The rising frequency of drug-resistant bacterial infections pose an imminent threat to human health. Attempts to generate novel antibiotics to address this problem often fail because the outer membrane (OM) of gram-negative bacteria acts as a barrier that blocks the entry of many small molecules. This OM barrier is densely packed with lipopolysaccharides (LPS) and outer membrane proteins (OMPs) that organize heterogeneously within the OM. Specifically, it has been demonstrated that OMPs form higher-order assemblies in the membrane termed ?islands?. The OM porins are trimeric and highly abundant OMPs but it is unknown how this oligomerization state influences their function, insertion into the OM, or the assembly of OMP islands. I hypothesize that OMP trimerization is critical for OMP function and fundamentally underlies the heterogenous organization of the OM. Using the model OMP, LamB, I have developed a set of tools that will allow me to examine this hypothesis. First, I will use bacterial genetic techniques to determine how OMP oligomeric state influences OMP function and partitioning of LPS. Second, I will use fluorescence microscopy techniques to characterize the role of OMP oligomerization in the OMP island assembly. Finally, I will determine how local secretion of OMPs affect OM organization. Together, my proposed work will define the molecular mechanisms that govern the organization of the OM. Importantly, this will inform strategies for disrupting the OM and in designing OM permeable antibiotics. The training goal of this fellowship is to continue to grow my scientific knowledge while further developing skills as a leader and mentor in order to fulfill my career goal of becoming a research group leader. Being a member of the Silhavy lab at Princeton University will allow me to develop a teaching philosophy and participate in the teaching and mentoring of undergraduate and graduate students. I will perfect my communication skills through ample opportunities to present my work at meetings and conferences. Additionally, the Molecular Biology Department at Princeton University is collaborative, interdisciplinary environment that promotes that development of ideas and sharing of expertise. Together, I believe that the completion of this training plan in the research environment of the Silhavy lab at Princeton University will allow me to develop the necessary skills to run my own research group.
The outer membrane of Gram-negative bacteria is an impermeable barrier that prevents the entry of large and hydrophobic molecules, effectively hindering the efficacy of available antibiotics. The studies proposed will characterize how outer membrane proteins assemble, function and organize in the E. coli outer membrane. A full understanding of how the outer membrane is organized and maintained will streamline antibiotic discovery and lead to sophisticated strategies to treating infections caused by Gram-negative bacteria.
