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.