Loss of <i>Ag85A</i> disrupts plasma membrane domains and promotes free mycolic acid accumulation in mycobacteria

Summary The mycomembrane of mycobacteria, composed primarily of long-chain mycolic acids, is critical for cell survival, structural integrity, and resistance to environmental stress, yet its underlying synthesis mechanisms remain incompletely understood. This study investigates the role of Ag85A, a key enzyme in mycomembrane synthesis, in regulating plasma membrane domains and cell envelope organization in Mycobacterium smegmatis . Using Δ Ag85A deletion mutants, we combined microscopy, biochemical assays, thin-layer chromatography, and lipid analysis to evaluate changes in membrane structure, chemical accumulation, and lipid composition. Ag85A deletion leads to altered plasma membrane domain organization, increased chemical accumulation, changes in cell envelope lipid composition. Unexpectedly, lipid analysis revealed accumulation—not depletion—of mycolic acids in the mutant, suggesting that increased permeability is not directly due to mycolic acid loss. These findings highlight a novel link between mycomembrane composition and plasma membrane domain stability. Our study not only advances understanding of mycobacterial cell envelope architecture but also identifies potential targets for enhancing drug penetration in resistant mycobacterial infections. Significance The unique cell envelope of mycobacteria is central to survival, enabling it to resist immune defenses and antibiotic treatment. In this study, we reveal a novel function of Ag85A, a synthase of outmost layer of mycobacteria beyond its known role in mycolyltransferase activity: it is essential for the formation of plasma membrane domains that orchestrate cell envelope synthesis. By structuring plasma membrane domains, Ag85A contributes directly to the resilience of the cell envelope, reinforcing mycobacterial survival mechanisms under hostile conditions. Our findings provide a pivotal insight into mycobacterial cell biology, with broad implications for therapeutic development. Disrupting Ag85A-dependent membrane domain formation could weaken the protective cell envelope, offering a promising approach to enhance current treatments.