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

## TL;DR

Amentoflavone reduces the harmful effects of a toxin from Aeromonas hydrophila, offering a new treatment for aquaculture infections.

## Contribution

Amentoflavone is shown to inhibit Aerolysin activity, providing a novel therapeutic strategy for A. hydrophila infections.

## Key findings

- Amentoflavone reduces AerA hemolysis and inhibits pore-forming activity.
- AMF binding to AerA was confirmed through molecular docking and fluorescence assays.
- AMF treatment increased survival rates in infected channel catfish by 35%.

## Abstract

Aeromonas hydrophila (A. hydrophila) is an opportunistic and foodborne pathogen widely spread in the environments, particularly aquatic environments. Diseases caused by A. hydrophila 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 A. hydrophila, has been identified as a promising target for developing drugs controlling A. hydrophila infection. Here, we found that the natural compound amentoflavone (AMF) with the MIC of 512 μg/mL against A. hydrophila 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 A. hydrophila 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 A. hydrophila infection in aquaculture.