Thursday, January 30, 2025
1/30/2025
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.
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