The urinary pathobiont Actinobaculum massiliense generates androgens via the dirAB pathway.

While overlooked during the Human Microbiome Project, characterizing the urinary microbiota in health and disease is a new frontier in microbiome science. Recent studies have associated differential abundance of bacterial taxa including Propionimicrobium lymphophilum and Actinobaculum / Actinotignum spp. with prostate cancer. In this study, we collected urine from subjects prior to prostate biopsy and applied a novel H uman S terolbiome D iscovery H igh-throughput (HSDH) assay to identify culturable urinary bacteria with the ability to generate androgens. Application of the HSDH assay to urine samples led to the isolation of eight P. lymphophilum strains positive for cortisol side-chain cleavage (steroid-17,20-desmolase), 17β-HSDH activity, or both. In addition, we isolated three strains of Actinobaculum massiliense that encode D HEA isomerase reductase (dir ) genes. The dirA gene encodes a novel 3β/17β-hydroxysteroid dehydrogenase/Δ 4,5 -isomerase and the dirB gene encodes a novel 17β-hydroxysteroid dehydrogenase isoform. Structural prediction and molecular dynamics reveal probable catalytic mechanisms based on the shared catalytic triad but distinct binding pocket geometries of the DirA and DirB that describe their respective reactions. Phylogenetic analysis of DirA and DirB revealed homologs in urinary tract commensals as well as bacteria associated with steroid degradation found in aquatic and terrestrial environments. Taken together, the development of the HSDH assay and the identification of the dir pathway genes is a significant advance in microbial endocrinology, laying the methodological foundation and providing the molecular basis for understanding the role of urinary tract bacteria in host endocrine physiology.