130个效应蛋白的集体活动推动的，这些蛋白是由 细菌点/ICM型IVB分泌系统在感染期间进入宿主细胞胞浆。尽管几年来 密集的研究，细胞靶标，生化活性，以及彼此之间的功能关系 伯氏梭菌编码的大部分Dot/ICM效应蛋白仍然未知。C.中的遗传操作 Burnetii，如靶向基因缺失突变体的产生，已被证明是一个具有挑战性的，漫长的， 以及效率极低的事业。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)平台有效沉默伯氏梭菌胞内效应基因的表达 复制。在目标1中，我们将构建一个全面的CRISPRi菌株文库，针对所有 预测分别编码布氏梭菌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……里面 目标2，我们将利用CRISPRi的强大优势，同时减少 同一菌株中的多个基因。其目标是开始梳理效应器之间的遗传交互作用。我们 将表达针对一组17个效应基因的sgRNA的成对组合，这些基因以前 被认为对CCV的生物发生和/或细胞内复制很重要。这些目标加在一起，将极大地 增强可用于研究伯氏梭菌细胞内复制和毒力基因的工具包 发病机制，并将开始阐明效应蛋白之间的上位关系，对 形成液泡状的壁龛。

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.