The Molecular Basis of Staphylococcus aureus SplB manipulation of the host

While bacteria broadly secrete multiple virulence factors during infection to modulate the host cell machinery for infection, S. aureus is unique in that most strains encode for a distinct protease family of up to six members, broadly referred to as serine proteases-like A-F (SplA-F). Spl family members do not respond to standard serine protease inhibitors, highlighting their unique active siteS. For example, the structures of Spl family members alone reveal such shallow active sites that they have been proposed to be reliant on conformational changes for target recognition. Furthermore, a novel conformational “switch” that allosterically couples the N-termini of Spl family members to their active sites has been proposed to underlie their zymogen activation {Pustelny, 2014 #3042}. Thus, conformational changes and allostery underlie Spl function through unknown mechanisms. Our lab has developed NMR-based methods to specifically identify the role of conformational changes and allostery in enzyme function. Based on our preliminary biological studies that have identified SplB as the most highly expressed Spl family member, we have begun identifying SplB host targets and applying NMR-based methods to elucidate its target interactions and underlying allosteric mechanism of activation. We hypothesize that S. aureus SplB targets multiple host proteins (Aim 1), which induce target- dependent conformational changes (Aim 2). Activation of the SplB zymogen via N-terminal cleavage leads to both global structural and dynamic changes that facilitate active site opening and target entry (Aim 3). This hypothesis will be tested through the following specific aims: Aim 1) Identify host protein targets of S. aureus SplB. Rational-based approaches together with unbiased methods will be used to broadly identify SplB host targets and their cleavage sites will be identified by mass spectrometry (MS). Target sequences will be biochemically validated by inserting them as linkers within a fusion protein. Aim 2) Elucidate the conformational changes required for SplB engagement with host targets. High- resolution NMR structural methods will be used to determine how SplB targets are engaged and whether there are target-specific conformational changes. Aim 3) Identify the allosteric mechanism of SplB activation and the role of dynamics in SplB function. High-resolution NMR structural methods will be used to determine the structural basis of SplB zymogen activation, i.e., the allosteric “switch,” while NMR relaxation methods will be used to determine the dynamic basis of activation and the role of dynamics in function. Project Number: 1R01AI189606-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: ELAN EISENMESSER | Institution: UNIVERSITY OF COLORADO DENVER, Aurora, CO | Award Amount: $390,000 | Activity Code: R01 | Study Section: Macromolecular Structure and Function A Study Section[MSFA] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R01AI18960601