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

项目总结 这个项目将检验细菌胆盐水解酶(BSH)活性调节的假设。 肠道神经系统中的信号通路以剂量依赖的方式促进肠道运动 (ENS)包括RET，一种对ENS的发育和功能至关重要的酪氨酸激酶受体， 以及TGR5，这是已知的唯一由肠道神经元表达的胆汁酸受体。去探索 在这个假设中，我将集成各种技术来产生函数读数，结合一个 饮食依赖胆汁酸介导的运动表型成团的灵知生菌小鼠模型 光谱学、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。第一，细菌菌株 从单个健康的孟加拉人的微生物群中培养出来的将被归类为 用建立的体外筛选法检测具有高、中或低BSH活性的。这些 菌株已经过初步筛选，以确定是否存在bsh；在这里，我 将用时间尺度分辨率低至3小时的时间尺度来表征BSH活动，并相对于 小鼠体内两种主要的胆汁酸底物(牛磺胆酸和牛磺酸-β-小鼠胆酸 酸)。然后，选择高/中/低bsh活性的菌株将被移植到GnotoBiotic中。 以确定(I)BSH活动与肠道运动的相关性程度以及(Ii)是否 BSH的运动效应对分类多样性是强大的(对于理解 结论的概括性)。在未来，我的目标是启动一项临床研究，以测试 腹泻患者肠道细菌BSH活性与运动功能相关的假说-或 便秘-主要的肠易激综合征，以确定哪些亚群 细菌胆汁酸代谢可作为治疗靶点。在目标2中，我建议确定 微生物组编码的bsh活性对肠道运动影响的关键ENS中介因子。至 确定TGR5胆汁酸受体是否负责调节这种胆汁酸- 依赖反应，我将使用常规饲养的和诺生菌TGR5-/-小鼠。要确定 关键的ENS分子介体和途径，我将采用TRAP-SEQ(翻译核糖体 亲和纯化测序)，这是一项研究不同种群的 小鼠的大脑，以描述灵知生菌小鼠的ENS。使用这种方法，我将阐明 促胆剂(姜黄素)和模拟人体肠道细菌群落的作用 确定了小肠和结肠ENS转录组上的BSH活性，相关 这些信号与测量的通过时间和胆汁酸特征有关。总而言之，这些数据获得 将有助于剖析细菌胆汁酸在多大程度上 新陈代谢调节肠道运动，并提供关于相互作用的机械性见解 在饮食成分、肠道微生物群和ENS信号通路之间调节 能动性。未来的后续研究将涉及将未培养的完整微生物群从 选择表型良好的患有运动障碍的人作为灵知生菌小鼠。

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.