Rutin Combats Salmonella infection through virulence suppression and gut barrier maintenance

• Rutin markedly inhibits Salmonella Typhimurium invasion of host cells without affecting bacterial viability. • Rutin binds HilA and suppresses T3SS-1 activity. • Rutin modulates biofilm formation and reduces Salmonella virulence in vivo. • Rutin preserves intestinal barrier integrity and partially restores microbiota balance. Salmonella Typhimurium ( S. Typhimurium) is a major zoonotic pathogen causing severe gastroenteritis and septicemia. Traditional antibiotics targeting bacterial growth face increasing resistance challenges, while the development of novel antimicrobials is slow and costly. Antivirulence therapy, which targets bacterial virulence factors, has emerged as a promising alternative strategy. To demonstrate that the natural flavonoid rutin can serve as an antivirulence agent against S. Typhimurium infection. A host-cell invasion assay was used to screen natural compounds. Bacteria treated with rutin were subjected to transcriptomic profiling (RNA-seq), followed by validation using qPCR, Western blot, a β-lactamase reporter system, intrinsic fluorescence spectroscopy, circular dichroism and electrophoretic mobility shift assays. Auto-aggregation and crystal-violet assays, together with confocal and scanning electron microscopy, were used to evaluate biofilm-associated phenotypes. An acute murine salmonellosis model was used to evaluate the therapeutic efficacy of rutin. Histological analysis, immunostaining and 16S rRNA gene sequencing were performed to assess intestinal barrier integrity and gut microbiota composition. Rutin significantly inhibited S. Typhimurium invasion of HeLa and Caco-2 cells without affecting bacterial growth or host cell viability. Integrated transcriptomic and functional analyses demonstrated that rutin targets HilA, the master regulator of type III secretion system 1 (T3SS-1), leading to reduced T3SS-1 effector expression and secretion. Rutin also decreased transcription of the biofilm master regulator CsgD, reduced bacterial auto-aggregation, and disrupted mature biofilm architecture. In infected mice, oral administration of rutin decreased bacterial burden and tissue injury, improved epithelial barrier integrity, attenuated intestinal inflammation, and alleviated gut microbiota imbalance. Rutin mitigates S. Typhimurium infection through coordinated suppression of HilA-dependent T3SS-1 activity and CsgD-mediated biofilm formation, while supporting intestinal barrier function and modulating gut microbial composition. Rutin shows potential as an adjunct or alternative to conventional antibiotics.