Cleansing the cytosol: Most human cells, not just those belonging to the immune system, mount protective responses to infection when activated by the immune cytokine interferon-gamma (IFN-γ). How IFN-γ confers this function in nonimmune cells and tissues is poorly understood. Gaudet et al. used genome-scale CRISPR/Cas9 gene editing to identify apolipoprotein L-3 (APOL3) as an IFN-γ–induced bactericidal protein that protects human epithelium, endothelium, and fibroblasts against infection (see the Perspective by Nathan). APOL3 directly targets bacteria in the host cell cytosol and kills them by dissolving their anionic membranes into lipoprotein complexes. This work reveals a detergent-like mechanism enlisted during human cell-autonomous immunity to combat intracellular pathogens. Science , abf8113, this issue p. eabf8113 ; see also abj5637, p. 276

Lysosomal damage is an endogenous danger signal, but its significance for innate immunity and the specific signaling pathways it engages remain unclear. Here, we uncover an immune-inducible pathway that connects lysosomal damage to mitochondrial DNA (mtDNA) efflux and type I IFN production. We find that transient lysosomal damage elicits sub-lethal mitochondrial outer membrane permeabilization (MOMP) via BAK/BAX macropores; however, the inner mitochondrial membrane (IMM) maintains a barrier against wholesale mtDNA release. Priming with type II IFN (IFN-γ) induced the antibacterial factor APOL3, which, upon sensing lysosomal damage, targets mitochondria undergoing MOMP to selectively permeabilize the IMM, enhance mtDNA release, and potentiate downstream cGAS signaling. Biochemical and cellular reconstitution revealed that, analogous to its bactericidal detergent-like mechanism, APOL3 permeabilized the IMM by solubilizing cardiolipin. Our findings illustrate how cells enlist an antibacterial protein to expedite the breakdown of endosymbiosis and facilitate a heightened response to injury and infection.

Actin is integral to eukaryotic physiology as a biomechanical polymer and as a structural barrier for cell-autonomous defense against infection. Some microbial pathogens exploit the actin cytoskeleton, however, to evade cell-autonomous immunity. Subversion of actin to enter host cells and for actin-based motility are often employed by intracellular pathogens to spread from cell-to-cell. Using RNA-sequencing and computational data mining, we identify the host actin-binding protein XIRP1 as commonly induced during infection. XIRP1 is expressed by fibroblasts and macrophages in response to immune cytokines such as interferon-gamma (IFN-γ) and infection with bacteria such as Listeria, Shigella, and Salmonella . Confocal and super-resolution structured illumination microscopy (SIM) found XIRP1 localizes to fibroblast focal adhesions and macrophages podosomes. Within human macrophages, XIRP1 is recruited to cytosolic Listeria monocytogenes in an ActA-dependent manner as it replicates and uses actin-based motility for host cell escape. Chromosomal removal of XIRP1 in mice impaired this dissemination and rendered them more resistant to Listeria infection than C57BL/6NJ wildtype controls in vivo . We propose that professional cytosolic pathogens like Listeria can co-opt XIRP1 to escape the hostile intracellular environment of IFN-γ-activated macrophages as part of the host-pathogen arms race during cell-autonomous immunity.

SUMMARY: Lysosomal damage is an endogenous danger signal to the cell, but its significance for innate immunity and how specific signaling pathways are engaged by this stressor remain unclear. Here, we uncover an immune-inducible pathway that connects lysosomal damage to mitochondrial DNA (mtDNA) efflux and type I IFN production. Lysosomal damage elicits mitochondrial outer membrane permeabilization (MOMP) via BAK/BAX macropores; however, the inner mitochondrial membrane (IMM) prevents wholesale mtDNA release in resting cells. Priming with type II IFN (IFN-γ) induced the antibacterial effector apolipoprotein L-3 (APOL3), which upon transient lysosomal damage, targets mitochondria undergoing MOMP and selectively permeabilizes the IMM to enhance mtDNA release and activate cGAS/STING signaling. Biochemical and cellular reconstitution revealed that analogous to its bactericidal detergent-like mechanism, APOL3 solubilizes cardiolipin to permeabilize the IMM. Our findings illustrate how cells use an antibacterial protein to expedite the breakdown of endosymbiosis and facilitate a heightened response to injury and infection.