Saetra, R. S. R.; Hansen, M.; Kappelhoff, S.; Bugge, M.; Marstad, A.; Ryan, L.; Devant, P.; Kagan, J. C.; Freude, K. K.; Beckwith, K. S.; FLO, T. H.

Lytic cell death is recognized as a driver of tissue damage and dissemination during Mycobacterium tuberculosis (Mtb) infection, yet its execution mechanisms remain incompletely defined. Here, we identify Ninjurin-1 (NINJ1), a mediator of plasma membrane rupture (PMR), as a central effector of macrophage lysis in response to Mtb. Infection triggered NINJ1 oligomerization, and genetic or pharmacological inhibition of NINJ1 markedly reduced lactate dehydrogenase (LDH) release. Surprisingly, individual or combined inhibition of pyroptosis, apoptosis, necroptosis, or ferroptosis failed to prevent PMR in Mtb-infected macrophages. Instead, the ESX-1-secreted effector EsxA directly induced NINJ1 oligomerization, implicating pathogen-driven membrane damage as the upstream trigger. NINJ1 activation was independent of calcium or high-MW PEG, although PEG reduced LDH-release, implicating swelling-induced membrane changes in PMR. Cytokine release was mostly NINJ1-independent, except for CXCL-10, which was diminished in NINJ1-deficient macrophages. These findings establish NINJ1 as a key executioner of lytic cell death during Mtb-infection.