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

项目摘要/摘要 早在1957年Nabarro等人的文献就表明，糖皮质激素，如皮质醇，可以 转化为有效的雄激素，如羟基雄烯二酮(11β-OHAD)(1，2)。然而，直到1981年， 皮质醇的侧链断裂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-脱解酶(SD)(4，5)的人体肠道细菌--scindens梭菌。睿龙 等人(2013)利用RNA-Seq鉴定了皮质醇诱导基因(DesAB)，这些基因后来被我们的 实验室对SD(6，7)进行编码。亲本R01试图研究两种人类肠道细菌的贡献 表达DesAB以及表达DesAB的工程菌对循环11β-OHAD的影响 这种11-氧-雄激素前体对肠道免疫功能和基因表达的影响。建议的目的是 多样性补充是通过表征人类微生物组基因来扩展父母拨款中的研究 编码酶能将11β-Ohad进一步代谢成二氢立体异构体衍生物11β- OH-5-α-二氢雄烷(雄激素前体)和11β-OH-5β-二氢雄烷(非雄激素前体) 并将这些基因的表达整合到灵知菌动物研究中。这些机械论研究代表了 首次尝试将肠道微生物皮质类固醇代谢与循环11-氧-雄激素水平和 胃肠功能。这一独特的培训机会融合了厌氧细菌学、微生物基因 发现和酶特性，CRISPR-Cas9重组工程，灵知生物学，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助学金写作课程(Ridlon联合讲师)和一对一助学金和 共同导师撰写手稿、生物信息学和统计课程以及HPCBio模块 转录本分析、营养学、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.