The role of pyruvate oxidase in nitrosative stress resistance by Streptococcus parasanguinis

项目总结 副血链球菌是一种口腔共生菌，它通过 丙酮酸氧化酶(POXL)活性。这种过氧化氢可以与亚硝酸盐(NO2)反应生成过氧亚硝酸盐。 (ONOO-)和其他活性亚硝酸类(RNS)是有效的抗菌剂。我们实验室以前的工作 已经证明副血链球菌介导的RNS的产生可以抑制病原体的生长，如 变形链球菌和铜绿假单胞菌。尽管RNS对多种细菌有抗菌作用 细菌，副血链球菌是唯一耐药的，副血链球菌在 NO2和ONOO-的存在。这种抗性的机制尚不清楚。副血链霉菌不会利用 过氧化氢酶介导氧化应激，不能还原硝酸盐或其衍生物。然而，以前的文献 研究表明，在一些链球菌中，丙酮酸氧化酶可以减少氧化负担。要确定Poxl是否扮演 在副血链球菌亚硝化应激反应中的类似作用，Poxl突变体在RNS中的作用被测试 抵抗。有趣的是，Poxl突变体已经损害了生物膜的生长，这可以在 NO2或ONOO-的存在，表明NO2和ONOO-补偿POXL依赖的损失 生物膜的形成，Poxl可能调节含氮中间体的感觉或代谢。此外， Poxl突变体增加了ATP的产生，表明Poxl是代谢的重要调节因子。 代谢组学分析表明，在NO2存在的情况下，副血链球菌在全球范围内发生了 许多氧化还原清道夫，表明对RNS的反应和氧化应激可能是耦合的。这个项目 旨在确定Poxl在RNS反应中的作用。这项提议的首要假设是 副血链霉菌通过丙酮酸氧化酶协调其亚硝化应激反应。目标1服务于 通过确定1)介导氧化应激的SpxR来确定RNS应激反应的调节 链球菌的应激反应，通过POXL调节亚硝酸盐参与亚硝化应激反应 摄取和代谢，以及2)如果其他利用丙酮酸作为底物的酶也介导RNS 回应。目的2通过测定1)磷脂酰肌醇在体内的调节和作用 以NO2依赖的方式介导定植，以及2)如果Pox1的表达被当地营养改变 在有或没有NO2的情况下的可用性。总而言之，我们将使用分子和系统生物学方法， 与感染模型相结合，以了解氮代谢和 副血链霉菌的亚硝化应激反应。从这项提案中产生的数据将使口头 通过了解一种非规范的方法来对抗亚硝化胁迫，并通过 了解含氮中间体在口腔共生体定植中的作用。

PROJECT SUMMARY Streptococcus parasanguinis is an oral commensal bacterium that produces hydrogen peroxide through pyruvate oxidase (PoxL) activity. This hydrogen peroxide can react with nitrite (NO2) to form peroxynitrite (ONOO-) and other reactive nitrosative species (RNS) that are potent antimicrobials. Previous work in our lab has demonstrated that S. parasanguinis-mediated RNS production can inhibit growth of pathogens such as Streptococcus mutans and Pseudomonas aeruginosa. Despite the antimicrobial effects of RNS on a variety of bacteria, S. parasanguinis is uniquely resistant, with S. parasanguinis having a growth advantage in the presence of NO2 and ONOO-. Mechanisms of this resistance are unknown. S. parasanguinis does not utilize catalase to mediate oxidative stress and cannot reduce nitrate or its derivatives. However, previous literature has shown that in some streptococci, pyruvate oxidase reduces oxidative burden. To determine if PoxL plays a similar role in the S. parasanguinis nitrosative stress response, a poxL mutant was tested for its role in RNS resistance. Interestingly, the poxL mutant had impaired biofilm growth that could be partially rescued in the presence of either NO2 or ONOO-, indicating that NO2 and ONOO- compensate for the loss of poxL-dependent biofilm formation, and that poxL may modulate sensing or metabolism of nitrogenous intermediates. Further, the poxL mutant has increased ATP production, indicating that poxL is an important mediator of metabolism. Metabolomics analysis revealed that in the presence of NO2, S. parasanguinis undergoes a global shift in many redox scavengers, indicating that the response to RNS and oxidative stress may be coupled. This project aims to determine the role PoxL has on the RNS response. The overarching hypothesis for this proposal is that S. parasanguinis coordinates its nitrosative stress response through pyruvate oxidase. Aim 1 serves to determine the regulation of the RNS stress response by determining 1) if SpxR, which mediates the oxidative stress response in streptococci, is involved in the nitrosative stress response through PoxL regulation, nitrite uptake, and metabolism, and 2) if other enzymes that utilize pyruvate as a substrate also mediate the RNS response. Aim 2 works to understand the regulation and effects of PoxL in vivo by determining 1) if PoxL mediates colonization in a NO2-dependent manner, and 2) if poxL expression is altered by local nutritional availability with or without NO2. Taken together, we will use a molecular and systems biology approach, coupled with infection models to understand the fundamental mechanisms involved in nitrogen metabolism and the nitrosative stress response in S. parasanguinis. Data generated from this proposal will benefit the oral microbiology field by understanding a noncanonical approach to nitrosative stress resistance and by understanding the role nitrogenous intermediates play in the colonization of an oral commensal.