Commensal-derived acetylcholine enhances mucosal immune education [PRESTO_Salsa_screen_InVio_Bu_corrected]

The microbiota produces thousands of potentially bioactive small molecules1-3. High-throughput bioactivity screens of in vitro commensal cultures have exposed microbiota metabolites that shape host physiology by activating diverse G protein-coupled receptors (GPCR)4-7. However, due to technical limitations, the GPCRome-wide bioactivities of in vivo metabolomes, which result from complex diet-microbe-host interactions, remain unexplored. Here we leveraged a novel multiplexed GPCR screening technology to assess GPCRome-wide bioactivities of 100 commensal strains grown in vivo in mono-associated germ-free mice or in vitro in bacterial culture media. In vivo and in vitro commensal metabolomes exhibited distinct GPCR activation patterns due to: 1) host-mediated metabolite degradation, 2) in vivo microbial metabolic reprogramming, and 3) biotransformation of dietary substrates. Notably, we identified multiple commensal strains that produced acetylcholine (ACh) in vivo via the conversion of dietary choline, including select Bifidobacteria strains that dominate the microbiome in early life and a probiotic Pediococcus strain. Mechanistically, we identified and characterized the bacterial enzymes mediating this biotransformation in Bifidobacterium breve and Pediococcus pentosaceus, and generated an isogenic mutant B. breve strain lacking ACh production. Mice colonized with an ACh-producing B. breve strain and treated with an FDA-approved acetylcholinesterase inhibitor exhibited enhanced intestinal immunoglobulin A (IgA) production, altered microbiota composition, and increased resistance to enteric infection. Together, these findings suggest that commensal-derived ACh can enhance intestinal IgA production, strengthen mucosal immune defenses, and reinforce host-microbiota mutualism.