Discovering a multi-protein lipid export system in Mycobacteria

About This Grant

ABSTRACT A fundamental gap in our understanding is how mycobacteria, including the important pathogen M. tuberculosis, export lipids from the inner membrane across the aqueous periplasm to the outer membrane. The mycobacterial outer membrane contains a uniquely complex array of lipids that are essential for their resilience, antibiotic resistance, and virulence. In Gram-negative bacteria, lipid transport is accomplished by protein complexes that span the cell envelope, but no such export machinery has been identified in mycobacteria. This study introduces MELO (Mycobacterial Exporter of Lipids to the Outer membrane), a novel protein complex that mediates lipid export. We will implement new genetic and proteomic approaches to identify MELO and use functional assays in M. smegmatis and M. tuberculosis to test its role in lipid transport. We have discovered that MELO mutants are resistant to the phage Bxz1, providing a unique selective pressure enabling a novel genetic screen to search for MELO mutants in M. smegmatis. We will then map the MELO protein interaction network using an affinity purification-crosslinking mass spectrometry (AP-XL-MS) approach we have developed. Piloting this approach has already identified three candidate MELO components. Based on this preliminary data, we will functionally test the role of MELO components in lipid transport in the pathogenic M. tuberculosis. Successful completion of this project will identify MELO, a new system critical for outer membrane biogenesis in mycobacteria with broad implications for our understanding of bacterial cell envelope biology.