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Clean the cytosol
Most human cells, and not just those belonging to the immune system, develop protective responses to infection when activated by the immune cytokine interferon-gamma (IFN-γ). How IFN-γ confers this function in cells and non-immune tissues is poorly understood. Gaudet et al. used genome-wide CRISPR / Cas9 gene editing to identify apolipoprotein L-3 (APOL3) as an IFN-γ-induced bactericidal protein that protects the epithelium, endothelium and cells. human fibroblasts against infections (see Perspective from Nathan). APOL3 directly targets bacteria in the host cell cytosol and kills them by dissolving their anionic membranes in lipoprotein complexes. This work reveals a detergent-like mechanism engaged during the autonomic immunity of human cells to fight against intracellular pathogens.
Science, abf8113, this issue p. eabf8113; see also abj5637, p. 276
Structured summary
INTRODUCTION
In the arms race between the pathogen and the host, infectious microbes often evade 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 resistance to pathogens in most nucleated cells through the transcriptional induction of hundreds of genes stimulated by l interferon (ISG) encoding putative antimicrobial restriction factors. Remarkably, despite the importance of IFN-γ against all taxonomic classes of intracellular pathogens, many restriction factors induced by this cytokine remain to be characterized, as well as their molecular activities.
REASONING
Identified as the main cytokine activating human macrophages in 1983, IFN-γ actually reprograms many types of host cells transcriptionally to clear infection. This includes non-immune epithelial cell populations, which lack many of the traditional phagocytic defenses attributed to IFN-γ stimulation, but still manage to mount protective immune cell autonomic responses. To find ISG effectors involved in saving mucosal and barrier tissue types, we performed a genome-wide CRISPR-Cas9 screen in human epithelial cells activated by IFN-γ for their ability to restrict agents. virulent intracellular pathogens such as Salmonella enterica Typhimurium serovar.
RESULTS
We identify ISG apolipoprotein L3 (APOL3) as a potent effector protein capable of killing invasive bacteria in the cytosol. Human APOL family is a group of six genes that evolved rapidly under positive selection in simian primates; however, other than founding member APOL1, a secreted extracellular protein that forms human serum trypanolytic factor, the function of intracellular members of the APOL family is unknown. Genetically modified human cells are lacking APOL3 failed to control the replication of several invasive Gram-negative bacteria in the cytosol after activation of IFN-γ. These results have been validated in primary human intestinal epithelial cells, intestinal myofibroblasts and venular endothelium, all cellular targets that are generally not considered to be part of the immune system. We followed APOL3 by live microscopy and found that it quickly moved to bacteria exposed to the cytosol, unlike other members of the APOL family. A combination of superresolution imaging, bioengineered reporters, and cell-free reconstitution revealed that when APOL3 targets pathogens inside IFN-γ-activated cells, it inflicts lethal aggression on the bacterial inner membrane (IM). Here, APOL3 synergizes with other proteins encoded by ISG, including guanylate binding protein 1 (GBP1), which disrupts the outer membrane permeability barrier (OM) of bacterial O antigen to allow APOL3 to access the IM below. Using a panel of compositionally distinct liposomal targets, we found that the membranolytic activity of APOL3 towards microbial endomembranes rather than that of the host arose from an ability to dissolve bacterial polyanionic lipid substrates lacking cholesterol in discoid lipoprotein complexes; Single-particle electron cryo-microscopy revealed that these complexes resembled apolipoprotein-scaffold “nanodisks”. By corroborating these results in living bacteria by native mass spectrometry, we found that APOL3 went from a partially disordered lipid-free state to tightly folded lipoprotein nanodisks during lipid extraction from MI, a process which led to the rapid death of the bacteria.
CONCLUSION
Detergents are highly effective antimicrobials used to decontaminate surfaces infected with deadly pathogens. Our results identify APOL3 as an IFN-γ-stimulated host defense protein that developed potent detergent-like activity to confer bactericidal protection in the cytosol of human cells. APOL3 synergizes with other ISG hosts in a multi-pronged attack against the double membrane of Gram-negative bacteria, a formidable barrier that confers resistance to many classes of antibiotics. This study reveals that antibacterial agents that break down this barrier during infection exist naturally inside human cells. The fact that these agents are encoded in the IFN-γ-inducible defense program reinforces the importance of this powerful antimicrobial network for autonomic cellular immunity in humans.
(A) Negative staining electron microscopy of recombinant APOL3 (bead) added to Salmonella Typhimurium (yellow pseudocolorated periplasm). The destruction of the bacterial membrane (inset bordered in blue) is triggered by the extraction of lipids by APOL3 to form lipoproteins (inset bordered in burgundy). (B) Bacterial mutants (ΔwaaL) expression of a truncated O antigen allows passage of APOL3 through the outer membrane (OM) to the inner membrane (IM); this passage inside cells is facilitated by the synergy of proteins encoded by ISG such as GBP1 which co-target bacteria exposed to the cytosol.
Abstract
Activation of autonomic cell defense by the immune cytokine interferon-γ (IFN-γ) is essential for the control of life-threatening infections in humans. IFN-γ induces the expression of hundreds of host proteins in all nucleated cells and tissues, but many of these proteins remain uncharacterized. We screened 19,050 human genes by CRISPR-Cas9 mutagenesis and identified the IFN-γ-induced apolipoprotein L3 (APOL3) as a potent bactericidal agent protecting several types of non-immune barrier cells against infection. Canonical apolipoproteins generally solubilize mammalian lipids for extracellular transport; Rather, APOL3 targeted cytosol invasive bacteria to dissolve their anionic membranes in human-bacterial lipoprotein nanodisks detected by native mass spectrometry and visualized by single particle electron cryo-microscopy. Thus, humans have exploited the detergent-like properties of extracellular apolipoproteins to fashion intracellular lysine, thereby endowing non-immune resident cells with a mechanism to achieve sterilizing immunity.
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