Membrane microdomains are crucial for Mycobacterium marinum EsxA-dependent damage and cytosol escape

Infection by pathogenic mycobacteria such as Mycobacterium tuberculosis disrupts the membrane of the Mycobacterium-Containing Vacuole (MCV) via its EsxA virulence factor. Here, we have identified how pathogenic Mycobacteriun marinum exploits host sterol-rich membrane microdomains to induce MCV damage during infection in Dictyostelium discoideum and mammalian microglial BV-2 cells.  Transcriptomic and protein analyses revealed that the vacuolin isoform VacC is specifically and strongly induced in response to Mm infection, correlating with bacterial pathogenicity and EsxA-mediated damage. Vacuolins initially associate with the MCV in a patchy distribution, coinciding with sterol-rich microdomain formation and ultimately entirely coating the vacuole. Functional assays demonstrate that sterols and vacuolins significantly potentiate both chemical (LLOMe) and EsxA-mediated membrane damaging activity. Knock-out of vacuolins and sterol depletion with methyl-β-cyclodextrin drastically reduce EsxA partitioning into membranes in vitro, decrease MCV damage and Mm escape to the cytosol, thereby significantly impairing Mm intracellular growth by phenocopying the attenuation of the DRD1 mutant. Similar mechanisms involving flotillin microdomains facilitate Mm infection in mammalian cells, emphasizing conserved infection mechanisms across evolutionarily distant host phagocytes. Our results highlight host membrane microdomains as critical platforms exploited by virulent mycobacteria, thus representing potential therapeutic targets against mycobacterial diseases.