Membrane microdomains are crucial forMycobacterium marinumEsxA-dependent membrane damage, escape to the cytosol and infection

Abstract

ABSTRACTInfection by pathogenic mycobacteria such asMycobacterium tuberculosisdisrupts the membrane of the Mycobacterium-Containing Vacuole (MCV). The key effector EsxA, secreted via the ESX-1 type-VII system, is pivotal in this process, yet its membranolytic activity is not fully elucidated. Infecting the amoebaDictyostelium discoideumwithMycobacterium marinum, we demonstrate that the composition of the MCV membrane, notably its sterol-rich microdomains, significantly influences damage and rupture. Disruption of these microdomains through the knockout of organizing proteins, termed vacuolins, or through sterol depletion, markedly diminishesM. marinum-induced membrane damage and cytosolic escape, thereby increasing cellular resistance to infection. Furthermore, we establish that vacuolins and sterols are essential for thein vitropartitioning of EsxA within membranes. Extending our findings to mammalian cells, we show that the role of microdomain organizers and sterols is evolutionarily conserved; specifically, flotillin knockdown and sterol depletion enhance the resistance of murine microglial cells toM. marinuminfection. Our results underscore the critical role of host membrane microdomains in facilitating mycobacterial membranolytic activity and subsequent cytosolic access, pivotal for a successful infection.