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

使用 CRISPRi 方法定义伯氏柯克斯体效应蛋白之间的上位相互作用

• 批准号: 10509592
• 负责人: Craig R. Roy
• 金额: $ 25.13万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10509592
• 关键词: Animal Model; Architecture; Bacteria; Biochemical; Biogenesis; Biological; Biological Assay; Biology; CRISPR interference; CRISPR library; Cell membrane; Cell physiology; Cells; Communicable Diseases; Coxiella; Coxiella burnetii; Cytosol; Development; Disease; Gene Deletion; Gene Expression; Gene Silencing; Generations; Genes; Genetic; Goals; Health; Human; Individual; Infection; Knowledge; Libraries; Light; Lysosomes; Mammalian Cell; Mammals; Molecular; Mutagenesis; Organelles; Pathogenesis; Pathway interactions; Phenotype; Plasmids; Play; Process; Proteins; Public Health; Q Fever; Research; Research Project Grants; Role; System; Testing; Toxic effect; Vacuole; Virulence; Zoonoses; base; cellular targeting; exhaustion; genetic approach; genetic manipulation; insight; knock-down; mutant; novel; novel therapeutics; novel vaccines; pathogen; pathogenic bacteria; protein function; tool

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.

项目摘要/摘要 Coxiella burnetii是哺乳动物的强制性细胞细菌病原体，是该病原体的原因 人畜共患病Q发烧。进入哺乳动物细胞后，伯内特（C. burnetii）被动地通过 在指示建立一个宽敞的溶酶体衍生的细胞器之前 允许细菌复制的含紫胶液泡（CCV）。 CCV生物发生和 成熟是由大约130个效应子蛋白的集体活动驱动的 在感染过程中，细菌点/ICM型IVB分泌系统进入宿主细胞胞质。尽管有几年 激烈的研究，细胞靶标，生化活动和彼此之间的功能关系 对于由C. burnetii编码的大多数DOT/ICM效应蛋白仍然未知。 C. 伯内蒂（Burnetii），例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，漫长的 和高效的工作。在这个项目中，我们建议开发可诱导和可逆的CRISPR 干涉（CRISPRI）平台有效地沉默C. burnetii效应子基因表达在细胞内表达 复制。在AIM 1中，我们将构建一个针对所有基因的综合Crispri菌株库 预测将分别编码C. burnetii Dot/ICM效应子蛋白。这种目标方法将使我们能够 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 AIM 2，我们将利用Crispri的强大优势同时减少 在同一菌株中的多个基因。目的是开始取笑效应子之间的遗传相互作用。我们 将表达针对一组17个效应基因的SGRNA的成对组合，这些基因以前是 确定对CCV生物发生和/或细胞内复制很重要。在一起，这些目标将大大 增强可用于研究参与细胞内复制梭菌的毒力基因的工具包 发病机理，并将开始阐明对效应蛋白之间对 液泡生态位的形成。