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，是导致显著发病率、死亡率和医疗成本增加的原因。尽管CDI具有重要意义，但我们对这种感染的发病机制的理解仍存在重大差距。抗生素破坏了本地肠道微生物群，降低了对艰难梭菌定植的抵抗力。然而，我们对肠道微生物群如何对CDI产生耐药性的了解还很初级，这对改善针对这种感染的预防和治疗方法提出了重大障碍。我的长期目标是了解胃肠道微生物群如何介导对艰难梭菌的定植抗性。该应用程序的总体目标是确定与肠道微生物群变化相关的代谢物，这些代谢物有助于艰难梭菌的定植和发病机制。使用非靶向代谢组学方法，我们已经证明抗生素治疗小鼠的肠道环境以代谢谱的重大变化为特征。使用抗生素后，我们检测到初级胆汁酸、碳水化合物和氨基酸增加，游离脂肪酸、二级胆汁酸和二肽减少；这反映了肠道微生物群代谢活性的降低。随后，我们证明艰难梭菌可以利用许多这些代谢物进行体外萌发和生长。中心假设是抗生素治疗后支持艰难梭菌生长的特定营养物质的可用性是观察到的定植抗性降低的原因。提出这项研究的基本原理是，了解胃肠代谢组在艰难梭菌发病机制中的作用，有可能改善这种感染的预防和治疗方法。在强有力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验证：1)确定胃肠道中的代谢物

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.