Abstract

Bilobalide inhibits S. typhimurium biofilm formation, motility, and virulence. • Bilobalide binds QseB and blocks DNA binding, repressing QS and T3SS gene expression. • Bilobalide reduces S. typhimurium –induced inflammation and boosts antibiotic efficacy. • Bilobalide shows potential as a natural antimicrobial against S. typhimurium . Background Salmonella typhimurium is a major food-borne pathogen whose virulence is regulated by quorum sensing (QS) and the type III secretion system (T3SS). The rise of antibiotic resistance highlights the need for anti-virulence strategies targeting bacterial communication. Bilobalide, a sesquiterpene trilactone from Ginkgo biloba , has antimicrobial potential, but its role in QS regulation remains unclear. This study investigates how bilobalide modulates QS and virulence in S. typhimurium to support the development of anti-infective strategies. Purpose This study aimed to elucidate the molecular mechanism by which bilobalide attenuates S. typhimurium virulence, including biofilm formation, motility, and cytotoxicity. Additionally, the study evaluated bilobalide's potential as a QS inhibitor and adjunct to antibiotic therapy. Methods Phenotypic assays assessed the effects of bilobalide on S. typhimurium biofilm formation, motility, and cytotoxicity. RNA sequencing (RNA-seq) and RT-qPCR analyses identified differentially expressed genes after bilobalide treatment. Microscale thermophoresis (MST), molecular docking, and molecular dynamics simulations characterised the interaction between bilobalide and QseB. Electrophoretic mobility shift assays were used to determine whether bilobalide affected QseB–DNA binding. The combined antibacterial effects of bilobalide and antibiotics were evaluated using microdilution assays. In vivo efficacy was tested using a mouse infection model. Results Bilobalide significantly inhibited biofilm formation, motility, and T3SS gene expression in S. typhimurium without affecting bacteria