Abstract
Tuberculosis remains a global health crisis due to the resilience of Mycobacterium tuberculosis ( Mtb ), largely attributed to its unique cell envelope. The impermeability and structural complexity of the outer membrane of this envelope, driven by mycolic acids and glycolipids, pose significant challenges for therapeutic intervention. Here, we present the first all-atom models of an Mtb outer membrane using molecular dynamics simulations. We demonstrate that α-mycolic acids adopt extended conformations to stabilize bilayers, with a phase transition near 338 K that underscores their thermal resilience. Lipids in the outer leaflet such as PDIM and PAT induce membrane heterogeneity, migrating to the interleaflet space and reducing lipid order. The simulated mycobacterial outer membrane has ordered inner leaflets and disordered outer leaflets, which contrasts with the outer membrane of Gram-negative bacteria. These findings reveal that PDIM- and PAT-driven lipid redistribution, reduced lipid order, and asymmetric fluidity gradients enable Mtb's outer membrane to resist host-derived stresses and limit antibiotic penetration, thereby promoting bacterial survival. Our work provides a foundational framework for targeting the mycobacterial outer membrane in future drug development. TEASER: Molecular dynamics simulations reveal the unique architecture of the mycobacterial outer membrane: a fluid outer leaflet and an ordered, tightly packed inner leaflet.