Microbiota-derived propionate suppresses Salmonella virulence gene expression via LuxS quorum sensing

Despite mounting evidence that commensal microbes enhance host defenses, whether and how they directly suppress pathogen virulence remains elusive. Here, we investigate metabolites from the gut microbiota of infection‑resistant Tibetan chickens for their ability to reduce Salmonella virulence gene expression and elucidate the molecular mechanism by which these compounds inhibit the LuxS/AI‑2 quorum‑sensing system.

Initially, we compared the expression of the quorum‑sensing gene luxS and biofilm-associated virulence genes in Tibetan chickens and broiler chickens post-Salmonella infection. Notably, Tibetan chickens exhibited significantly lower virulence gene expression than broiler chickens. Subsequently, fecal microbiota transplantation (FMT) from Tibetan chickens to broiler chickens reduced virulence gene expression in infected recipients. Further, 16S rRNA gene sequencing of cecal contents revealed that FMT enhanced microbial diversity and altered composition in infected broiler chickens, specifically enriching short-chain fatty acids (SCFA)-producing beneficial bacteria (e.g., Bacteroides, Rikenellaceae_RC9_gut_group, Phascolarctobacterium, Desulfovibrio). Critically, using Transwell chambers to separate microbes and metabolites, we identified metabolites as mediators of this effect. Subsequent liquid chromatography-mass spectrometry (LC-MS) quantification demonstrated significantly elevated propionate concentrations in both uninfected and infected Tibetan chickens, and FMT-recipient broiler chickens. Propionate levels correlated negatively with key virulence factor expression. Moreover, in vitro experiments showed that propionate inhibited Salmonella biofilm formation, reduced autoinducer-2 (AI-2) activity, and downregulated the expression of virulence genes. In vivo, we further confirmed that propionate decreased the expression of Salmonella virulence genes. Taken together, these results support that propionate suppresses Salmonella virulence gene expression by targeting the LuxS/AI-2 quorum-sensing pathway. To validate this mechanism, we generated a luxS knockout strain by homologous recombination; strikingly, propionate failed to attenuate virulence gene expression in this mutant, thereby establishing the essential role of LuxS/AI-2. Finally, molecular docking identified propionate-LuxS binding sites (Ile53), and site-directed mutagenesis validated critical functional residues, highlighting structural determinants for virulence gene expression regulation.

These findings underscore the role of the gut-derived metabolite propionate in directly modulating pathogen virulence gene expression by targeting the LuxS/AI-2 quorum‑sensing system, offering novel insights into microbiota-based strategies for infectious disease management.