Novel Insights into the Therapeutic Effect of Amentoflavone Against Aeromonas hydrophila Infection by Blocking the Activity of Aerolysin

<i>Aeromonas hydrophila</i> (<i>A. hydrophila</i>) is an opportunistic and foodborne pathogen widely spread in the environments, particularly aquatic environments. Diseases caused by <i>A. hydrophila</i> in freshwater aquaculture result in huge economic losses every year. The increasing emergence of antibiotic resistance has limited the application of antibiotics in aquaculture. Aerolysin (AerA), the main virulence factor produced by <i>A. hydrophila,</i> has been identified as a promising target for developing drugs controlling <i>A. hydrophila</i> infection. Here, we found that the natural compound amentoflavone (AMF) with the MIC of 512 μg/mL against <i>A. hydrophila</i> could dose-dependently reduce the hemolysis of AerA, ranging from 0.5 to 4 μg/mL. Molecular docking and dynamics simulation results predicted that AMF could directly bind to domain 3 of AerA via Pro333 and Trp375 residues. Then, the binding sites were confirmed by fluorescence quenching assays. The results of heptamer formation demonstrated that the binding of AMF could affect the formation of oligomers and result in the loss of pore-forming activity. Cell viability assay showed that AerA after treatment with AMF ranging from 0.5 to 4 μg/mL could significantly reduce AerA-mediated cytotoxicity. Moreover, experimental therapeutics results showed that channel catfish infected with <i>A. hydrophila</i> and then administered with 20 mg/kg AMF at intervals of 12 h for 3 days could increase the survival rate by 35% compared with the positive control after a 10-day observation. These findings provided a novel approach to developing anti-infective drugs and a promising candidate for controlling <i>A. hydrophila</i> infection in aquaculture.