Project Summary/Abstract Despite its historical importance as a plague on humankind, syphilis remains among the most poorly understood of all human infections. This is a direct result of severe research constraints imposed by the historic inability to cultivate Treponema pallidum (Tp) continuously in vitro. In a departure from more conventional approaches, about 15 years ago we embarked on a bold structural biology-based initiative to characterize Tp’s lipoproteins (LPs), molecules critical to the membrane biology, bioenergetics, and intermediary metabolism of Tp, as a means of unlocking the mechanistic evolutionary “secrets” of Tp infection and syphilis pathogenesis. This progressive research avenue has become a very successful discovery platform, yielding many highly novel findings, including establishing a number of new bacterial molecular paradigms. For example, we discovered a novel bi-functional FAD pyrophosphatase/FMN transferase in Tp; this, in turn, led us to identify a post-translational protein flavinylation pathway in Tp’s periplasm, yielding flavoproteins that ostensibly influence cellular redox reactions. We then obtained evidence for Tp encoding an atypical flavin-based Rhodobacter Nitrogen Fixation (RNF)-type redox pump, likely representing the longstanding missing link between Tp’s membrane electrochemical gradient, redox balance, ATP generation, and an acetogenic energy conservation pathway. Historically, Tp has been thought not to encode such systems. Our contention of a flavin-based redox system not only addresses a number of longstanding unexplained metabolic dilemmas for Tp, but it also engenders a paradigm shift by now establishing Tp as a flavin auxotroph. We also have demonstrated that TP0572, a putative FMN- dependent ferric reductase, is flavinylated by Ftp (TP0796), likely an essential prerequisite for Tp’s reductive iron assimilation pathway(s). In addition, predicted cytosolic flavoproteins must play prominently in protecting Tp from oxidative stress and in maintaining the balance of NAD+/NADH. These collective notions support that, with limited potential for ATP generation in the absence of quinones, Tp has evolved a “flavin-centric metabolic lifestyle” to fulfill its metabolic requirements for human infection. This project shall address three core metabolic features relevant to Tp’s flavin biology: protein flavinylation and flavoprotein biogenesis (Aim 1), reductive iron assimilation and Fe-S protein biogenesis (Aim 2), and redox balance/energy conservation (via acetogenesis) (Aim 3). We also shall evaluate a small-molecule inhibitor(s) targeting Tp’s flavin auxotrophy as a potential new research tool(s) and/or new antimicrobial(s) against Tp and other pathogenic spirochetes (Aim 4). Taken together, this project shall elucidate key features concerning how Tp has evolved to exploit flavins as an underpinning of its stealth pathogenicity, potentially leading to new strategies to thwart human infection.