Understanding redox-regulated mechanisms of environmental adaptation in gastrointestinal symbionts

长期定居在宿主体内的细菌，如肠道菌群，必须适应宿主环境中各种形式的压力。细菌用来感知和响应这些环境信号的分子机制对于维持与宿主细胞的共生关系至关重要。氧化应激是宿主-微生物相互作用的一个标志，以其在宿主免疫反应中的作用而闻名；然而，上皮屏障在与微生物接触时也会产生活性氧（ROS）。我的实验室使用化学和遗传工具来定义细菌适应氧化应激的分子机制。我们使用常见的胃共生体幽门螺杆菌来模拟细菌对生理ROS的反应。幽门螺杆菌是胃菌群的一种正常成员，尽管持续暴露于产生ros的上皮细胞，但它可以在宿主体内持续存在数十年，类似于许多共生微生物。利用幽门螺杆菌与胃上皮细胞共培养，我们开发了一种化学蛋白质组学策略，可以识别宿主-微生物界面上ROS的蛋白质靶点。与分析基因表达变化的传统方法不同，我们的方法检测翻译后氧化修饰，即使蛋白质水平没有变化，也可以改变细胞信号。这使我们能够揭示宿主-微生物界面上的氧化还原信号事件，这些事件在很大程度上尚未被探索，并且可能介导细菌对氧化应激的适应。同时，我们正在研究含有硫醇的小分子维持细菌细胞内氧化还原平衡。虽然几乎所有的生命形式都能合成这些低分子量（LMW）硫醇，但某些种类的细菌缺乏低分子量硫醇生物合成所需的典型酶。因此，这些细菌如何在宿主-微生物界面解毒活性氧仍然是一个悬而未决的问题。我们最近发现了一种新的麦角硫因（EGT）细菌转运体，这是一种在动物组织中大量存在的具有强抗氧化特性的LMW硫醇。这种转运体在通常定植于胃肠道的细菌中广泛保守；因此，EGT摄取可能代表了宿主-微生物界面微生物氧化还原调控的新机制。在这个提议中，我们将确定蛋白质氧化和低分子量硫醇运输如何影响细菌对宿主环境的适应。首先，我们将鉴定微生物与产生ROS的真核细胞接触后被氧化的细菌蛋白，并阐明使细菌适应生理ROS的氧化还原信号通路。其次，我们将描述细菌中负责EGT运输的蛋白质，以增加对这一高度保守过程及其在微生物氧化还原生物学中的作用的理解。我们还将使用动物模型来确定EGT摄取如何影响体内微生物定植和EGT代谢。总之，这些研究将定义有助于维持宿主-微生物界面稳态的基本氧化还原信号通路（项目1）和运输机制（项目2）。从长远来看，我们的工作将为研究其他微生物中的这些过程提供一个框架，并可能为合理设计抗感染疗法提供新的靶点。

Bacteria that chronically colonize the host, such as the gut microbiota, must adapt to various forms of stress in the host environment. The molecular mechanisms bacteria use to sense and respond to these environmental signals are crucial for maintaining symbiotic associations with host cells. Oxidative stress is a hallmark of host-microbe interaction best known for its role in the host immune response; however, epithelial barriers also generate reactive oxygen species (ROS) in response to microbial contact. My lab uses chemical and genetic tools to define molecular mechanisms of bacterial adaptation to oxidative stress. We use the common gastric symbiont Helicobacter pylori to model bacterial responses to physiological ROS. H. pylori is a normal member of the gastric flora that can persist for decades in the host despite constant exposure to ROS-generating epithelial cells, similar to many commensal microbes. Using H. pylori co-cultured with gastric epithelial cells, we have developed a chemical proteomic strategy that can identify protein targets of ROS at the host-microbe interface. Unlike conventional methods for analyzing changes in gene expression, our approach detects post-translational oxidative modifications that can alter cell signaling even when protein levels do not change. This allows us to uncover redox-signaling events at the host-microbe interface that are largely unexplored and likely mediate bacterial adaptation to oxidative stress. In parallel, we are investigating thiol-containing small molecules that maintain redox balance within bacterial cells. While these low-molecular-weight (LMW) thiols are synthesized by nearly all life forms, certain classes of bacteria lack the canonical enzymes required for LMW-thiol biosynthesis. Consequently, how these bacteria detoxify ROS at the host-microbe interface remains an open question. We recently discovered a novel bacterial transporter of ergothioneine (EGT), an LMW thiol with potent antioxidant properties that is abundant in animal tissues. This transporter is broadly conserved in bacteria that commonly colonize the gastrointestinal tract; thus, EGT uptake could represent a new mechanism of microbial redox regulation at the host-microbe interface. In this proposal, we will determine how protein oxidation and LMW-thiol transport shape bacterial adaptation to the host environment. First, we will identify bacterial proteins that are oxidized following microbial contact with ROS-generating eukaryotic cells and elucidate the redox-signaling pathways that enable bacterial adaptation to physiological ROS. Second, we will characterize the proteins responsible for EGT transport in bacteria to increase understanding of this highly conserved process and its role in microbial redox biology. We will also use animal models to determine how EGT uptake influences microbial colonization and EGT metabolism in vivo. Together, these studies will define fundamental redox-signaling pathways (project 1) and transport mechanisms (project 2) that help maintain homeostasis at the host-microbe interface. In the long term, our work will provide a framework for investigating these processes in other microbes and could reveal new targets for the rational design of anti-infective therapies.