The flavin-centric metabolic lifestyle of Treponema pallidum

项目总结/摘要 尽管梅毒作为人类的一种瘟疫在历史上具有重要意义，但它仍然是最糟糕的疾病之一。 了解所有人类感染。这是由严格的研究限制强加的直接结果。 历史上不能在体外连续培养梅毒螺旋体（Tp）。在一个背离更多 大约15年前，我们开始了一项大胆的基于结构生物学的倡议， 表征Tp的脂蛋白（LP），膜生物学，生物能量学的关键分子， TP的中间代谢，作为解锁TP的机械进化“秘密”的手段 感染和梅毒发病机制。这一进步的研究途径已经成为一个非常成功的 发现平台，产生了许多非常新颖的发现，包括建立了一些新的细菌 分子模式例如，我们发现了一种新的双功能FAD焦磷酸酶/FMN， 这反过来又使我们确定了一个翻译后蛋白黄素化途径， 周质，产生表面上影响细胞氧化还原反应的黄素蛋白。然后我们得到了 Tp编码非典型的基于黄素的红细菌固氮（RNF）型氧化还原泵的证据， 可能代表TP的膜电化学梯度、氧化还原 平衡、ATP生成和产乙酸能量守恒途径。从历史上看，TP一直是 不被认为编码这样的系统。我们对基于黄素的氧化还原系统的争论不仅解决了 一些长期无法解释的代谢困境的TP，但它也产生了一个范式转变， 现在确定Tp为黄素营养缺陷型。我们还证明了TP 0572，一个假定的FMN- 依赖性铁还原酶，被Ftp（TP 0796）黄素化，可能是TP的基本先决条件 还原铁同化途径。此外，预测的胞质黄素蛋白必须发挥突出的作用， 保护Tp免受氧化应激和维持NAD+/NADH平衡。这些集体 概念支持，在没有醌的情况下，ATP产生的潜力有限，Tp已经进化 一种“黄素为中心的代谢生活方式”，以满足其对人类感染的代谢要求。这个项目 将解决三个核心代谢特征相关的TP的黄素生物学：蛋白质黄素化和 黄素蛋白生物合成（Aim 1）、还原铁同化和Fe-S蛋白生物合成（Aim 2）、氧化还原 平衡/能量守恒（通过产乙酸）（目标3）。我们还将评估一种小分子 靶向Tp的黄素营养缺陷型的抑制剂作为潜在的新研究工具和/或新的 抗Tp和其他病原性螺旋体的抗菌剂（目标4）。总的来说，该项目将 阐明TP如何进化以利用黄素作为其隐形的基础的关键特征 致病性，可能导致新的战略，以阻止人类感染。

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.