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.

项目摘要 金黄色葡萄球菌（Staphylococcus aureus）是一种普遍存在的病原体，可导致人类的侵袭性疾病。7 b型分泌物 系统（T7 bSS）。金黄色葡萄球菌将一组特定的因子穿过细菌包膜，这是必需的。 对于鼠伤寒沙门氏金黄色葡萄球菌的毒力和持久性在感染的宿主组织。这些蛋白质的分泌依赖于 ess操纵子的表达，其编码T7 bSS结构蛋白，所述结构蛋白组装成转运复合物 在细菌的包膜内。T7 bSS的表达和组装机制还不清楚。博士 Bobrovskyy的研究建立了T7 bSS复合物的纯化方案并鉴定了辅助基因 调节子（Agr）和肽聚糖水解酶β-D-半乳糖苷酶H作为支持T7 b分泌所必需的因子。因此， 本提案的目的是揭示纯化的T7 bSS的组成、化学计量和组装 复合物（Aim 1），确定肽聚糖水解酶β H支持T7 b分泌的机制（Aim 2），阐明Agr对T7 bSS的调控作用（目的3）。总之，这些目标将检验一个总体假设， ess基因座。金黄色葡萄球菌受Agr途径调节，导致T7 bSS复合物的组装， 跨越包膜并允许底物移位。在目的1中，纯化T7 bSS复合物偶联 单粒子电子低温显微镜（cryo-EM）和交联质谱法将用于 研究能够使底物穿过葡萄球菌细胞包膜移位的结构组分。 在目标2中，将使用遗传和生物化学方法的组合来研究以下因素的贡献： T7 bSS复合物的组装和底物穿过厚肽聚糖细胞壁的易位。在 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， 和其他中间因子的作用，将被检测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， 使他能够成功地过渡到细菌生理学和致病机理领域的独立性。的 合作和跨学科的研究环境，在微生物学系在大学 芝加哥，并获得最先进的设备在核心设施，如先进的电子 显微镜设施，非常适合候选人的建议。候选人的导师Missiakas博士和 赵先生将确保研究和培训目标的进展。米萨卡斯博士是国际上 葡萄球菌蛋白分泌领域公认的科学家，有很强的指导学员的历史， 他们中的许多人后来在学术界和工业界有了自己的事业。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EM分析，并将为 候选人在这个领域。Bobrovskyy博士还组建了一个咨询委员会，由Phoebe Rice博士，Jim 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 博士Bobrovskyy实现他的目标，并推动他的职业生涯走向独立。