Multi-disciplinary analysis of the role of PagC for outer membrane vesicle biogenesis

PROJECT SUMMARY/ABSTRACT Bacterial outer membrane vesicles (OMVs) are small, spherical protrusions composed of the inner and outer leaflets of the Gram-negative outer membrane. OMV diameters typically range from 20 to 200 nanometers (nm), although the mean size varies widely across species. Biogenesis of these vesicles requires induction of positive membrane curvature at a point of origin, after which expansion and subsequent scission of these vesicles proceed in an ATP-independent manner. Functionally, OMVs are primarily produced as response elements to environmental stresses, including large temperature shifts, pH perturbation, and divalent cation limitation. Despite numerous studies supporting the biological significance of OMVs, the underlying mechanisms of biogenesis remain elusive. In Salmonella enterica subsp Typhymurium (STm), recent evidence suggests that PagC, a product of the PhoPQ regulon, is sufficient to induce vesiculation when produced during low pH and divalent cation conditions. PagC is an eight-stranded ß-barrel that spans the outer membrane with four extracellular loops (EL1-4), two of which (EL2 and EL3) are reported as critical drivers of PagC-mediated OMV biogenesis. In vivo experiments guided by molecular dynamic simulations identified several key residues that may mediate a conformational change that induces positive membrane curvature required for OMV production. Accordingly, we hypothesize that PagC induces OMV biogenesis by introducing positive membrane curvature through conformational shifts in extracellular loops 2 and 3. Using an integrative approach of biophysical, biochemical, ultrastructural, and microbiological techniques, we will define the molecular underpinnings of PagC-mediated OMV biogenesis. As outlined in Aim 1, we will leverage solid-state nuclear magnetic resonance to monitor the dynamics of PagC in a native OMV environment. Using a combination of solid-state nuclear magnetic resonance and proteomics as described in Aim 2, we will define interactions of PagC with outer membrane components to clarify the physical requirements for enhancement of OMV production by PagC. Finally, we will use the proposed experimentation in Aim 3 to optimize studies for screening the phenotypic consequences of wild-type and mutant PagC isoforms. Taken together, our studies will utilize an integrative approach to describe the mechanisms of PagC-mediate. Although the extracellular loops of PagC appear to be unique to STm, we expect that the induction of membrane curvature through a protein conformational shift may provide a framework for studies in other bacteria. Given the challenges of high- throughput screening for key factors involved in OMV biogenesis, our studies may provide a basis for mining OMV proteomic studies to identify candidate outer membrane proteins that facilitate OMV production in other bacterial species.