PDIM drives the transition from repairable to catastrophic EsxA-mediated vacuole membrane damage during Mycobacterium marinum infection

During infection, pathogenic mycobacteria damage the membrane of the Mycobacterium-containing vacuole (MCV), triggering host repair responses that preserve an intracellular permissive niche before bacteria escape to the cytosol at later stages. How the MCV transitions from repairable injury to catastrophic rupture remains poorly understood. Here, we dissected the respective contributions of the ESX-1 effectors EsxA/EsxB and the cell-envelope lipid phthiocerol dimycocerosate (PDIM) during Mycobacterium marinum infection of the amoeba Dictyostelium discoideum. Combining host reporters for membrane damage and repair with single and double bacterial mutants, we show that EsxA/EsxB and PDIM are both required for full intracellular virulence but act at distinct stages of MCV damage progression. Loss of EsxA/EsxB strongly reduced recruitment of ESCRT and autophagy reporters to the MCV, demonstrating that EsxA initiates repairable membrane lesions. In contrast, PDIM-deficient bacteria retained the ability to recruit early damage reporters and ESCRT machinery but showed reduced autophagy-associated repair, failed to efficiently acquire cytosolic perilipin coating, and remained largely confined within the MCV. Genetic disruption of host autophagy restored cytosolic access and intracellular growth of PDIM-deficient bacteria, indicating that PDIM is specifically required to overcome host repair capacity rather than to initiate damage. Importantly, the sequential requirement for EsxA and PDIM was conserved during infection of murine microglial BV-2 cells. Remarkably, PDIM-defective mutants induced lysenin recruitment, a reporter of sphingomyelin exposure, but progression to autophagy engagement was strongly decreased. Together, our results support a two-step model in which EsxA initiates MCV membrane damage, while PDIM amplifies these lesions towards catastrophic rupture, enabling escape to the cytosol and dissemination.