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

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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.