Biophysical investigation of bacterial lipid trafficking proteins for novel antibiotic development

About This Grant

Project Summary/Abstract Gram-negative bacteria, like Escherichia coli, greatly contribute to the ongoing crisis of antimicrobial-resistant infections, which impact millions annually. These bacteria have a unique cellular architecture as they are surrounded by a double membrane barrier. The efficient flux of lipids between these membranes is required for bacterial growth and survival; two conserved Mammalian Cell Entry (MCE) protein family members have been implicated in facilitating this translocation. This work will investigate the functional mechanisms of these MCE- proteins, as they are unrealized targets of novel antibiotic development. We will utilize an in vitro reconstitution assay to reconstruct the lipid-trafficking function of these proteins using synthetic membrane mimetics to better understand substrate specificity, lipid translocation rates, and how inclusion of MCE-protein binding partners affects activity (Specific Aim 1). These experiments will be complemented by a thorough investigation of MCE- protein membrane binding, which will be interrogated using a novel microscopy-based assay (Specific Aim 2). Together, these aims will identify integral protein regions for function, along with critical protein-protein and membrane-protein interfaces for potential drug targeting. Finally, we will construct the first computational model of MCE-mediated lipid trafficking and validate this model extensively with reproduction of genetic experiments (Specific Aim 3). This model will be fundamental in understanding how newly designed drugs will affect the accumulation and depletion of lipids under various conditions and will predict how to best target lipid trafficking for bacterial death. These aims will be primarily completed by undergraduate student-scientists, directly providing them with transformative independent research experiences which will further their technical, analytical, and communication skills in preparation for their future careers in the biomedical sciences. This work seamlessly combines biophysical characterization of lipid trafficking proteins with computational simulations to provide a three-fold approach for the identification of novel antibiotic targets. Accordingly, these aims directly address the voids in our current understanding of the mechanisms of bacterial lipid trafficking and the role of MCE-proteins in this critically important process, while providing several unique avenues for antibiotic drug development.