Characterization of bacterial reductases acting on the A-ring of 11-oxy-androgens

项目概要/摘要 Nabarro 等人早在 1957 年的文献就证明，皮质醇等糖皮质激素可以 转化为强效雄激素，如羟基雄烯二酮 (11β-OHAD) (1,2)。然而直到1981年 皮质醇的侧链裂解首次在人类粪便悬浮液中观察到 (3)。 1984年，研究人员 分离出具有类固醇 17,20-去氨酶 (SD) 能力的人类肠道细菌 Clostridium scindens (4,5)。里德隆 等人 (2013) 利用 RNA-Seq 来鉴定皮质醇诱导基因 (desAB)，后来我们的研究人员证实了这一点 实验室对 SD (6,7) 进行编码。母体 R01 试图研究人类肠道细菌的贡献 表达 DesAB 以及表达 DesAB 的工程化大肠杆菌对循环 11β-OHAD 的影响，以及 这种 11-氧雄激素前体对肠道免疫特征和基因表达的影响。拟议的目标 多样性补充是通过表征人类微生物组基因来扩展母基金中的研究 编码能够将11β-OHAD进一步代谢成二氢立体异构衍生物11β-的酶 OH-5α-二氢雄甾烷（雄激素前体）和 11β-OH-5β-二氢雄甾烷（非雄激素前体） 并将这些基因的表达整合到限生动物研究中。这些机制研究代表 首次尝试将肠道微生物皮质类固醇代谢与循环 11-氧雄激素水平建立因果关系 胃肠功能。这种独特的培训机会整合了厌氧细菌学、微生物基因 发现和酶表征、CRISPR-Cas9 重组工程、微生物学、免疫学、宿主和 微生物转录组学（RNA-Seq）。培训还将包括多样化的职业发展和教学 课程包括 NUTR 550 资助写作课程（Ridlon 联合讲师）和一对一资助和 共同导师、生物信息学和统计课程以及 HPCBio 模块的手稿写作发展 转录组分析、营养、微生物和哺乳动物生物化学课程以及微生物学。

Project Summary/Abstract Literature from as early as 1957 from Nabarro et al demonstrated that glucocorticoids such as cortisol can be converted into potent androgens like hydroxyandrostenedione (11β-OHAD) (1,2). However, it wasn't until 1981 that side-chain cleavage of cortisol was first observed in human stool suspensions (3). In 1984, researchers isolated the human gut bacterium, Clostridium scindens, capable of steroid-17,20-desmolase (SD) (4,5). Ridlon et al (2013) utilized RNA-Seq to identify the cortisol-inducible genes (desAB), which were later confirmed by our laboratory to encode SD (6,7). The parent R01 seeks to study the contribution of both human gut bacteria expressing DesAB as well as engineered E. coli expressing DesAB to circulating 11β-OHAD, and the effect of this 11-oxy-androgen precursor on intestinal immune profile and gene expression. The aim of the proposed diversity supplement is to extend the studies in the parent grant by characterizing human microbiome genes encoding enzymes capable of further metabolism of 11β-OHAD into dihydro-stereoisomeric derivatives 11β- OH-5α-dihydroandrostane (androgen-precursor) and 11β-OH-5β-dihydroandrostane (non-androgen precursor) and integrating expression of these genes into gnotobiotic animal studies. These mechanistic studies represent the first attempt to causally link gut microbial corticosteroid metabolism to circulating 11-oxy-androgen levels and gastrointestinal function. This unique training opportunity integrates anaerobic bacteriology, microbial gene discovery and enzyme characterization, CRISPR-Cas9 recombineering, gnotobiology, immunology, host and microbial transcriptomics (RNA-Seq). Training will also consist of diverse career development and didactic coursework including NUTR 550 Grant Writing Course (Ridlon co-instructor) and one-on-one grant and manuscript writing development by the co-mentors, bioinformatics and statistical courses and HPCBio modules on transcriptomic analysis, nutrition, microbial and mammalian biochemistry courses, and microbiology.