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

Tuberculosis (TB), caused by Mycobacterium tuberculosis ( Mtb ), 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 Mtb biology during infection remains incomplete, limiting the development of new diagnostics, therapeutic and vaccine strategies. The Mtb cell envelope and its associated secretion systems are central to virulence and immune evasion. Mtb 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 Mtb mutants with altered EV production using a 96‐well filtration platform combined with a lipophilic dye or an Mtb ‐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 Mtb .