Multi-Omics Unveils Strain-Specific Neuroactive Metabolite Production Linked to Inflammation Modulation byBacteroidesand Their Extracellular Vesicles

Bacteroides species are key members of the human gut microbiome and play crucial roles in gut ecology, metabolism, and host-microbe interactions. This study investigated the strain-specific production of neuroactive metabolites by 18 Bacteroidetes (12 Bacteroides , 4 Phocaeicola , and 2 Parabacteroides ) using multi-omics approaches. Genomic analysis revealed a significant potential for producing GABA, tryptophan, tyrosine, and histidine metabolism-linked neuroactive compounds. Using untargeted and targeted metabolomics, we identified key neurotransmitter-related or precursor metabolites, including GABA, L-tryptophan, 5-HTP, normelatonin, kynurenic acid, L-tyrosine, and norepinephrine, in a strain- and media-specific manner, with GABA (1-2 mM) being the most abundant. Additionally, extracellular vesicles (EVs) produced by Bacteroides harbor multiple neuroactive metabolites, mainly GABA, and related key enzymes. We used CRISPR/Cas12a-based gene engineering to create a knockout mutant lacking the glutamate decarboxylase gene ( gadB ) to demonstrate the specific contribution of Bacteroides finegoldii -derived GABA in modulating intestinal homeostasis. Cell-free supernatants from wild-type (WT, GABA+) and Δ gadB (GABA-) provided GABA-independent reinforcement of epithelial membrane integrity in LPS-treated Caco-2/HT29-MTX co-cultures. EVs from WT and Δ gadB attenuated inflammatory immune response of LPS-treated RAW264.7 macrophages, with reduced pro-inflammatory cytokines (IL-1β and IL-6), downregulation of TNF-α, and upregulation of IL-10 and TGF-β. GABA production by B. finegoldii had a limited impact on gut barrier integrity but a significant role in modulating inflammation. This study is the first to demonstrate the presence of a myriad of neuroactive metabolites produced by Bacteroides species in a strain- and media-specific manner in supernatant and EVs, with GABA being the most dominant metabolite and influencing immune responses. Importance Bacteroides is a keystone gut symbiont that largely influences gut ecological dynamics and intestinal homeostasis. While previous studies highlighted the contribution of Bacteroides to human health, the mechanisms by which these species interact with the gut-brain axis are still emerging. This study highlights the remarkable potential of Bacteroides species to produce a wide spectrum of neurotransmitter-related or precursor metabolites, such as γ-aminobutyric acid (GABA), L-tryptophan, 5-hydroxytryptophan (5-HTP), tyramine, normelatonin, L-tyrosine, norepinephrine, and spermine. Bacteroides neurometabolic signaling to the host may involve extracellular vesicles (EVs), potentially modulating the gut-brain axis and host immune responses. Notably, B. finegoldii exhibit distinct anti-inflammatory characteristics resulting from different molecular patterns, including GABA and EV production. Our findings suggest that Bacteroides and their EVs hold great promise as next-generation psychobiotics.