Molecular determinants of oxidative stress in Salmonella pathogenesis

项目概要/摘要 我们意外地发现发酵有助于沙门氏菌的抗氧化防御， 这一观察结果对防御氧化应激具有广泛的意义，远远超出了细菌。 感染性腹泻每年折磨着10亿人，占人类死亡总数的4%。许多这些 感染是由2,500种非伤寒沙门氏菌血清型中的一种引起的，它可以造成生命， 在非常年轻、非常年老和HIV感染者中威胁全身性并发症。氧化应激 由NADPH氧化酶的酶活性产生的抗氧化剂是最有效的宿主防御之一 沙门氏菌在与专业吞噬细胞结合期间面临的问题。遗传毒性， Fenton介导的DNA双链断裂与细胞功能障碍， 蛋白质中的半胱氨酸残基和金属辅因子构成了氧化应激如何杀死 沙门氏菌和许多其他细菌病原体。然而，尽管他们在抵抗战争中发挥了核心作用， 沙门氏菌病，各种机制的相对重要性，其中活性氧造成抗- 沙门氏菌的活性知之甚少。我们对保护沙门氏菌的适应性反应的理解 对抗氧化应激的能力也同样肤浅筛选对过氧化氢有反应的突变体， 最重要的效应NADPH氧化酶，揭示了以前未预料到的作用，中央代谢 以及过氧化氢介导的沙门氏菌杀灭中的电子传递链。我们的初步数据 表明细胞包膜蛋白的氧化和浆细胞增生样病变（即，内外分离 膜）作为先前在氧化应激期间杀死沙门氏菌的未知步骤。这些 研究为NADPH氧化酶如何产生有效的抗沙门氏菌活性提供了一个创新的框架 在巨噬细胞的先天反应中。我们将检验发酵有助于 沙门氏菌的抗氧化防御，通过协助ATP合成，平衡氧化还原，并使二硫键 形成周质蛋白，从而保护细胞包膜免受活性氧的致命损伤 由NADPH氧化酶产生的物质。具体而言，我们将描述发酵在 抗氧化防御伤寒和非伤寒沙门氏菌，阐明机制，氧化 压力促进发酵，并确定细胞内沙门氏菌是如何被NADPH氧化酶杀死的。不 这一知识不仅将阐明沙门氏菌发病机理的关键方面，而且还将为以下方面提供见解： 各种沙门氏菌血清型的独特和共享的抗氧化防御。我们的研究最终可能会 对微生物发病机理、衰老、糖尿病或癌症生物学等领域的影响， 应力是一种内在成分。特异性抑制细菌糖酵解酶和发酵 这些途径可能导致新的抗生素治疗的发展。未来的沙门氏菌对策可以 也探索增加呼吸活动作为一种手段，煽动氧化杀死的战略。

PROJECT SUMMARY/ ABSTRACT We have made the unexpected discovery that fermentation contributes to Salmonella's antioxidant defenses, an observation with wide ranging implications for defense against oxidative stress, well beyond bacteria. Infectious diarrhea afflicts a billion people a year and is responsible for 4% of all human deaths. Many of these infections are caused by one of the 2,500 serovars of nontyphoidal Salmonella enterica, which can inflict life- threatening systemic complications in the very young, very old, and HIV-infected individuals. Oxidative stress emanating from the enzymatic activity of the NADPH oxidase is one of the most potent host defenses Salmonella face during their associations with professional phagocytic cells. Genotoxicity that ensues from Fenton-mediated DNA double strand breaks together with cellular malfunctions associated with the oxidation of cysteine residues and metal cofactors in proteins constitute the paradigm for how oxidative stress kills Salmonella and numerous other bacterial pathogens. However, despite their central role in resistance to salmonellosis, the relative importance of the various mechanisms by which reactive oxygen species inflict anti- Salmonella activity is poorly understood. Our understanding of the adaptive responses that protect Salmonella against oxidative stress is similarly superficial. A screen of mutants in response to hydrogen peroxide, one of the most important effectors of the NADPH oxidase, revealed previously unanticipated roles for central metabolism and the electron transport chain in the hydrogen peroxide-mediated killing of Salmonella. Our preliminary data suggest oxidation of cell envelope proteins and plasmolysis-like lesions (i.e., separation of inner and outer membranes) as previously unsuspected steps in the killing of Salmonella during oxidative stress. These investigations offer an innovative framework for how NADPH oxidase inflicts potent anti-Salmonella activity during the innate response of macrophages. We will test the hypothesis that fermentation contributes to Salmonella's antioxidant defenses by assisting with ATP synthesis, balancing redox, and enabling disulfide bond formation in periplasmic proteins, thereby protecting the cell envelope from lethal damage by reactive oxygen species generated by the NADPH oxidase. Specifically, we will characterize the role fermentation plays in the antioxidant defenses of typhoidal and nontyphoidal Salmonella, elucidate the mechanism by which oxidative stress promotes fermentation, and determine how intracellular Salmonella is killed by the NADPH oxidase. Not only will this knowledge illuminate key aspects of Salmonella pathogenesis, but should also provide insights into unique and shared antioxidant defenses of various Salmonella serovars. Our research could ultimately have an impact on fields as diverse as microbial pathogenesis, aging, diabetes, or cancer biology for which oxidative stress is an intrinsic component. Drugs that specifically inhibit bacterial glycolytic enzymes and fermentative pathways may lead to the development of novel antibiotic treatments. Future Salmonella countermeasures could also explore strategies that increase respiratory activity as a means to foment oxidative killing.