Staphylococcus aureus Type 7b Secretion System assembly and regulation

金黄色葡萄球菌 7b 型分泌系统的组装和调节

• 批准号: 10507394
• 负责人: Maksym Bobrovskyy
• 金额: $ 10.43万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-18 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10507394
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Abscess; Academia; Advisory Committees; Area; Award; Bacillus cereus; Bacterial Physiology; Base Pairing; Biochemical; Blood Circulation; Cell Density; Cell Wall; Chicago; Clinical; Communities; Complex; Core Facility; Coupled; Cryoelectron Microscopy; Defect; Development; Disease; Disease model; Electron Microscopy Facility; Elements; Environment; Equipment; Future; Gene Cluster; Gene Expression Regulation; Genes; Genetic; Genetic Screening; Genetic Transcription; Genus staphylococcus; Goals; Gram-Positive Bacteria; Growth; Human; Hydrolase; Immune response; Industry; Infection; Infectious Skin Diseases; Inflammatory; Interdisciplinary Study; International; Laboratories; Lesion; Light; Listeria monocytogenes; Mass Spectrum Analysis; Membrane; Membrane Proteins; Mentors; Messenger RNA; Microbiology; Molecular; Mus; N-Acetylmuramoyl-L-alanine Amidase; Operon; Osteomyelitis; Pathogenesis; Pathogenicity; Pathway interactions; Peptidoglycan; Phase; Phenocopy; Post-Transcriptional Regulation; Production; Protein Family; Protein Secretion; Proteins; Protocols documentation; RNA; RNAIII; Recording of previous events; Regulation; Regulator Genes; Research; Research Training; Resolution; Rice; Risk Factors; Role; Scientist; Sepsis; Set protein; Site; Staphylococcus aureus; Streptococcus; Structural Biologist; Structural Protein; Structure; System; Testing; Thick; Tissues; Training; Training Support; Translations; United States; Universities; Virulence; Virulence Factors; Work; antimicrobial; career; career development; cell envelope; crosslink; experience; high risk; in silico; insight; mutant; particle; pathogen; protein complex; protein protein interaction; quorum sensing; soft tissue; stem; stoichiometry; tool; training opportunity

PROJECT SUMMARY Staphylococcus aureus is a pervasive pathogen that causes invasive disease in humans. The type 7b secretion system (T7bSS) of S. aureus transports a specific set of factors across the bacterial envelope, which are required for S. aureus virulence and persistence in infected host tissues. Secretion of these proteins is dependent on the expression of the ess operon, which encodes T7bSS structural proteins that assemble into a transport complex within the bacterial envelope. The mechanisms of T7bSS expression and assembly are not well understood. Dr. Bobrovskyy's research established a purification protocol for the T7bSS complex and identified accessory gene regulator (Agr) and peptidoglycan hydrolase EssH as factors necessary to support T7b secretion. Thus, the overall objectives of this proposal are to reveal the composition, stoichiometry and assembly of the purified T7bSS complex (Aim 1), determine the mechanism whereby peptidoglycan hydrolase EssH supports T7b secretion (Aim 2), and elucidate T7bSS regulation by Agr (Aim 3). Together, these aims will test an overarching hypothesis that the ess locus of S. aureus is regulated by the Agr pathway, leading to the assembly of the T7bSS complex that spans the envelope and permits substrate translocation. In Aim 1, purification of the T7bSS complex coupled with single-particle electron cryomicroscopy (cryo-EM) and cross-linking mass spectrometry will be utilized to investigate the structural components that enable translocation of substrates across staphylococcal cell envelope. In Aim 2, a combination of genetic and biochemical approaches will be used to investigate the contribution of EssH to the assembly of T7bSS complex and substrate translocation across a thick peptidoglycan cell wall. In Aim 3, the role of the post-transcriptional regulator RNAIII, a component of the staphylococcal Agr pathway, and of other intermediate factors, will be examined for ess gene regulation. In addition, the proposed training and career development activities are intended to provide Dr. Bobrovskyy with the experience and tools that will allow him to successfully transition to independence in the field of bacterial physiology and pathogenesis. The collaborative and interdisciplinary research environment in the Department of Microbiology at the University of Chicago, and access to the state-of-the-art equipment at the core facilities, such as the Advanced Electron Microscopy Facility, are well suited for the candidate's proposal. The candidate's mentors Drs. Missiakas and Zhao, will assure the progress of the research and training objectives. Dr. Missiakas is an internationally recognized scientist in the field of staphylococcal protein secretion, with a strong history of mentoring trainees, many of whom went onto having careers in academia and industry. Dr. Zhao is a structural biologist who specializes in cryo-EM analysis of membrane protein complexes and will provide training and support to the candidate in this area. Dr. Bobrovskyy also assembled an advisory committee consisting of Drs. Phoebe Rice, Jim Slauch and Sam Light, who will assist the candidate in his research and training. Overall, this award will enable Dr. Bobrovskyy to attain his goals and propel his career towards independence.

