LAUSR

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 Interferon-induced APOL3 is a detergent-like protein that kills cytosolic pathogens to protect human cells against infection. INTRODUCTION In the arms race between pathogen and host, infecting microbes often escape extracellular defense mechanisms to exploit the nutrient-rich intracellular environment as a replicative niche. In humans, this is countered by the interferon-γ (IFN-γ) response, which confers widespread pathogen resistance in most nucleated cells through the transcriptional induction of hundreds of interferon-stimulated genes (ISGs) encoding putative antimicrobial restriction factors. Remarkably, despite the importance of IFN-γ against all taxonomic classes of intracellular pathogens, many restriction factors elicited by this cytokine remain to be characterized, as do their molecular activities. RATIONALE Identified as the major human macrophage-activating cytokine in 1983, IFN-γ in fact transcriptionally reprograms numerous host cell types to eliminate infection. This includes nonimmune epithelial cell populations, which lack many traditional phagocytic defenses ascribed to IFN-γ stimulation, yet still manage to mount protective cell-autonomous immune responses. To find ISG effectors involved in safeguarding mucosal and barrier tissue types, we conducted a genome-wide CRISPR-Cas9 screen in IFN-γ–activated human epithelial cells for their ability to restrict virulent intracellular pathogens such as Salmonella enterica serovar Typhimurium. RESULTS We identify the ISG apolipoprotein L3 (APOL3) as a potent effector protein capable of killing cytosol-invasive bacteria. The human APOL family is a cluster of six genes that have evolved rapidly under positive selection in simian primates; however, aside from the founding member APOL1, a secreted extracellular protein that forms the trypanolytic factor of human serum, the function of the intracellular APOL family members is unknown. Human cells genetically engineered to lack APOL3 failed to control the replication of multiple cytosol-invasive Gram-negative bacteria after IFN-γ activation. Such findings were validated in primary human intestinal epithelial cells, intestinal myofibroblasts, and venular endothelium—all cellular targets not typically considered part of the immune system. We tracked APOL3 by live microscopy and found that it rapidly relocated to cytosol-exposed bacteria, whereas other APOL family members did not. A combination of superresolution imaging, bioengineered reporters, and cell-free reconstitution revealed that when APOL3 targets pathogens inside IFN-γ–activated cells, it inflicts a lethal insult to the bacterial inner membrane (IM). Here APOL3 synergizes with other ISG-encoded proteins, including guanylate-binding protein 1 (GBP1), that perturb the bacterial O-antigen outer membrane (OM) permeability barrier to allow APOL3 access to the IM underneath. Using a panel of compositionally distinct liposome targets, we found that APOL3 membranolytic activity toward microbial rather than host endomembranes stemmed from an ability to dissolve bacterial polyanionic lipid substrates lacking cholesterol into discoidal lipoprotein complexes; single-particle cryo–electron microscopy found that these complexes resembled apolipoprotein-scaffold “nanodiscs.” Corroborating these findings in live bacteria by native mass spectrometry, we found that APOL3 transitioned from a partially disordered lipid-free state to tightly folded lipoprotein nanodiscs upon extracting lipid from the IM—a process that resulted in rapid death of the bacterium. CONCLUSION Detergents are highly effective antimicrobials used to decontaminate surfaces infected by deadly pathogens. Our results identify APOL3 as an IFN-γ–stimulated host defense protein that has evolved potent detergent-like activity to bestow bactericidal protection in the cytosol of human cells. APOL3 synergizes with other host ISGs in a multipronged attack against the double membrane of Gram-negative bacteria—a formidable barrier that imparts resistance to many classes of antibiotics. This study reveals that antibacterial agents that dismantle this barrier during infection naturally exist inside human cells. That these agents are encoded within the IFN-γ–inducible defense program reinforces the importance of this powerful antimicrobial network for cell-autonomous immunity in humans. APOL3 kills intracellular bacteria. (A) Negative-stain electron microscopy of recombinant APOL3 (bead) added to Salmonella Typhimurium (periplasm pseudocolored yellow). Destruction of bacterial membrane (blue-bordered inset) is triggered by APOL3 extracting lipid to form lipoproteins (burgundy-bordered inset). (B) Bacterial mutants (ΔwaaL) expressing a truncated O-antigen permit passage of APOL3 through the outer membrane (OM) to the inner membrane (IM); this passage inside cells is facilitated by synergizing ISG-encoded proteins such as GBP1 that co-target cytosol-exposed bacteria. Activation of cell-autonomous defense by the immune cytokine interferon-γ (IFN-γ) is critical to the control of life-threatening infections in humans. IFN-γ induces the expression of hundreds of host proteins in all nucleated cells and tissues, yet many of these proteins remain uncharacterized. We screened 19,050 human genes by CRISPR-Cas9 mutagenesis and identified IFN-γ–induced apolipoprotein L3 (APOL3) as a potent bactericidal agent protecting multiple non–immune barrier cell types against infection. Canonical apolipoproteins typically solubilize mammalian lipids for extracellular transport; APOL3 instead targeted cytosol-invasive bacteria to dissolve their anionic membranes into human-bacterial lipoprotein nanodiscs detected by native mass spectrometry and visualized by single-particle cryo–electron microscopy. Thus, humans have harnessed the detergent-like properties of extracellular apolipoproteins to fashion an intracellular lysin, thereby endowing resident nonimmune cells with a mechanism to achieve sterilizing immunity.