ACTIVITY-BASED PROFILING OF BILE SALT HYDROLASES IN THE GUT MICROBIOME IN HEALTH AND DISEASE

项目摘要/摘要 人类的肠道被数以万亿计的微生物定居，这些微生物被称为肠道微生物区系，被认为是 与我们自己的细胞数量相匹敌。这些微生物共同代谢肠道内的小分子。 管腔通过执行生化转换的细菌酶的活动来实现。越来越多的证据 表明这些小分子代谢物对宿主免疫和生理有很大的好处。然而， 产生这些分子的酶和生化途径仍然知之甚少。 这项提议寻求开发化学方法来了解肠道的新陈代谢活动 微生物组，以更好地了解肠道中代谢物的产生及其对健康和疾病的贡献。 指导这项工作的最重要的假设是，基于活动的分析可以用来识别活跃的胆汁 肠道微生物体内的盐水解酶(BSHS)，它产生细菌修饰的胆汁酸，具有 在生理学和疾病中的重要作用。我们将通过以下研究来解决这一假设： 开发标记活性胆盐水解酶的选择性化学探针。建立在我们强大的 初步数据基于一种新的基于活动的探针可以标记活性BSH，我们将开发改进的 对这种关键酶的不同亚型表现出更高的选择性和特异性的探针 不同的底物偏好，由不同的细菌菌株产生。这块化学探针板 将使人们能够更好地了解肠道微生物群中不同细菌菌株的BSH活性。 从健康和疾病的小鼠和人类肠道微生物群中提取活性胆盐水解酶。 基于初步数据显示结肠炎中BSH活性的变化，这与生物失调有关， 我们将把我们的一组优化的探针应用于小鼠和人类肠道微生物群，以分析活性BSHS在 健康和疾病，使用结肠炎的小鼠模型和人类患者样本。这些结果将在 受肠道微生物群影响的健康和炎症性疾病期间BSH活性的变化。 显示健康和疾病中小鼠和人类肠道中胆盐水解酶的活性。我们会 应用化学探针对健康小鼠和人类肠道组织内活性BSH进行成像 和疾病。这些研究将确定活跃代谢的肠道细菌生态位的定位。 健康期间的胆汁酸和受肠道微生物群失调影响的炎症性疾病，如结肠炎。 目前基于元基因组学的技术在报告以下基因方面能力有限 存在于微生物群中。我们的化学方法将定义肠道内酶的活性 微生物组进行重要的小分子代谢物的代谢，调节宿主的生理和 病理学。总的来说，我们的工具将有助于更深入地了解肠道中宿主-微生物群的相互作用 以及这种关系如何影响人类健康和疾病。

Project Summary/Abstract The human intestines are colonized by trillions of microorganisms, termed the gut microbiota, which are thought to rival the number of our own cells. Together, these microbes metabolize small molecules within the intestinal lumen through the activities of bacterial enzymes that carry out biochemical transformations. Growing evidence suggests that these small-molecule metabolites confer major benefits to host immunity and physiology. However, the enzymes and biochemical pathways that produce these molecules remain poorly understood. This proposal seeks to develop chemical approaches to understand the metabolic activity of the gut microbiome to better understand metabolite production in the gut and how it contributes to health and disease. The overarching hypothesis guiding this work is that activity-based profiling can be used to identify active bile salt hydrolases (BSHs) within the gut microbiome, which produce bacterially-modified bile acids that have important functions in physiology and disease. We will address this hypothesis with the following studies: Develop selective chemical probes for labeling active bile salt hydrolases. Building on our strong preliminary data based on a novel activity-based probe that can label active BSH, we will develop improved probes that exhibit greater selectivity and specificity for different isoforms of this critical enzyme that have different substrate preferences and are produced by various strains of bacteria. This panel of chemical probes will enable a greater understanding of BSH activities from diverse bacterial strains within the gut microbiome. Profile active bile salt hydrolases from mouse and human gut microbiomes in health and disease. Building on preliminary data demonstrating changes in BSH activity in colitis, which is associated with dysbiosis, we will apply our panel of optimized probes to mouse and human gut microbiomes to profile active BSHs in health and disease, using both mouse models of colitis and human patient samples. These results will inform on changes in BSH activity during health and inflammatory diseases that are influenced by the gut microbiome. Visualize bile salt hydrolase activity in mouse and human intestines in health and disease. We will apply the chemical probes to image active BSH within the intestinal tissue from mice and humans in both health and disease. These studies will determine the localizations of gut bacterial niches that are actively metabolizing bile acids during health and inflammatory diseases that are affected by gut microbiome dysbiosis, e.g., colitis. Current technologies based on metagenomics are limited in their ability to report on genes that are present within the microbiome. Our chemical approach will define how activities of enzymes within the gut microbiome carry out metabolism of important small-molecule metabolites that regulate host physiology and pathology. Broadly, our tools will contribute to a deeper understanding of host-microbiome interactions in the gut and how this relationship influences human health and disease.