Bioassay-Driven, Fractionation-Empowered, Focused Metabolomics for Discovering Bacterial Activators of Aryl Hydrocarbon Receptor

Aryl hydrocarbon receptor (AhR) is a transcription factor that regulates gene expression upon ligand activation, enabling microbiota-dependent induction, training, and function of the host immune system. A spectrum of metabolites, encompassing indole and tryptophan derivatives, have been recognized as activators. This work introduces an integrated, mass spectrometry-centric workflow that employs a bioassay-guided, fractionation-based methodology for the identification of AhR activators derived from human bacterial isolates. By leveraging the workflow efficiency, the complexities inherent in metabolomics profiling are significantly reduced, paving the way for an in-depth and focused mass spectrometry analysis of bioactive fractions isolated from bacterial culture supernatants. A pivotal discovery from our study is the identification of N-formylkynurenine (NFK) — a transient intermediate of tryptophan metabolism — as an AhR activator, a finding previously undocumented. The workflow reported provides a roadmap and significantly improves the efficiency of identifying bioactive metabolites using mass spectrometry-based metabolomics. Mass spectrometry datasets are accessible at the National Metabolomics Data Repository (PR001479, Project DOI: 10.21228/M8JM7Q).