Cell Envelope Remodeling and Lipid Metabolism Coordinate Extracellular Vesicle Output in Mycobacterium Tuberculosis

Tuberculosis (TB), caused by <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>), remains a leading global cause of death due to the pathogen's highly adaptive physiology and its ability to manipulate host immunity. Despite extensive study, our understanding of <i>Mtb</i> biology during infection remains incomplete, limiting the development of new diagnostics, therapeutic and vaccine strategies. The <i>Mtb</i> cell envelope and its associated secretion systems are central to virulence and immune evasion. <i>Mtb</i> releases extracellular vesicles (EVs) that carry immunomodulatory molecules, including lipoproteins, which influence immune responses. Although individual genes and environmental cues have been shown to alter vesiculogenesis, a genome-wide dissection of EV biogenesis has not been attempted. Here, we establish a high-throughput screening system to identify <i>Mtb</i> mutants with altered EV production using a 96-well filtration platform combined with a lipophilic dye or an <i>Mtb</i>-EV (MEV)-specific antibody. Our genome-wide screen revealed genes in cell envelope remodeling and fatty acid metabolism are major regulators of vesiculogenesis. These findings provide a robust platform for defining the molecular determinants of MEV production and lay the groundwork for mechanistic characterization of vesicle biogenesis in <i>Mtb</i>.