The interplay between nutrient availability and secondary bile acid metabolism in commensal Clostridia mediates colonization resistance against C. difficile

项目摘要/摘要 在Theriot实验室，我们应用尖端技术和高通量方法来分析肠道 使用一系列实验技术和动物模型的微生物组和代谢组。 我们利用了许多 方法横跨多个领域，包括细菌遗传学、细菌生理学、蛋白质工程、 生物化学，并应用各种组学方法(微生物学、转录组学、蛋白质组学和 代谢组学)在体外和体内确定肠道微生物区系如何提供 对艰难梭菌的定植抗性。 本土肠道微生物区系的许多基本功能之一是其维持定殖性的能力 抗药性，并防止病原体在肠道内建立和生长。已经有了大量的 这一领域的研究试图确定肠道微生物区系调节定植的机制 抵抗。潜在的机制包括争夺养分，占据物理空间或生物量， 产生抑制产物，并塑造宿主的免疫反应。一种流行的模式生物，用来 艰难梭状芽孢杆菌是一种对肠道变化非常敏感的细菌 微生物区系的结构和功能。艰难梭菌是一种厌氧、芽胞形成、革兰氏阳性细菌。 1935年分离出来，是艰难梭菌感染(CDI)的病原体。艰难梭菌是如何 从肠道中丧失的定植抵抗力中受益对发展具有重大意义 CDI防治的治疗学。 我的长期目标是了解肠道微生物区系如何调节对艰难梭菌的定植抵抗。 该应用程序的总体目标是确定养分利用率(氨基)之间的关系 在对艰难梭菌的定植抗性背景下的胆汁酸代谢。基于 初步数据我们的假设是，氨基酸的可用性通过以下方式影响次级胆汁酸的产生 共生梭状芽孢杆菌，这将改变对艰难梭菌的定植耐药性。为了调查这件事 假设，我们计划在体外改变限定和丰富介质中的氨基酸丰度，并使用限定饮食 活体以了解这如何影响共生梭状芽胞杆菌的次生胆汁酸的产生。利用我们的 健壮且可复制的无菌和抗生素处理的CDI小鼠模型，我们将确定这些 代谢过程影响艰难梭菌的建立和生长，以及周围的肠道微生物 社区。使用LC-IMS-MS和Protein-SIP等新平台，我们将定义肠道代谢组和 在殖民抵抗的背景下的代谢蛋白质组。 这项拟议的研究的贡献是重大的，因为它寻求摆脱非靶向治疗。 像FMT一样，并朝着有针对性的方法发展，由此我们可以使用饮食(氨基酸)来控制次要的 共生梭状芽孢杆菌产生胆汁酸，恢复对艰难梭菌的定植抗性。调查结果 将促进对微生物-微生物相互作用、宿主-微生物相互作用和 改进以微生物为基础的治疗方法。除了艰难梭菌之外，它还有可能让我们智能地 设计个性化的治疗干预措施，针对复杂生态环境中的人类健康状况 人体肠道的环境。

Project Summary/Abstract In the Theriot laboratory we apply cutting-edge technology and high-throughput methods to analyze the gut microbiome and metabolome using a range of experimental techniques and animal models. We leverage many approaches that span diverse fields including bacterial genetics, bacterial physiology, protein engineering, biochemistry, and apply a variety of omic approaches (microbiomics, transcriptomics, proteomics, and metabolomics) in vitro and in vivo to define the mechanisms behind how the gut microbiota provides colonization resistance against C. difficile. One of the many essential functions of the indigenous gut microbiota is its ability to maintain colonization resistance and to prevent establishment and growth of pathogens in the gut. There has been a great deal of research in this area trying to define the mechanisms by which the gut microbiota mediates colonization resistance. Potential mechanisms include competition for nutrients, taking up physical space or biomass, production of inhibitory products, and shaping the host immune response. A popular model organism used to interrogate these mechanisms is Clostridioides difficile due to its exquisite sensitivity to changes in the gut microbiota structure and function. C. difficile is an anaerobic, spore-forming, Gram-positive bacterium first isolated in 1935 and the causative agent for C. difficile infection (CDI). Unlocking how C. difficile is able to benefit from the loss of colonization resistance in the gut has major implications for development of therapeutics for prevention and treatment of CDI. My long-term goal is to understand how the gut microbiota mediates colonization resistance against C. difficile. The overall objective of this application is to determine the relationship between nutrient availability (amino acids) and bile acid metabolism in the context of colonization resistance against C. difficile. Based on preliminary data our hypothesis is that amino acid availability influences secondary bile acid production by commensal Clostridia, which will alter colonization resistance against C. difficile. In order to investigate this hypothesis, we plan to alter amino acid abundances in defined and rich media in vitro, and use defined diets in vivo to understand how this impacts secondary bile acid production of commensal Clostridia. Leveraging our robust and reproducible germfree and antibiotic treated mouse models of CDI, we will determine how these metabolic processes affect the establishment and growth of C. difficile, as well as the surrounding gut microbial community. Using novel platforms like LC-IMS-MS and Protein-SIP, we will define the gut metabolome and metaproteome in the context of colonization resistance. The contribution of the proposed research is significant as it seeks to move away from untargeted therapies like FMT and move toward a targeted approach, whereby we can use diet (amino acids) to control secondary bile acid production by commensal Clostridia, restoring colonization resistance against C. difficile. The findings in this proposal will advance understanding of microbe-microbe interactions, host-microbe interactions, and improve microbiome-based therapeutics. Beyond C. difficile it has the potential to allow us to intelligently design customized therapeutic interventions to target human health conditions in the complex ecological environment of the human intestine.