Inhibitory mechanism of phloretin on the AgrA LytTR domain-agr operon complex formation and its application in beef

Inhibitory mechanism of phloretin on the AgrA LytTR- agr complex was revealed. • The AgrA LytTR domain protein was purified successfully. • EMSA confirmed phloretin may disrupt the binding of AgrA LytTR domain and P3 promoter. • MD simulations revealed that phloretin sterically hindered AgrA- agr binding, validated by mixed fluorescence quenching. • Phloretin acts as QSI to inhibit the secretion of enterotoxin and hemolysin in beef. Staphylococcus aureus ( S. aureus ) represents a major foodborne pathogen whose enterotoxin production poses significant challenges to food safety due to its high environmental resistance and limited efficacy of conventional sterilization. Since the expression of enterotoxins is predominantly governed by the agr quorum sensing system, targeting this regulatory pathway has become a strategic choice for virulence control. This study elucidated the mechanism by which phloretin, a potential quorum sensing inhibitor, interferes with the agr system to attenuate virulence. To achieve this, the recombinant AgrA LytTR domain was expressed and purified, and its interaction with phloretin was characterized using thermal shift assays (TSA), electrophoretic mobility shift assays (EMSA), and molecular dynamics (MD) simulations. The results showed that phloretin specifically binds to the AgrA LytTR domain, enhancing its thermal stability and disrupting AgrA LytTR- agr operon binding by reducing the free energy of interaction between them, without causing significant structural rearrangement. Mechanistic analysis indicated that phloretin sterically hinders key β -sheet turn residues (HIS169, ASN201, ARG233), thereby impairing DNA recognition, downregulating RNAIII transcription, and inhibiting agr signaling. In cooked beef, phloretin significantly inhibited the secretion of enterotoxins and α -hemolysin, while delaying lipid oxidation and protein degradation, and maintaining the meat texture. These findings suggested that phloretin is a multifunctional substance with anti-virulence, antioxidant, and preservative properties, demonstrating its potential as a natural food preservative. Download: Download high-res image (118KB) Download: Download full-size image