The gut microbiota, bile acid-mediated enteric nervous system signaling, and modulation of gastrointestinal motility

项目摘要 本项目将测试细菌胆盐水解酶（BSH）活性调节 通过肠神经系统中的信号传导途径以剂量依赖性方式影响肠道运动 (ENS)包括RET，一种对ENS发育和功能至关重要的酪氨酸激酶受体， 和TGR 5，已知由肠神经元表达的唯一胆汁酸受体。探讨 这个假设，我将整合各种技术，以产生功能读数，结合 饮食依赖性胆汁酸介导的运动表型的无菌小鼠模型 光谱分析、下一代测序和生物信息学。在目标1中，我建议确定 肠道运动受细菌BSH活性调节的程度。首先，细菌菌株 从一个健康的孟加拉国人的微生物群中培养出来的细菌将被归类为 使用已建立的体外筛选具有高、中水平或低BSH活性。这些 已对菌株进行了初步筛选，以确定是否存在BSH;在此， 将以低至3小时的时标分辨率表征BSH活动， 小鼠中两种主要的主要胆汁酸底物（牛磺胆酸和牛磺-β-鼠胆酸 酸）。然后，选择具有高/中/低BSH活性的菌株将被移植到gnotobiotic 小鼠，以确定（i）BSH活性与肠道运动性相关的程度和（ii）是否 BSH的运动效应对分类多样性是稳健的（对于理解 结论的普遍性）。将来，我的目标是启动一项临床研究来测试 假设肠道细菌BSH活性与腹泻患者的运动相关-或 便秘为主的肠易激综合征，以确定亚群，其中 细菌胆汁酸代谢可以作为治疗靶点。在目标2中，我建议确定 微生物组编码的BSH活性对肠道运动的影响的关键ENS介导剂。到 确定TGR 5胆汁酸受体是否负责介导这种胆汁酸- 依赖性反应，我将使用常规饲养的和无菌Tgr 5-/-小鼠。以识别 关键ENS分子介质和途径，我将采用TRAP-Seq（翻译核糖体 亲和纯化测序），这是一种开发用于研究不同群体的 小鼠脑，以在gnotobiotic小鼠中描绘ENS。通过这种方法，我将阐明 胆汁动力剂（姜黄）和模型人肠道细菌群落的作用， 在小肠和结肠中ENS转录组上定义的BSH活性， 这些信号与测量的通过时间和胆汁酸谱。这些数据一起， 将有助于分析细菌胆汁酸 代谢调节肠道运动，并提供有关相互作用的机制见解 饮食成分、肠道微生物组和调节 能动性未来的后续研究将涉及将完整的未培养的微生物群从 选择具有运动障碍的良好表型的人到gnotobiotic小鼠中。

PROJECT SUMMARY This project will test the hypothesis that bacterial bile salt hydrolase (BSH) activity regulates gut motility in a dose-dependent manner via signaling pathways in the enteric nervous system (ENS) that include RET, a tyrosine kinase receptor critical for ENS development and function, and TGR5, the only bile acid receptor known to be expressed by enteric neurons. To explore this hypothesis, I will integrate various technologies to produce functional readouts, combining a gnotobiotic mouse model of diet-dependent bile acid-mediated motility phenotypes with mass spectrometry, next-generation sequencing, and bioinformatics. In Aim 1, I propose to determine the extent to which gut motility is regulated by bacterial BSH activity. First, bacterial strains cultured from the microbiota of a single healthy Bangladeshi individual will be classified as possessing high, mid-level, or low BSH activity using an established in vitro screen. These strains have undergone preliminary screening to establish presence or absence of BSH; here I will characterize BSH activity with time-scale resolution down to 3 hours and with respect to the two predominant primary bile acid substrates in mice (taurocholic acid and tauro-beta-muricholic acid). Then, selected strains with high/mid/low BSH activity will be transplanted into gnotobiotic mice to determine (i) the extent to which BSH activity correlates with gut motility and (ii) whether BSH's motility effects are robust to taxonomic diversity (essential for understanding generalizability of the findings). In the future, I aim to initiate a clinical study to test the hypothesis that gut bacterial BSH activity correlates with motility in patients with diarrhea- or constipation-predominant irritable bowel syndrome, in order to identify subsets in whom bacterial bile acid metabolism could serve as a therapeutic target. In Aim 2, I propose to identify key ENS mediators of the effects of microbiome-encoded BSH activity on gut motility. To determine whether the TGR5 bile acid receptor is responsible for mediating this bile acid- dependent response, I will use conventionally raised and gnotobiotic Tgr5-/- mice. To identify key ENS molecular mediators and pathways, I will adopt TRAP-Seq (translating ribosome affinity purification sequencing), a technology developed to study distinct populations of the mouse brain, to profile the ENS in gnotobiotic mice. Using this approach, I will elucidate the effects of a cholekinetic agent (turmeric) and model human gut bacterial communities with defined BSH activity on the ENS transcriptome in the small intestine and colon, correlating these signals with measured transit times and bile acid profiles. Together, these data obtained from humanized gnotobiotic mice will help dissect the extent to which bacterial bile acid metabolism regulates gut motility and provide mechanistic insights regarding interactions between dietary ingredients, the gut microbiome, and ENS signaling pathways that regulate motility. Follow-up future studies will involve transplanting intact uncultured microbiota from selected well-phenotyped humans with motility disorders into gnotobiotic mice.