Activity-based profiling of bile salt hydrolases in the gut microbiome in health and disease

项目总结/摘要 人类肠道内有数万亿种微生物，称为肠道微生物群， 与我们自身细胞的数量相匹敌。这些微生物一起在肠道内代谢小分子， 通过进行生化转化的细菌酶的活动形成管腔。越来越多的证据 表明这些小分子代谢物赋予宿主免疫和生理学主要益处。然而，在这方面， 产生这些分子的酶和生化途径仍然知之甚少。 该提案旨在开发化学方法来了解肠道的代谢活动 微生物组，以更好地了解代谢产物在肠道中的生产，以及它如何有助于健康和疾病。 指导这项工作的首要假设是，基于活性的分析可用于识别活性胆汁 肠道微生物组内的盐水解酶（BSH），其产生细菌修饰的胆汁酸， 在生理学和疾病中的重要功能。我们将通过以下研究来解决这一假设： 开发选择性化学探针标记活性胆盐水解酶。建立在我们强大的 基于一种新的基于活性的探针，可以标记活性BSH的初步数据，我们将开发改进的 探针对这种关键酶的不同亚型表现出更大的选择性和特异性， 不同的底物偏好，并由各种细菌菌株产生。这组化学探针 将使人们能够更好地了解肠道微生物组中不同细菌菌株的BSH活性。 从健康和疾病中的小鼠和人类肠道微生物组中分析活性胆盐水解酶。 建立在初步数据的基础上，表明结肠炎中BSH活性的变化，这与生态失调有关， 我们将把我们的优化探针组应用于小鼠和人类肠道微生物组，以分析 健康和疾病，使用结肠炎的小鼠模型和人类患者样品。这些结果将提供信息， 在健康和炎症性疾病期间BSH活性的变化受肠道微生物组的影响。 可视化胆盐水解酶活性在小鼠和人类肠道在健康和疾病。我们将 应用化学探针对健康的小鼠和人类肠道组织中的活性BSH进行成像， 和疾病这些研究将确定积极代谢的肠道细菌小生境的定位 胆汁酸在健康和炎性疾病期间受到肠道微生物组生态失调的影响，例如，结肠炎 目前基于宏基因组学的技术在报告基因的能力方面是有限的， 存在于微生物组中。我们的化学方法将确定肠道内酶的活性 微生物组进行调节宿主生理学的重要小分子代谢物的代谢， 病理总的来说，我们的工具将有助于更深入地了解肠道中宿主-微生物组的相互作用。 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.