Nitric Oxide Cytotoxicity in Salmonellosis

性状（由申请人提供）：宿主细胞通过产生一氧化氮（NO.）抑制多种微生物。本项目的目标是了解NO.如何抑制细菌以及病原菌如何抵抗NO.。我们的假设是宿主来源的NO与细菌含金属和硫醇的靶点相互作用，产生阻止细菌生长的基本代谢改变。该提案的具体目标是通过分析特定的新型NO.靶标来回答重要的机制问题：[1]细菌如何感知和响应宿主来源的NO.？NsrR，一种亚硝酸盐敏感的抑制剂，用于防御亚硝化应激; [2] NO如何限制细菌代谢？LpdA或硫辛酰胺脱氢酶，TCA循环和氨基酸生物合成所需的酶的重要亚基[3] NO如何破坏氧化还原稳态？PFL或丙酮酸-甲酸裂解酶，可将丙酮酸转化为乙酰辅酶A和甲酸，以促进NAD+在限氧条件下的再生NO。将在两种重要的细菌病原体中分析细胞毒性。NsrR和LpdA将在肠道细菌鼠伤寒沙门氏菌中进行研究。PFL将在革兰氏阳性菌金黄色葡萄球菌中进行研究，该菌采用独特的策略绕过NO对PFL的抑制。这个项目将研究各种潜在的分子机制，通过铁硫簇，铁-亚硝基形成，S-亚硝基化，或甘氨酰自由基淬灭不稳定的蛋白质失活NO。这些目标将通过我们实验室常规使用的遗传、生物化学和体内分析相结合来实现。从这些研究中获得的见解将提供一个综合的画面，即宿主来源的NO如何改变入侵细菌的代谢以损害生长，以及病原体如何通过解毒NO或绕过关键的抑制位点来克服这些作用。人体的免疫细胞产生一氧化氮来限制微生物的生长。该研究项目分析了一氧化氮对细菌产生毒性作用的机制，以及沙门氏菌和葡萄球菌等致病菌抵抗这种重要宿主防御机制的机制。这些研究将有助于确定预防或治疗感染的新策略。

DESCRIPTION (provided by applicant): Host cells inhibit a broad range of microbes by producing nitric oxide (NO.). The goal of this project is to understand how NO. inhibits bacteria and how pathogenic bacteria resist NO.. Our hypothesis is that host-derived NO. interacts with bacterial metal- and thiol-containing targets to produce fundamental metabolic alterations that prevent bacterial growth. The specific aims of this proposal are to answer important mechanistic questions by analyzing specific novel NO. targets: [1] How do bacteria sense and respond to host-derived NO.?-- NsrR, a nitrite- sensitive repressor of defenses against nitrosative stress; [2] How does NO. constrain bacterial metabolism?-- LpdA, or lipoamide dehydrogenase, an essential subunit of enzymes required for the TCA cycle and amino acid biosynthesis [3] How does NO. disrupt redox homeostasis?-- PFL, or pyruvate-formate lyase, which converts pyruvate to acetyl CoA and formate to facilitate the regeneration of NAD+ under oxygen-limited conditions NO. cytotoxicity will be analyzed in two important bacterial pathogens. NsrR and LpdA will be studied in the enteric bacterium Salmonella Typhimurium. PFL will be investigated in the gram-positive bacterium Staphylococcus aureus, which employs a unique strategy to bypass PFL inhibition by NO.. This project will examine a variety of potential molecular mechanisms for protein inactivation by NO through destabilization of iron-sulfur clusters, iron-nitrosyl formation, S-nitrosylation, or glycyl radical quenching. The aims will be achieved by a combination of genetic, biochemical and in vivo analyses in routine use in our laboratory. Insights from these studies will provide an integrated picture of how host- derived NO. alters the metabolism of invading bacteria to impair growth and how pathogens can overcome these actions by detoxifying NO. or bypassing crucial sites of inhibition. PROJECT NARRATIVE The body's immune cells produce nitric oxide to limit the growth of microorganisms. This research project analyzes the mechanisms by which nitric oxide exerts toxic effects on bacteria and the mechanisms by which pathogenic bacteria such as Salmonella and Staphylococcus resist this important host defense mechanism. Such studies will help to identify novel strategies for the prevention or treatment of infection.