Shifts in the Gastrointestinal Metabolome During Clostridium difficile Infection

描述(由申请人提供)：艰难梭菌感染(CDI)是抗生素相关性结肠炎的主要原因，并导致显著的发病率、死亡率和医疗费用增加。尽管CDI很重要，但在我们对这种感染的发病机制的理解上仍存在重大差距。抗生素破坏了本土肠道微生物区系，降低了对艰难梭菌定植的抵抗力。然而，我们对肠道微生物区系如何对CDI产生抵抗力的了解是基本的，这为改进预防和治疗这种感染的方法提供了一个重要的障碍。我的长期目标是了解胃肠道微生物区系如何调节对艰难梭菌的定植耐药性。这项应用的总体目标是定义与肠道微生物区系变化相关的代谢物，这些代谢物有助于艰难梭菌的定植和发病。使用非靶向代谢组学方法，我们已经表明，抗生素治疗的小鼠的肠道环境的特征是代谢谱发生了重大变化。在给予抗生素后，我们检测到初级胆汁酸、碳水化合物和氨基酸的增加，而游离脂肪酸、次级胆汁酸和二肽的减少；反映了肠道微生物组代谢活动的减弱。随后，我们证明艰难梭菌可以利用这些代谢物中的许多进行体外萌发和生长。中心假设是，在抗生素治疗后，支持艰难梭菌在GT生长的特定营养物质的可用性是观察到的定植耐药性下降的原因。这项拟议研究的基本原理是，了解艰难梭菌的胃肠代谢组在致病机制中所起的作用，有可能改进这种感染的预防和治疗方法。在强大的初步数据的指导下，这一假设将通过追求两个特定目标来检验：1)识别胃肠道中的代谢物 与艰难梭菌的定植和致病有关；以及2)确定调节艰难梭菌致病的肠道代谢物的生理浓度。在第一个具体项下 目的：我们将使用非靶向代谢组学方法从CDI之前和感染的不同阶段的小鼠胃肠道中识别候选生物标记物。在第二个特定目标下，我们将使用靶向代谢组学方法来确认和定量在特定目标1、CDI之前和感染不同阶段受到显著影响的代谢物。我们还将使用体外研究来确认它们在艰难梭菌的萌发、生长和毒素产生中所起的作用。这种方法是创新的，因为我们正在以一种不同的方式使用新的质谱学技术，以帮助解决将改善公众健康的重要生物学问题。这项拟议的研究意义重大，因为它将导致识别新的生物标记物和潜在的治疗干预目标，以预防或治疗CDI。我的整个职业目标是建立一个独立的研究生涯，连接代谢组学和生物医学传染病领域，重点是了解艰难梭菌的发病机制。我的长期研究兴趣一直包括研究疾病的影响以及它如何影响人类健康。随着“组学”技术的出现，复杂的社区，包括胃肠道，可以被定义。文森特·杨博士和查尔斯·伯兰特博士的专业知识和共同指导确保了这一研究项目的成功，并确保了我作为研究科学家继续取得成功。我的导师和合作者共享的综合资源将使我能够访问艰难梭菌感染的小鼠模型和代谢组学核心设施，其中包括访问最先进的质谱学设备和训练有素的代谢组学专家。最后，这些研究将使我有机会学习与代谢组谱分析这一新兴领域相关的方法，包括研究设计、样品制备、代谢物提取和分析，以及数据分析和获得数据的生物信息学解释的过程。这项建议中详述的导师计划，以及进一步的伦理学、生物信息学、统计学和代谢组学研讨会等教学课程，将帮助我成为生物医学传染病和代谢组学领域的独立研究员。

DESCRIPTION (provided by applicant): Clostridium difficile infection (CDI) is the leading cause of antibiotic-associated colitis and is responsible for significant morbidity, mortality and increased healthcare costs. Despite the significance of CDI, there are major gaps in our understanding of the pathogenesis of this infection. Antibiotics disrupt the indigenous gut microbiota, reducing resistance to C. difficile colonization. However, our knowledge of how the gut microbiota confers resistance to CDI is rudimentary, presenting a significant roadblock to improving preventative and therapeutic approaches against this infection. My long-term goal is to understand how the gastrointestinal tract microbiota mediates colonization resistance against C. difficile. The overall objective of this application is to define metabolites associated with changes in the gut microbiota that contribute to C. difficile colonization and pathogenesis. Using an untargeted metabolomics approach, we have shown that the intestinal environment of antibiotic-treated mice was characterized by major shifts in metabolic profiles. Following antibiotic administration, we detected increases in primary bile acids, carbohydrates, and amino acids and decreased free fatty acids, secondary bile acids and dipeptides; reflecting the diminished metabolic activity of the gut microbiome. Subsequently, we demonstrated that C. difficile could utilize many of these metabolites for in vitro germination and growth. The central hypothesis is that the availability of specific nutrients that support C. difficile growth in the gt after antibiotic treatment is responsible for the observed decrease in colonization resistance. The rationale for the proposed research is that understanding the role the gastrointestinal metabolome plays in C. difficile pathogenesis has the potential to improve preventative and therapeutic approaches for this infection. Guided by strong preliminary data, this hypothesis will be tested by pursuing two specific aims: 1) Identify metabolites in the gastrointestinal tract that contribute to C. difficile colonization and pathogenesis; and 2) Determine the physiological concentrations of gut metabolites that modulate C. difficile pathogenesis. Under the first specific aim, we will use an untargeted metabolomics approach to identify candidate biomarkers from the murine gastrointestinal tract prior to CDI and during different stages of infection. Under the second specific aim, we will use a targeted metabolomics approach to confirm and quantitate metabolites that were significantly affected in specific aim 1, prior to CDI and during different stages of infection. We will also use in vitro studies to confirm their role in C. difficile germination, growth and toxin production. The approach is innovative, because we are using new mass spectrometry technology in a different way, to help solve important biological questions that will improve public health. The proposed research is significant, because it will lead to the identification of novel biomarkers and potential targets for therapeutic interventions to prevent or treat CDI. My overall career goal is to establish an independent research career bridging the field of metabolomics and biomedical infectious diseases, with emphasis on understanding Clostridium difficile pathogenesis. My long-term research interests have always included studying the impact of disease and how it impacts human health. With the advent of "omics" technologies complex communities, including the gastrointestinal tract, can be defined. The expertise and co-mentorship of both Dr. Vincent Young and Dr. Charles Burant ensures success of this research project and my continued success a research scientist. The combined resources that my mentors and collaborators share will allow me access to mouse models of C. difficile infection and the Metabolomics Core Facility, which includes access to state of the art mass spectrometry equipment and trained experts in metabolomics. Finally, these studies will provide me with the opportunity to learn the methodologies related to the emerging field of metabolomic profiling, including the design of studies, sample preparation, metabolite extraction and analysis by the latest mass spectrometer based methods and the processes of data analysis and bioinformatics interpretation of the acquired data. The mentorship plan detailed in this proposal and further didactic coursework in ethics, bioinformatics, statistics, and workshops on metabolomics will help me to become an independent researcher in the field of biomedical infectious diseases and metabolomics.