Pregnane X receptor (PXR)-activating gut bacterial metabolites

肠道微生物区系通常通过产生小分子来调节人类的健康和疾病。 代谢物；然而，大多数肠道微生物代谢物的特性及其生物学作用 在很大程度上仍然不为人知。孕烷X受体(PXR)是一种配体激活的转录因子 通过结构多样化的化学物质。PXR调节药物代谢编码基因的表达 酶包括细胞色素P450 3A4(CYP3A4)。此外，肠道和肝脏中的PXR已被 与维持肠道屏障和免疫功能以及能量平衡有关。 越来越多的证据表明，肠道微生物群会产生PXR配体。到目前为止，几乎没有细菌 据报道，代谢物是小鼠或人的PXR配体，系统地努力识别PXR- 活性、肠道微生物代谢物一直缺乏。这种知识的缺乏构成了在 确定肠道微生物区系如何改变人类健康和疾病，特别是通过PXR。我们的长期目标是 研究的目的是识别和描述影响肠道微生物-宿主相互作用的因素。总体目标 这一应用的目的是识别肠道细菌代谢产物，激活配体激活的转录因子PXR 具有多种生物学功能，包括调节药物代谢酶和 维持肠道内环境平衡。我们的中心假设是肠道细菌产生的代谢物能够激活 PXR.我们的假设基于以下初步结果：(1)在高表达人 PXR(HepG2/hPXR)，小鼠盲肠内容物有机提取物显著诱导hPXR反式激活 PXR靶基因(即CYP3A4)启动子的缺失，表明肠道微生物中存在hPXR激活子 产品。(2)从10种常见的人体肠道细菌中筛选出核梭杆菌。 作为hPXR激活剂的生产者(S)；在HepG2/hPXR细胞中，核盘藻的有机提取物培养物 培养上清可显著诱导CYP3A4启动子的hPXR反式激活。(3)我们提高了玉米产量 通过改变生长介质，获得了核藻的活性代谢物，并获得了质子核磁共振和高密度脂蛋白。 一种活性代谢物富集组分的分辨率质谱仪结果。结果表明，PXR- 核盘藻活性代谢产物(S)可能是新化合物。总而言之，我们的数据表明 细菌产生未知的代谢物(S)来激活PXR。基于我们的发现，我们建议(1) 确定肠道细菌衍生的代谢物促进hPXR反式激活CYP3A4表达和(2) 确定核盘藻中产生hPXR激活子(S)的遗传决定因素。成功完成 这些研究将为未来的调查奠定基础，为调查提供急需的工具集 肠道微生物区系调节PXR活性的潜在分子机制。总而言之，这些努力 将加深我们对肠道微生物区系如何控制宿主生理的理解。

The gut microbiota modulates human health and disease often via the production of small molecule metabolites; however, the identities of the majority of gut microbial metabolites and their biological actions remain largely unknown. Pregnane X receptor (PXR) is a ligand-activated transcription factor that is activated by structurally diverse chemicals. PXR regulates the expression of genes encoding drug-metabolizing enzymes including cytochrome P450 3A4 (CYP3A4). Additionally, PXR in the intestine and liver has been implicated in the maintenance of gut barrier and immune functions as well as energy homeostasis. Accumulating evidence suggests that the gut microbiota produces PXR ligands. To date, few bacterial metabolites have been reported as mouse or human PXR ligands, and systematic efforts to identify PXR- activating, gut microbial metabolites have been lacking. This lack of knowledge constitutes a substantial gap in defining how the gut microbiota alters human health and disease especially via PXR. The long-term goal of our research is to identify and characterize factors mediating gut microbes-host interactions. The overall objective of this application is to identify gut bacterial metabolites activating PXR, a ligand-activated transcription factor with pleiotropic biological functions including the regulation of drug-metabolizing enzymes and the maintenance of gut homeostasis. Our central hypothesis is that gut bacteria produce metabolites that activate PXR. Our hypothesis is based on the following preliminary results: (1) In HepG2 cells overexpressing human PXR (HepG2/hPXR), the organic extracts of mouse cecum contents significantly induced hPXR transactivation of PXR target gene (i.e., CYP3A4) promoter, indicating the presence of hPXR activators among gut microbial products. (2) A screening of 10 common human gut bacteria led to identification of Fusobacterium nucleatum as a producer of hPXR activator(s); in HepG2/hPXR cells, the organic extracts of F. nucleatum culture supernatants significantly induced hPXR transactivation of the CYP3A4 promoter. (3) We increased the yield of the active metabolite from F. nucleatum by changing the growth media and obtained a proton NMR and high- resolution mass spectrometry results of an active metabolite-enriched fraction. The results suggest that PXR- activating metabolite(s) of F. nucleatum are likely novel compounds. Together, our data indicate that gut bacteria produce as-yet-unknown metabolite(s) that activate PXR. Based our findings, we propose to (1) identify gut bacteria-derived metabolites that promote hPXR transactivation of CYP3A4 expression and (2) define genetic determinants for the production of hPXR activator(s) in F. nucleatum. Successful completion of these studies will set the stage for future investigation, providing much-needed tool sets to investigate underlying molecular mechanisms for how the gut microbiota modulates PXR activity. Together, these efforts will enhance our understanding of how the gut microbiota controls host physiology.