Biogenesis of the gram-negative bacterial cell envelope

PROJECT SUMMARY The outer membrane in Gram-negative bacteria is a dynamic interface that mediates bacterial interactions with the host by displaying proteins on its surface. The recently discovered surface- exposed lipoproteins (SLPs) play critical roles in pathogenesis, including iron and nutrient acquisition, adhesion, immune evasion, and serve as valuable vaccine targets. Despite their biomedical importance, the mechanism underlying lipoprotein localization to the cell surface is the least understood aspect of bacterial envelope biogenesis. The long-term goal of research in my laboratory is to define the mechanism of lipoprotein targeting and export to the bacterial cell surface. Improving our understanding of the molecular determinants for export will enable the development of predictive computational models for lipoprotein localization and genomic identification of SLPs. One SLP subfamily includes lipoproteins that depend on a partner β- barrel outer membrane protein (OMP) for surface exposure. We discovered the RcsF lipoprotein in Escherichia coli as the first example of this type. Furthermore, we discovered that the highly conserved and essential β-barrel assembly machinery (Bam) complex plays a critical role in the biogenesis of RcsF, uncovering the novel function of the Bam complex in lipoprotein biogenesis. Here, we propose to use an interdisciplinary approach to identify the molecular mechanism by which the Bam complex displays lipoproteins on the cell surface. For in-depth mechanistic investigations, we continue our work in E. coli, focusing on how the Bam complex recognizes SLPs and coordinates lipoprotein surface exposure with OMP assembly and the specific role of Bam lipoproteins in this process. However, we are also expanding the scope to include other gram-negative model systems to facilitate studies of conserved and variable aspects of envelope and SLP biogenesis, enriching our understanding of these processes across diverse species. Completing the proposed studies will substantially expand our understanding of the biogenesis of SLPs and, more broadly, Gram-negative cell envelope biogenesis. The knowledge gained from the proposed studies will enable the formulation of computational models for identifying novel SLPs and much-needed vaccine targets.