PDF(Pubmed)
Abstract：
The ability of Mycobacterium tuberculosis (Mtb) to tolerate nitric oxide (•NO) and superoxide (O2•-) produced by phagocytes contributes to its success as a human pathogen. Recombination of •NO and O2•- generates peroxynitrite (ONOO-), a potent oxidant produced inside activated macrophages causing lethality in diverse organisms. While the response of Mtb toward •NO and O2•- is well established, how Mtb responds to ONOO- remains unclear. Filling this knowledge gap is important to understand the persistence mechanisms of Mtb during infection. We synthesized a series of compounds that generate both •NO and O2•-, which should combine to produce ONOO-. From this library, we identified CJ067 that permeates Mtb to reliably enhance intracellular ONOO- levels. CJ067-exposed Mtb strains, including multidrug-resistant (MDR) and extensively drug-resistant (XDR) clinical isolates, exhibited dose-dependent, long-lasting oxidative stress and growth inhibition. In contrast, Mycobacterium smegmatis (Msm), a fast-growing, non-pathogenic mycobacterial species, maintained redox balance and growth in response to intracellular ONOO-. RNA-sequencing with Mtb revealed that CJ067 induces antioxidant machinery, sulphur metabolism, metal homeostasis, and a 4Fe-4S cluster repair pathway (suf operon). CJ067 impaired the activity of the 4Fe-4S cluster-containing TCA cycle enzyme, aconitase, and diminished bioenergetics of Mtb. Work with Mtb strains defective in SUF and IscS involved in Fe-S cluster biogenesis pathways showed that both systems cooperatively protect Mtb from intracellular ONOO- in vitro and inducible nitric oxide synthase (iNOS)-dependent growth inhibition during macrophage infection. Thus, Mtb is uniquely sensitive to intracellular ONOO- and targeting Fe-S cluster homeostasis is expected to promote iNOS-dependent host immunity against tuberculosis (TB).
摘要：
结核分枝杆菌（Mtb）耐受吞噬细胞产生的一氧化氮（·NO）和超氧化物（O2·-）的能力有助于其作为人类病原体的成功。•NO和O2•-的重组产生过氧亚硝酸盐（ONOO-），一种在激活的巨噬细胞内部产生的有效氧化剂，在多种生物体中引起致死性。虽然Mtb对·NO和O2·-的反应已经确立，Mtb对ONOO的反应尚不清楚。填补这一知识空白对于了解感染期间Mtb的持续机制很重要。我们合成了一系列同时产生·NO和O2·-的化合物，它们应该结合起来产生ONOO-。从这个图书馆，我们确定了CJ067渗透Mtb以可靠地增强细胞内ONOO-水平。CJ067暴露的Mtb菌株，包括多药耐药（MDR）和广泛耐药（XDR）临床分离株，表现出剂量依赖性，持久的氧化应激和生长抑制。相比之下,耻垢分枝杆菌（Msm），一个快速增长的，非致病性分枝杆菌物种，维持氧化还原平衡和生长以响应细胞内ONOO-。用Mtb进行RNA测序显示CJ067诱导抗氧化机制，硫代谢，金属稳态，和4Fe-4S簇修复途径(suf操纵子)。CJ067损害了含4Fe-4S簇的TCA循环酶的活性，乌头酸酶,和减少Mtb的生物能学。对参与Fe-S簇生物发生途径的SUF和IscS缺陷的Mtb菌株的研究表明，两种系统共同保护Mtb免受巨噬细胞感染期间细胞内ONOO-体外和诱导型一氧化氮合酶（iNOS）依赖性生长抑制的影响。因此，Mtb对胞内ONOO-独特敏感，靶向Fe-S簇稳态有望促进iNOS依赖性宿主对结核病（TB）的免疫。
