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

摘要 一些微生物组衍生的代谢物与人类疾病有关，但微生物区系 产生数以百计的其他代谢物，这些代谢物没有得到很好的研究。此外，这些化学物质中的许多 拥有尚未被详细探讨的疾病联系。在未发表的合作研究中 在使用非靶向代谢组学作为发现平台的项目1中，我们发现了两种这样的代谢物 成员水平与患心脏代谢性疾病风险相关的类别：芳香族 硫酸盐和次级胆汁酸。 芳基硫酸盐，目标1的焦点，包括吲哚硫酸盐(IS)和对甲酚硫酸盐(PC)，衍生 仅由肠道微生物区系产生的代谢物及其血浆水平与心血管相关 疾病(CVD)风险。通过在肾功能正常的受试者中进行非靶向代谢组学研究，我们发现 血浆中新的(以前未知的)芳基硫酸盐，是微生物来源的，与心血管疾病有关。 次级胆汁酸，目标2：胆酸池非常集中，几乎全部由 (98%+)微生物来源的胆汁酸。因为胆汁酸的浓度很高，一种化合物 即使占这个池的1%，也存在于生物相关的浓度中。在筛查研究中使用 项目1和2(Hazen&Brown)在T2 DM患者中，我们发现两种胆汁酸亚组水平 与疾病风险、石胆酸(与心血管疾病直接相关)和 IsDCA/isoLCA(与T2 DM呈负相关)。到目前为止，还很难直接测试Gut的作用 微生物群产生了像芳基硫酸盐和次级胆汁酸这样的代谢物，这是因为他们不能“切换” 个体代谢物在宿主体内的开启/关闭。利用我们在代谢途径发现和开发方面的专业知识 微生物组基因编辑使我们能够研究两类关键肠道的因果关系和机制 微生物衍生分子、芳基硫酸盐和次级胆汁酸。 我们在目标1和目标2中采取了类似的方法，从强大的人类代谢组学数据开始，将 微生物衍生的代谢物导致心血管疾病和T2 DM(AIMS 1a/2a)。我们将预测生物合成途径 从这些分析中产生的代谢物(目标1b/2b)会在这些基因中产生功能增减突变。 途径，并在体外验证它们(目标1c/2c)。然后我们将通过在体内定植来验证这些途径 无菌小鼠，其品系或群落对仅在感兴趣代谢物的产生上有所不同 (目标为1d/2d)。然后我们将使用这些精心控制的小鼠来研究微生物酶和 与心血管疾病和2型糖尿病相关的表型的感兴趣代谢物(目标1e-f/2e-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 In unpublished collaborative studies with Project 1 using untargeted metabolomics as a discovery platform, we uncovered two such metabolite classes with members whose levels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).