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

项目摘要 副血链球菌是一种口腔粘膜细菌，其产生过氧化氢， 丙酮酸氧化酶（PoxL）活性。这种过氧化氢可以与亚硝酸盐（NO2）反应形成过氧亚硝酸盐 （ONOO-）和其他活性亚硝化物质（RNS）是有效的抗菌剂。我们实验室以前的工作 证明了S.副血吸虫介导的RNS产生可以抑制病原体的生长， 变形链球菌和绿脓杆菌。尽管RNS对多种微生物具有抗菌作用， 细菌、S. parasanginis是唯一的抗性，S.副血吸虫具有生长优势， NO2和ONOO-的存在。这种抗性的机制尚不清楚。S.副血吸虫不利用 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 已经表明，在某些链球菌中，丙酮酸氧化酶降低氧化负荷。为了确定PoxL是否扮演了一个 在S. poxL突变体在RNS中的作用 阻力有趣的是，poxL突变体具有受损的生物膜生长，这可以在细胞中部分挽救。 NO2或ONOO-的存在，表明NO2和ONOO-补偿了poxL依赖性的损失。 生物膜形成，且poxL可以调节含氮中间体感测或代谢。此外，本发明还 poxL突变体增加了ATP的产生，表明poxL是代谢的重要介质。 代谢组学分析表明，在NO2存在下，S. parasanguinis经历了全球性的转变， 许多氧化还原清除剂，表明RNS和氧化应激的反应可能是耦合的。这个项目 旨在确定PoxL对RNS反应的作用。这一提议的首要假设是， S. parasanguinis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）SpxR，其介导氧化应激反应， 链球菌的应激反应，通过PoxL调节，亚硝酸盐 摄取和代谢，以及2）如果利用丙酮酸作为底物的其他酶也介导RNS 反应目的2通过测定1）PoxL是否与细胞增殖有关，以了解PoxL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poxL表达被局部营养改变， 有或没有NO2。总之，我们将使用分子和系统生物学方法， 再加上感染模型，以了解氮代谢的基本机制， S.副血吸虫从这个提案中产生的数据将有利于口头 微生物学领域通过理解亚硝化胁迫抗性的非经典方法， 了解含氮中间体在口腔黏膜定植中的作用。

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.