Understanding redox-regulated mechanisms of environmental adaptation in gastrointestinal symbionts

了解胃肠道共生体环境适应的氧化还原调节机制

基本信息

批准号：
10222730
负责人：
Stavroula Hatzios
金额：
$ 49.68万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-01 至 2025-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10222730
关键词：
Anabolism Animal Model Anti-Infective Agents Antioxidants Bacteria Bacterial Infections Bacterial Model Bacterial Proteins Biology Cells Chemicals Chronic Diagnosis Environment Enzymes Epithelial Epithelial Cells Equilibrium Eukaryotic Cell Event Exposure to Gene Expression Goals Helicobacter pylori Homeostasis Immune response Life Mediating Metabolism Methods Microbe Modification Molecular Molecular Weight Oxidation-Reduction Oxidative Stress Oxides Pathway interactions Physiological Prevention Process Property Proteins Proteomics Reactive Oxygen Species Regulation Role Shapes Signal Pathway Signal Transduction Stomach Stress Sulfhydryl Compounds Work animal tissue chemical genetics commensal microbes design environmental adaptation gastrointestinal gut colonization gut microbiota host colonization host-microbe interactions in vivo member microbial microbial colonization novel oxidation response small molecule symbiont tool uptake

项目摘要

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.

长期定植宿主的细菌，例如肠道菌群，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联至关重要。氧化应激是宿主 - 微生物相互作用的标志，以其在宿主免疫反应中的作用而闻名。但是，上皮屏障还会响应微生物接触而产生活性氧（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型幽门螺杆菌对生理ROS的细菌反应进行建模。幽门螺杆菌是胃菌群的正常成员，尽管持续暴露于ROS生成的上皮细胞，但类似于许多共生微生物，但仍可以在宿主中持续数十年。使用幽门螺杆菌与胃皮细胞共培养，我们开发了一种化学蛋白质组学策略，可以鉴定在宿主微叶界面上ROS的蛋白质靶标。与分析基因表达变化的常规方法不同，我们的方法检测到翻译后的氧化修饰，即使蛋白质水平不变，也可以改变细胞信号传导。这使我们能够在很大程度上未开发的宿主微粒界面上发现氧化还原的信号事件，并可能介导细菌适应氧化应激。同时，我们正在研究含硫醇的小分子，这些分子在细菌细胞中维持氧化还原平衡。尽管这些低分子量（LMW）硫醇几乎由所有生命形式合成，但某些类别的细菌缺乏LMW-硫醇生物合成所需的规范酶。因此，这些细菌如何在宿主微型界面上排毒ROS仍然是一个空旷的问题。我们最近发现了一种新型的细菌转运蛋白（EGT），EGT（EGT）是一种具有有效抗氧化特性的LMW硫醇，在动物组织中很丰富。该转运蛋白在细菌中广泛保守，通常在胃肠道上定居。因此，EGT摄取可以代表宿主微生物界面上微生物氧化还原调节的新机制。在此提案中，我们将确定蛋白质氧化和LMW-硫醇如何将细菌适应对宿主环境的适应。首先，我们将鉴定细菌蛋白与生成Ros的真核细胞接触后被氧化的细菌蛋白，并阐明氧化还原信号途径，从而使细菌适应生理ROS。其次，我们将表征负责细菌中EGT转运的蛋白质，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。这些研究将共同定义基本的氧化还原信号途径（项目1）和运输机制（项目2），这些途径有助于维持在宿主微球界面的体内平衡。从长远来看，我们的工作将提供一个框架来研究其他微生物中的这些过程，并可以揭示抗感染疗法合理设计的新目标。