Discovery of a Small-Molecule Inhibitor Targeting the Biofilm Regulator BrpT in <i>Vibrio vulnificus</i>

<i>Vibrio vulnificus</i>, an opportunistic human pathogen, employs biofilm formation as a key survival and virulence mechanism. BrpT, a transcriptional regulator, is essential for <i>V. vulnificus</i> biofilm development by regulating the expression of biofilm-related genes. In this study, we aimed to identify a small molecule inhibitor of BrpT to combat <i>V. vulnificus</i> biofilm formation. High-throughput screening of 7,251 compounds using an <i>Escherichia coli</i> reporter strain carrying the arabinose-inducible <i>brpT</i> gene and a BrpT-activated promoter fused to the <i>luxCDABE</i> operon identified a hit compound, BTI (BrpT Inhibitor). BTI potently inhibited BrpT activity in <i>V. vulnificus</i> (EC₅₀ of 6.48 μM) without affecting bacterial growth or host cell viability. Treatment with BTI significantly reduced the expression of the BrpT regulon and impaired biofilm formation and colony rugosity in <i>V. vulnificus</i>, thus increasing its susceptibility to antibiotics. In vitro biochemical analyses revealed that BTI directly binds to BrpT and inhibits its transcriptional regulatory activity. The identification of BTI as a specific inhibitor of BrpT that effectively diminishes <i>V. vulnificus</i> biofilm formation provides a promising foundation for the development of novel anti-biofilm strategies, with the potential to address the growing challenge of antibiotic resistance and improve the treatment of biofilm-associated infections.