The gut microbiota modulates human health and disease often via the production of small molecule metabolites; however, the identities of the majority of gut microbial metabolites and their biological actions remain largely unknown. Pregnane X receptor (PXR) is a ligand-activated transcription factor that is activated by structurally diverse chemicals. PXR regulates the expression of genes encoding drug-metabolizing enzymes including cytochrome P450 3A4 (CYP3A4). Additionally, PXR in the intestine and liver has been implicated in the maintenance of gut barrier and immune functions as well as energy homeostasis. Accumulating evidence suggests that the gut microbiota produces PXR ligands. To date, few bacterial metabolites have been reported as mouse or human PXR ligands, and systematic efforts to identify PXR- activating, gut microbial metabolites have been lacking. This lack of knowledge constitutes a substantial gap in defining how the gut microbiota alters human health and disease especially via PXR. The long-term goal of our research is to identify and characterize factors mediating gut microbes-host interactions. The overall objective of this application is to identify gut bacterial metabolites activating PXR, a ligand-activated transcription factor with pleiotropic biological functions including the regulation of drug-metabolizing enzymes and the maintenance of gut homeostasis. Our central hypothesis is that gut bacteria produce metabolites that activate PXR. Our hypothesis is based on the following preliminary results: (1) In HepG2 cells overexpressing human PXR (HepG2/hPXR), the organic extracts of mouse cecum contents significantly induced hPXR transactivation of PXR target gene (i.e., CYP3A4) promoter, indicating the presence of hPXR activators among gut microbial products. (2) A screening of 10 common human gut bacteria led to identification of Fusobacterium nucleatum as a producer of hPXR activator(s); in HepG2/hPXR cells, the organic extracts of F. nucleatum culture supernatants significantly induced hPXR transactivation of the CYP3A4 promoter. (3) We increased the yield of the active metabolite from F. nucleatum by changing the growth media and obtained a proton NMR and high- resolution mass spectrometry results of an active metabolite-enriched fraction. The results suggest that PXR- activating metabolite(s) of F. nucleatum are likely novel compounds. Together, our data indicate that gut bacteria produce as-yet-unknown metabolite(s) that activate PXR. Based our findings, we propose to (1) identify gut bacteria-derived metabolites that promote hPXR transactivation of CYP3A4 expression and (2) define genetic determinants for the production of hPXR activator(s) in F. nucleatum. Successful completion of these studies will set the stage for future investigation, providing much-needed tool sets to investigate underlying molecular mechanisms for how the gut microbiota modulates PXR activity. Together, these efforts will enhance our understan...