Microbial neurosteroid biosynthesis: Holdemania filiformis generates isopregnanolone that reaches the brain via gut-to-brain transport

5-neurosteroids such as allopregnanolone and isopregnanolone play critical roles in neurological health and mood regulation, yet current therapeutic production faces significant limitations. We demonstrate that specific gut microbes represent a previously unrecognized source of bioavailable 5-neurosteroids that reach the central nervous system via the gut-brain axis. Through integrated metabolomic and genomic analyses of progesterone-amended fecal cultures, we identified Holdemania as a major producer of isopregnanolone via microbial steroid 5-reductase (BaiJ type 2) and 3{beta}-hydroxysteroid dehydrogenase/reductase. Phylogenetic analysis revealed that BaiJ-like sequences cluster predominantly within Firmicutes, with Holdemania species forming a distinct clade. In female C57BL/6 mice administered progesterone and H. filiformis, 5-neurosteroids including isopregnanolone predominated in gut tissues while allopregnanolone was the major hepatic neurosteroid. Critically, using stable isotope tracing with [3,4-{superscript 1}3C2]progesterone, we detected {superscript 1}3C-labeled isopregnanolone in brain tissue, providing direct evidence for gut-to-brain transport of microbiota-derived neurosteroids. High-fat diet significantly enhanced brain 5-neurosteroid accumulation. Global meta-analysis reveals reduced Holdemania abundance in PCOS patients (n = 346) compared to healthy women (n = 321). These findings identify gut microbiota as pharmacologically relevant neurosteroid producers and position H. filiformis as a promising probiotic candidate for enhancing endogenous neurosteroid production to treat mood disorders and other neuropsychiatric conditions. HighlightsO_LIHoldemania was identified as a major producer of 5-neurosteroids, particularly isopregnanolone, in the intestinal tract C_LIO_LIHoldemania 5-reductase (BaiJ type 2) belongs to a distinct phylogenetic clade compared to characterized Clostridium BaiJ (type 1) C_LIO_LI5-neurosteroids occurred predominantly in the cecum of female mice administered H. filiformis, progesterone, and high-fat diet C_LIO_LI{superscript 1}3C-labeled 5-neurosteroids were detected in brain tissue of female mice orally administered [3,4-{superscript 1}3C2]progesterone and H. filiformis, demonstrating gut-to-brain transport C_LIO_LIGut microbes such as H. filiformis represent promising probiotic candidates for enhancing 5-neurosteroid production and circulation via the gut-brain axis C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=146 HEIGHT=200 SRC="FIGDIR/small/628284v3_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1b85caaorg.highwire.dtl.DTLVardef@cbb352org.highwire.dtl.DTLVardef@169f7fcorg.highwire.dtl.DTLVardef@17c638_HPS_FORMAT_FIGEXP M_FIG C_FIG