Membrane microdomains are crucial for Mycobacterium marinum EsxA-dependent membrane damage, escape to the cytosol, and infection.

Mycobacterium tuberculosis disrupts the membrane of the Mycobacterium-containing vacuole (MCV) via its EsxA virulence factor, secreted via the ESX-1 system. We identified how pathogenic Mycobacterium marinum exploits host sterol-rich membrane microdomains to induce MCV damage during infection of Dictyostelium discoideum and murine microglial BV-2 cells. Transcriptomic and protein analyses revealed that vacuolinC is specifically induced in response to EsxA-mediated damage. Vacuolins initially associate with the MCV in a patchy distribution, coinciding with sterol-rich microdomain formation before entirely coating the MCV. Functional assays demonstrate that membrane microdomains potentiate l-leucyl-l-leucine methyl ester and EsxA-mediated membrane damage. Knockout of vacuolins and sterol depletion drastically reduce EsxA partitioning into membranes in vitro and decrease MCV damage and Mm escape to the cytosol, thereby substantially impairing Mm intracellular growth and phenocopying in Dd and microglial cells the attenuation of the Mm ΔRD1 mutant lacking ESX-1. Our results highlight host membrane microdomains as critical platforms exploited by virulent mycobacteria across evolutionary distant host phagocytes, thus representing potential therapeutic targets.