Project 3: Microbiota generated aryl sulfates and secondary bile acids in cardiometabolic disease

摘要 一些微生物群衍生的代谢物与人类疾病有关，但微生物群 产生数百种尚未被充分研究的代谢物。此外，许多化学品 拥有尚未被详细探索的疾病联系。在未发表的合作研究中， 项目1使用非靶向代谢组学作为发现平台，我们发现了两种这样的代谢物 其水平与发生心脏代谢疾病的风险相关的成员类别：芳基 硫酸盐和二级胆汁酸。 目标1的焦点是芳基硫酸酯，包括硫酸吲哚酚（IS）和对甲酚硫酸酯（pCS）， 仅来自肠道微生物群产生的代谢物，并且它们的血浆水平与心血管疾病相关。 疾病（CVD）风险。通过肾功能保留受试者的非靶向代谢组学， 血浆中新的（以前未知的）芳基硫酸盐，来源于微生物，与CVD相关。 次级胆汁酸，目的2：胆汁酸池显著浓缩，几乎完全由 (98%+）的微生物组来源的胆汁酸。由于胆汁酸浓度高， 即使占这个池的1%，也是以生物相关浓度存在的。在筛选研究中， 项目1和2（哈岑& Brown）在T2 DM患者中，我们发现两种胆汁酸的血浆水平低于 类与疾病风险显著相关，石胆酸（与CVD直接相关）， isoDCA/isoLCA（与T2 DM呈负相关）。到目前为止，很难直接测试肠道的作用， 由于不能“切换”，微生物群产生代谢物如芳基硫酸盐和次级胆汁酸， 个体代谢物在宿主内的开/关。利用我们在代谢途径发现和 微生物组基因编辑我们已经能够研究两种关键类型的肠道疾病的因果关系和机制 微生物衍生的分子、芳基硫酸盐和次级胆汁酸。 我们在目标1和2中采取了一种平行的方法，从强大的人类代谢组学数据开始， 微生物组衍生的代谢产物对CVD和T2 DM的作用（目的1a/2a）。我们将预测生物合成途径 从这些分析中出现的代谢物（目的1b/2b），在这些突变体中产生功能获得和功能丧失突变体。 途径，并在体外验证它们（目的1c/2c）。然后，我们将在体内通过定殖 无菌小鼠，其品系或群落对仅在感兴趣的代谢产物的产生方面存在差异 (Aims 1 d/2 d）。然后，我们将使用这些精心控制的小鼠来研究微生物酶的作用， 与CVD和T2 DM相关的表型中的目标代谢物（目的1 e-f/2 e-f）。

Abstract A few microbiome-derived metabolites have been implicated in human disease, but the microbiota produce hundreds of additional metabolites that are not well studied. Moreover, many of these chemicals possess disease connections which have not yet been explored in detail. In unpublished collaborative studies with Project 1 using untargeted metabolomics as a discovery platform, we uncovered two such metabolite classes with members whose levels are correlated with the risk of developing cardiometabolic disease: aryl sulfates and secondary bile acids. Aryl sulfates, the focus of Aim 1, including indoxyl sulfate (IS) and p-cresol sulfate (pCS), derive exclusively from gut microbiota generated metabolites and their plasma levels correlate with cardiovascular disease (CVD) risk. Through untargeted metabolomics in subjects with preserved renal function we identified new (previously unknown) Aryl sulfates in plasma that are microbial in origin and associated with CVD. Secondary bile acids, Aim 2: The bile acid pool is remarkably concentrated and consists almost entirely (98%+) of microbiome-derived bile acids. Because bile acids are present in high concentrations, a compound that makes up even 1% of this pool is present at biologically relevant concentrations. In screening studies with Projects 1 & 2 (Hazen & Brown) among T2DM patients we discovered plasma levels of two bile acid sub- classes strikingly correlate with disease risks, lithocholic acid (directly correlated with CVD) and isoDCA/isoLCA (inversely correlated with T2DM). To date, it has been difficult to directly test the role of gut microbiota generated metabolites like aryl sulfates and secondary bile acids due to the inability to `toggle' individual metabolites on/off within a host. Leveraging our expertise in metabolic pathway discovery and microbiome gene editing we have enabled studies of causality and mechanism for two key classes of gut microbiota-derived molecules, aryl sulfates and secondary bile acids. We take a parallel approach in Aims 1 and 2 that starts with powerful human metabolomics data that links microbiome-derived metabolites to CVD and T2DM (Aims 1a/2a). We will predict biosynthetic pathways for metabolites that emerge from these analyses (Aims 1b/2b), create gain- and loss-of-function mutants in these pathways, and validate them in vitro (Aims 1c/2c). We will then validate the pathways in vivo by colonizing germ-free mice with strain or community pairs that differ only in the production of the metabolite of interest (Aims 1d/2d). We will then use these carefully controlled mice to study the role of the microbial enzyme and metabolite of interest in phenotypes relevant to CVD and T2DM (Aims 1e-f/2e-f).