Inducible nitric oxide synthase (iNOS) is necessary for GBP-mediated T. gondii restriction in murine macrophages via vacuole nitration and intravacuolar network collapse.
Inducible nitric oxide synthase (iNOS) is necessary for GBP-mediated T. gondii restriction in murine macrophages via vacuole nitration and intravacuolar network collapse.
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诱导型一氧化氮合酶 (iNOS) 对于 GBP 介导的弓形虫通过液泡硝化和液泡内网络崩溃对小鼠巨噬细胞的限制是必需的。
DOI：
10.1101/2023.07.24.549965
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Ewald,SarahE
中科院分区：
文献类型：
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作者：
Zhao,Xiao-Yu;Lempke,SamanthaL;UrbánArroyo,JanC;Yin,Bocheng;Holness,NadiaK;Smiley,Jamison;Ewald,SarahE
弓形虫是啮齿动物和人类的专性细胞内原生动物病原体。弓形虫在细胞内生长和逃避细胞自主免疫的能力依赖于寄生虫液泡(PV)的完整性。干扰素诱导的鸟苷结合蛋白(GBP)是弓形虫清除的中心介质，然而，GBP募集与PV和弓形虫限制的确切机制尚不清楚。这种知识差距与大量空泡中的异源GBP靶向有关，以及缺乏有选择地提纯完整PV的工具。为了确定与GBP2阳性空泡相关的寄生虫清除的介体，我们使用了一种新的蛋白质发现工具自动空间靶向光学微蛋白质组学(AutoSTOMP)。这种方法确定了诱导型一氧化氮合酶(INOS)在受感染的骨髓来源的髓系细胞中的含量与GBPS相似。髓系细胞上iNOS的表达是小鼠体内控制弓形虫生长和存活急性感染所必需的。弓形虫感染干扰素γ诱导的巨噬细胞足以诱导诱导型一氧化氮合酶表达。INOS通过一氧化氮的合成而不是精氨酸的消耗来抑制弓形虫的感染，导致PV的强健和选择性的硝化。诱导型一氧化氮合酶和液泡硝化对寄生虫的最适抑制作用取决于染色体3Gbps。值得注意的是，在iNOS基因敲除中发生了GBP2的募集和PV膜的褶皱，然而，这些空泡中含有分裂的寄生虫。INOS活性是连接寄生虫和寄主细胞质的纳米管膜的液泡内网络崩溃所必需的。基于这些数据，我们得出结论，iNOS产生的活性氮物种与染色体3 Gbps合作，靶向PV的不同生物学，这是最佳清除小鼠髓系细胞寄生虫所必需的。
Toxoplasma gondii is an obligate intracellular, protozoan pathogen of rodents and humans. T. gondii’s ability to grow within cells and evade cell-autonomous immunity depends on the integrity of the parasitophorous vacuole (PV). Interferon-inducible guanylate binding proteins (GBPs) are central mediators of T. gondii clearance, however, the precise mechanism linking GBP recruitment to the PV and T. gondii restriction is not clear. This knowledge gap is linked to heterogenous GBP-targeting across a population of vacuoles and the lack of tools to selectively purify the intact PV. To identify mediators of parasite clearance associated with GBP2-positive vacuoles, we employed a novel protein discovery tool automated spatially targeted optical micro proteomics (autoSTOMP). This approach identified inducible nitric oxide synthetase (iNOS) enriched at levels similar to the GBPs in infected bone marrow-derived myeloid cells. iNOS expression on myeloid cells was necessary for mice to control T. gondii growth in vivo and survive acute infection. T. gondii infection of IFNγ-primed macrophage was sufficient to robustly induce iNOS expression. iNOS restricted T. gondii infection through nitric oxide synthesis rather than arginine depletion, leading to robust and selective nitration of the PV. Optimal parasite restriction by iNOS and vacuole nitration depended on the chromosome 3 GBPs. Notably, GBP2 recruitment and ruffling of the PV membrane occurred in iNOS knockouts, however, these vacuoles contained dividing parasites. iNOS activity was necessary for the collapse of the intravacuolar network of nanotubular membranes which connects parasites to each other and the host cytosol. Based on these data we conclude reactive nitrogen species generated by iNOS cooperate with the chromosome 3 GBPs to target distinct biology of the PV that are necessary for optimal parasite clearance in murine myeloid cells.