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: 10686233
• 负责人: Maksym Bobrovskyy
• 金额: --
• 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 2023-08-06
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10686233
• 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; 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; Resolution; Rice; Risk Factors; Role; Scientist; Sepsis; Set protein; Site; Staphylococcus aureus; Streptococcus; Structural Biologist; Structural Protein; Structure; System; Testing; Thick; Tissues; Training; Translation Initiation; Translations; United States; Universities; Virulence; Virulence Factors; Work; antimicrobial; career; career development; cell envelope; crosslink; experience; high risk; in silico; insight; mutant; particle; pathogen; posttranscriptional; 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.

项目总结 金黄色葡萄球菌是一种普遍存在的病原体，会导致人类侵袭性疾病。7b型分泌物 金黄色葡萄球菌的系统(T7bSS)跨细菌被膜运输一组特定的因子，这是必需的 金黄色葡萄球菌在受感染宿主组织中的毒力和持久性。这些蛋白质的分泌依赖于 ESS操纵子的表达，该操纵子编码T7bSS结构蛋白，组装成运输复合体 在细菌的包膜内。T7bSS的表达和组装机制尚不清楚。Dr。 Bobrovsky的研究建立了T7bSS复合体的纯化方案，并鉴定了辅助基因 调节剂(AGR)和肽聚糖水解酶EssH作为支持T7b分泌所必需的因素。因此，总体上， 本方案的目的是揭示纯化的T7bSS的组成、化学计量比和组装 复合体(目标1)，确定肽聚糖水解酶EssH支持T7b分泌的机制(目标1) 2)，并阐明了AGR对T7bSS的调控(目标3)。总之，这些目标将检验一个压倒一切的假设 金黄色葡萄球菌的ESS基因座受AGR途径的调控，导致T7bSS复合体的组装 跨越包膜并允许底物转移。在目标1中，偶联的T7bSS络合物的纯化 通过单粒子电子冷冻显微镜(CRYO-EM)和交联质谱学将用于 研究使底物能够跨葡萄球菌细胞膜转位的结构成分。 在目标2中，将结合遗传和生化方法来研究 EssH对T7bSS复合体的组装和底物跨厚的肽聚糖细胞壁的转运。在……里面 目的3，葡萄球菌AGR途径的一个组成部分，转录后调节因子RNAIII的作用， 以及其他中间因素，将被检测为ESS基因调控。此外，拟议的培训 职业发展活动的目的是为博布罗夫斯基博士提供经验和工具， 让他在细菌生理和发病机制领域成功过渡到独立。这个 加州大学微生物学系的协作和跨学科研究环境 芝加哥，以及使用核心设施的最先进设备，如Advanced Electron 显微镜设备，非常适合候选人的提议。候选人的导师米西亚卡斯博士和 赵，将保证研究和培训目标的进展。米西亚卡斯博士是一位国际专家 葡萄球菌蛋白分泌领域的公认科学家，有指导学员的丰富历史， 他们中的许多人后来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体的冷冻-EM分析，并将为 这一领域的候选人。博布罗夫斯基博士还召集了一个咨询委员会，由菲比·赖斯博士、吉姆 Slauch和Sam Light，他们将帮助候选人进行研究和培训。总体而言，该奖项将使 博布罗夫斯基博士，以实现他的目标，并推动他的事业走向独立。