Microbial regulation of DNA repair in intestinal epithelial cells

背景 肠道含有丰富的微生物和肠道上皮细胞(IEC)，它们对调节这些微生物与宿主的相互作用至关重要。IEC表达大量的模式识别受体(PRR)，使它们能够感知微生物并对其做出反应。这一点至关重要，因为微生物可以产生破坏DNA的产物，而IEC非常容易积累突变，因为它们经常分裂，每次都必须复制它们的DNA。虽然许多保护性DNA修复途径在IEC中运作，但这些修复机制如何由肠道微生物产物调节的基本问题仍然没有得到回答，尽管这种调节途径的存在可能会对基本肠道生理学产生重要影响。 目标和目的 该计划的总体目标是确定肠道微生物区系在调节宿主细胞DNA修复中的作用。我们将通过解决三个目标来实现这一目标。 目的1.确定不同PRR在IEC上的激活是否调节DNA修复机制。我们的工作将集中在PRR的Toll样受体(TLR)类别上。我们将把IEC暴露在可激活不同TLR的微生物分子中，并评估这如何改变DNA修复蛋白的表达和每条DNA修复途径的效率。 目的2.确定微生物代谢产物如何改变DNA修复和诱导DNA损伤。微生物代谢的副产物对IEC有多种作用，但对IEC DNA修复的影响尚不清楚。我们将检测IEC在接触特定微生物代谢物时不同DNA修复途径的活性，并确定IEC是否更容易受到可能改变其功能的突变的影响。 目的3.确定饮食如何调节微生物区系引起的肠道DNA损伤。饮食在决定肠道微生物区系产生哪些代谢物方面起着重要作用。然而，人们对哪些饮食因素调节直接针对DNA的微生物产品的产生或IEC DNA修复的效率知之甚少。我们将使用细胞培养和小鼠实验来评估不同的营养物质是否直接改变了这些化合物的产生，或者改变了微生物调节DNA修复的能力。 冲击 DNA修复是一个基本的生物学过程，在快速分裂的IEC中尤为重要。拟议的项目将为我们的团队和其他人未来的研究建立一个必要的框架，因为它将从根本上了解特定的微生物物种和饮食成分如何影响调节肠道生理的Lynchpin细胞类型。这个项目还将使我团队中的学生实习生接触到令人兴奋的新技术和合作，这将帮助他们发展技能和人际关系，这将有助于他们未来的科学职业生涯。

BACKGROUND The intestine contains an abundant colony of microorganisms and intestinal epithelial cells (IEC) that are critical for regulating the interaction of these microbes with the host. IEC express numerous pattern recognition receptors (PRR) that allow them to sense and respond to microbes. This is critical since microbes can produce products that damage DNA and IEC are very susceptible to accumulating mutations because they divide frequently and must reproduce their DNA each time. While numerous protective DNA repair pathways operate within IEC, the basic question of how these repair mechanisms are regulated by intestinal microbial products remains unanswered despite the fact that the existence of such regulatory pathways could have important consequences for fundamental intestinal physiology. OBJECTIVE AND AIMS The overall objective of the proposed program is to characterize the role of intestinal microbiota in regulating DNA repair in host cells. We will accomplish this by addressing three aims. Aim 1. Determine if the activation of different PRR on IEC regulates DNA repair mechanisms. We will focus our work on the Toll-like receptors (TLR) category of PRR. We will expose IEC to microbial molecules known to activate different TLR and evaluate how this changes DNA repair protein expression and the efficiency of each DNA repair pathway. Aim 2. Determine how microbial metabolic products alter DNA repair and induce DNA damage. The byproducts of microbial metabolism exert many effects on IEC yet their influence on IEC DNA repair is unknown. We will examine the activity of different DNA repair pathways in IEC when exposed to specific microbial metabolites and determine if the IEC become more or less susceptible to the development of mutations that could alter their function. Aim 3. Determine how diet regulates microbiota-induced DNA damage in the intestine. Diet plays a significant role in determining what metabolites are produced by intestinal microbiota. However, little is known about what dietary factors regulate the generation of microbial products that directly target DNA or the efficiency of IEC DNA repair. We will use cell culture and mouse experiments to evaluate if different nutrients directly alter production of these compounds or alter the ability of microbes to regulate DNA repair. IMPACT DNA repair is a fundamental biological process that is especially important in rapidly dividing IEC. The proposed project will establish a necessary framework for future research by our team and by others since it will generate a fundamental understanding of how specific microbial species and dietary components can influence the lynchpin cell type regulating intestinal physiology. This project will also expose student trainees in my group to exciting new techniques and collaborations that will help them develop both skills and relationships that will benefit their future scientific careers.