项目概要 金黄色葡萄球菌是一种普遍存在的病原体，可引起人类侵袭性疾病。 7b型分泌 金黄色葡萄球菌系统（T7bSS）在细菌包膜上运输一组特定的因子，这是必需的 用于检测金黄色葡萄球菌在受感染宿主组织中的毒力和持久性。这些蛋白质的分泌取决于 ess 操纵子的表达，编码组装成转运复合物的 T7bSS 结构蛋白 在细菌包膜内。 T7bSS 表达和组装的机制尚不清楚。博士。 Bobrovskyy 的研究建立了 T7bSS 复合物的纯化方案并鉴定了辅助基因 调节因子 (Agr) 和肽聚糖水解酶 EssH 作为支持 T7b 分泌所必需的因子。因此，总体 该提案的目的是揭示纯化 T7bSS 的组成、化学计量和组装 复合物（目标 1），确定肽聚糖水解酶 EssH 支持 T7b 分泌的机制（目标 2)，并阐明 Agr 对 T7bSS 的调节（目标 3）。这些目标将共同检验一个总体假设： 金黄色葡萄球菌的 ess 基因座受 Agr 途径调节，导致 T7bSS 复合物的组装 跨越包膜并允许底物易位。在目标 1 中，T7bSS 复合物的纯化耦合 单粒子电子冷冻显微镜 (cryo-EM) 和交联质谱法将用于 研究使底物跨葡萄球菌细胞包膜易位的结构成分。 在目标 2 中，将结合使用遗传和生化方法来研究 EssH 参与 T7bSS 复合物的组装以及穿过厚肽聚糖细胞壁的底物易位。在 目标 3，转录后调节因子 RNAIII（葡萄球菌 Agr 途径的一个组成部分）的作用， 以及其他中间因子，将检查 ess 基因调控。此外，拟议的培训 和职业发展活动旨在为 Bobrovskyy 博士提供经验和工具，使他能够 让他成功过渡到细菌生理学和发病机制领域的独立。这 大学微生物学系的协作和跨学科研究环境 芝加哥，并可使用核心设施的最先进设备，例如 Advanced Electron 显微镜设施非常适合候选人的提案。候选人的导师博士。米西亚卡斯和 赵先生将保证研究和培训目标的进展。 Missiakas 博士是一位国际 葡萄球菌蛋白分泌领域公认的科学家，具有指导学员的悠久历史， 其中许多人继续在学术界和工业界从事职业生涯。赵博士是一位结构生物学家 专门从事膜蛋白复合物的冷冻电镜分析，并将为 该领域的候选人。 Bobrovskyy 博士还组建了一个由 Drs. 组成的咨询委员会。菲比·赖斯、吉姆 Slauch 和 Sam Light，他们将协助候选人进行研究和培训。总体而言，该奖项将使 Bobrovskyy 博士实现了他的目标并推动他的职业生涯走向独立。