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.

项目概要/摘要 伯纳特柯克斯体 (Coxiella burnetii) 是哺乳动物的专性细胞内细菌病原体，也是该病的病原体。 人畜共患病Q热。进入哺乳动物细胞后，C. burnetii 被动地通过 内溶酶体途径，然后指导建立一个宽敞的、源自溶酶体的细胞器，称为 允许细菌复制的含有柯克斯体的液泡（CCV）。 CCV生物合成过程 成熟是由大约 130 种效应蛋白的集体活动驱动的，这些效应蛋白由 感染过程中，细菌 Dot/Icm 型 IVB 分泌系统进入宿主细胞胞质溶胶。尽管多年来 深入研究，细胞靶标、生化活性以及彼此之间的功能关系 对于伯氏念珠菌编码的大多数 Dot/Icm 效应蛋白仍然未知。 C. 基因操作 伯内特菌，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、 以及工作效率极低的情况。在这个项目中，我们建议开发一种可诱导且可逆的 CRISPR 干扰 (CRISPRi) 平台可有效沉默 C. burnetii 细胞内效应基因表达 复制。在目标 1 中，我们将构建一个针对所有基因的综合 CRISPRi 菌株库。 预测单独编码 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，同时降低 同一菌株中的多个基因。目标是开始梳理效应器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，这些基因之前已被 被认为对 CCV 生物发生和/或细胞内复制很重要。这些目标共同努力将极大地促进 增强可用于研究伯氏梭菌细胞内复制相关毒力基因的工具包， 发病机制，并将开始揭示对效应蛋白重要的上位关系 液泡生态位的形成。