A human apolipoprotein L with detergent-like activity kills intracellular pathogens.
A human apolipoprotein L with detergent-like activity kills intracellular pathogens.
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DOI：
10.1126/science.abf8113
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发表时间：
2021-07-16
期刊：
影响因子：
--
通讯作者：
MacMicking JD
中科院分区：
文献类型：
--
作者：
Gaudet RG;Zhu S;Halder A;Kim BH;Bradfield CJ;Huang S;Xu D;Mamiñska A;Nguyen TN;Lazarou M;Karatekin E;Gupta K;MacMicking JD
在病原体和宿主之间的军备竞赛中，感染微生物通常会逃避细胞外防御机制，利用富含营养的细胞内环境作为复制生态位。在人类中，这被干扰素-γ（IFN-γ）应答抵消，其通过编码推定的抗微生物限制因子的数百个干扰素刺激基因（ISG）的转录诱导在大多数有核细胞中赋予广泛的病原体抗性。值得注意的是，尽管IFN-γ对细胞内病原体的所有分类类别都很重要，但由这种细胞因子引起的许多限制因子仍有待表征，其分子活性也是如此。IFN-γ在1983年被鉴定为主要的人类巨噬细胞活化细胞因子，事实上，IFN-γ转录重编程许多宿主细胞类型以消除感染。这包括非免疫上皮细胞群体，其缺乏归因于IFN-γ刺激的许多传统吞噬防御，但仍设法建立保护性细胞自主免疫应答。为了找到参与保护粘膜和屏障组织类型的ISG效应子，我们在IFN-γ激活的人上皮细胞中进行了全基因组CRISPR-Cas9筛选，以确定其限制毒性细胞内病原体（如沙门氏菌）的能力。我们确定ISG载脂蛋白L3（APOL 3）作为一种有效的效应蛋白能够杀死细胞溶质侵入细菌。人类APOL家族是一个由6个基因组成的基因簇，在猿类灵长类动物中，这些基因在正选择下迅速进化;然而，除了创始成员APOL 1（一种分泌的细胞外蛋白，形成人血清的锥虫溶解因子）外，细胞内APOL家族成员的功能尚不清楚。经基因工程改造以缺乏APOL 3的人类细胞在IFN-γ活化后未能控制多种细胞溶质侵入性革兰氏阴性细菌的复制。这些发现在原代人肠上皮细胞、肠肌成纤维细胞和小静脉内皮细胞中得到了验证，这些细胞靶点通常不被认为是免疫系统的一部分。我们通过活体显微镜追踪APOL 3，发现它迅速转移到细胞溶质暴露的细菌中，而其他APOL家族成员则没有。超分辨率成像、生物工程报告基因和无细胞重建的组合揭示，当APOL 3靶向IFN-γ激活细胞内的病原体时，它会对细菌内膜（IM）造成致命伤害。在这里，APOL 3与其他ISG编码的蛋白质协同作用，包括鸟苷酸结合蛋白1（GBP 1），干扰细菌O抗原外膜（OM）渗透屏障，使APOL 3进入下面的IM。使用一组组成不同的脂质体靶点，我们发现APOL 3对微生物而不是宿主内膜的膜溶解活性源于将缺乏胆固醇的细菌聚阴离子脂质底物溶解成盘状脂蛋白复合物的能力;单颗粒冷冻电子显微镜发现这些复合物类似于载脂蛋白支架“纳米盘”。通过天然质谱法证实了活细菌中的这些发现，我们发现APOL 3在从IM中提取脂质时从部分无序的无脂质状态转变为紧密折叠的脂蛋白纳米盘-这是导致细菌快速死亡的过程。去污剂是用于净化被致命病原体感染的表面的高效抗菌剂。我们的研究结果将APOL 3鉴定为IFN-γ刺激的宿主防御蛋白，其已经进化出有效的去污剂样活性以在人类细胞的胞质溶胶中赋予杀菌保护。APOL 3与其他宿主ISG协同作用，多管齐下攻击革兰氏阴性菌的双膜，这是一种强大的屏障，可对许多类抗生素产生耐药性。这项研究表明，在感染过程中拆除这一屏障的抗菌剂自然存在于人体细胞内。这些试剂在IFN-γ诱导的防御程序中编码，这加强了这种强大的抗微生物网络对人类细胞自主免疫的重要性。APOL 3杀死细胞内细菌。(A)添加到鼠伤寒沙门氏菌中的重组APOL 3（珠）的负染电子显微镜检查（周质假黄色）。细菌膜的破坏（蓝色边框的插图）由APOL 3提取脂质以形成脂蛋白（勃艮第边框的插图）触发。(B)表达截短的O-抗原的细菌突变体（ΔwaaL）允许APOL 3通过外膜（OM）到达内膜（IM）;通过协同ISG编码的蛋白质（如GBP 1）促进细胞内的这种通过，这些蛋白质共同靶向细胞溶质暴露的细菌。免疫细胞因子干扰素-γ（IFN-γ）激活细胞自主防御对于控制人类危及生命的感染至关重要。IFN-γ在所有有核细胞和组织中诱导数百种宿主蛋白质的表达，然而这些蛋白质中的许多仍然未被表征。我们通过CRISPR-Cas9诱变筛选了19，050个人类基因，并将IFN-γ诱导的载脂蛋白L3（APOL 3）鉴定为保护多种非免疫屏障细胞类型免受感染的有效杀菌剂。典型的载脂蛋白通常溶解哺乳动物脂质用于细胞外转运; APOL 3相反靶向细胞溶质侵入细菌，以将其阴离子膜溶解成通过天然质谱检测的人细菌脂蛋白纳米盘，并通过单颗粒冷冻电子显微镜观察。因此，人类已经利用细胞外载脂蛋白的去污剂样特性来形成细胞内溶素，从而赋予驻留的非免疫细胞以实现杀菌免疫的机制。
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. 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. 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. 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.
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结构细胞是器官特异性免疫反应的关键调节剂。
影响因子：
64.8
作者：
Krausgruber T;Fortelny N;Fife-Gernedl V;Senekowitsch M;Schuster LC;Lercher A;Nemc A;Schmidl C;Rendeiro AF;Bergthaler A;Bock C
通讯作者：
Bock C
影响因子：
14.8
作者：
Kelley LA;Mezulis S;Yates CM;Wass MN;Sternberg MJ
通讯作者：
Sternberg MJ
影响因子：
4.4
作者：
Ernst, WA;Thoma-Uszynski, S;Modlin, RL
通讯作者：
Modlin, RL
影响因子：
56.9
作者：
Kim, Bae-Hoon;Shenoy, Avinash R.;MacMicking, John D.
通讯作者：
MacMicking, John D.
影响因子：
5.8
作者：
Gautier, Romain;Douguet, Dominique;Drin, Guillaume
通讯作者：
Drin, Guillaume