Virtual Screening and Mechanistic Elucidation of Myricetin as a Quorum Sensing Inhibitor Targeting AhyI in Aeromonas hydrophila

Abstract

The rise of antibiotic-resistant Aeromonas hydrophila threatens global aquaculture, necessitating the development of anti-virulence strategies such as quorum sensing (QS) inhibition. This study aimed to identify natural quorum sensing inhibitors targeting the AHL synthase AhyI, building upon ongoing research that has demonstrated the importance of such inhibitors in controlling bacterial communication and potential applications in treating bacterial infections. We utilized computer-aided drug design techniques, employing AlphaFold2 to model the structure of AhyI, followed by Through virtual screening of 6,079 natural compounds using molecular docking with Schrödinger Glide and AutoDock, The flavonoid myricetin was identified as a top candidate and validated in vitro. At sub-inhibitory concentrations, myricetin significantly suppressed key virulence phenotypes in A. hydrophila AH72, including motility, extracellular enzyme secretion, hemolysis, and biofilm formation. Target validation using a heterologous expression system and UPLC-MS/MS confirmed that myricetin directly inhibits AhyI activity, reducing the production of multiple AHL signaling molecules. Transcriptomic analysis revealed a concentration-dependent mechanism: lower concentrations disrupted iron transport, while higher concentrations imposed a broad metabolic burden by downregulating central energy and amino acid pathways. Recent studies have shown that myricetin functions as a multi-targeted anti-virulence agent, effectively disrupting quorum sensing, metabolic pathways, and stress response mechanisms, thereby showcasing its significant potential. as a lead compound for developing sustainable disease management strategies in aquaculture.