Proteomics reveals the role of the EamB transporter from Aeromonas hydrophila LP-2 in biofilm formation.

Biofilms play a pivotal role in the survival and persistence of microorganisms, endowing them with heightened resistance to environmental stressors and antimicrobial agents. The EamB protein, which encodes an inner membrane transporter, acted as a negative regulator of biofilm formation, and the gene eamB deletion in the pathogen Aeromonas hydrophila LP-2 resulted in a significant increase in biofilm formation. Proteomic analysis revealed a total of 616 differentially abundant proteins between the ΔeamB and wild-type (WT) strains, with 308 downregulated and 308 upregulated. RT-qPCR was employed to verify the stability and accuracy of the proteomics data. Bioinformatic analysis indicated that EamB is involved in critical bacterial biological processes, including flagellar assembly, amino acid metabolism, and fatty acid degradation. Biofilm formation assays further revealed that supplementation with exogenous lysine significantly inhibited biofilm formation in the ΔeamB strain, conversely, exogenous cysteine and O-acetylserine obviously increased biofilm formation in the ΔeamB strain. These findings demonstrated that EamB may modulate bacterial biofilm formation in A. hydrophila through the regulation of amino acid metabolism. This finding provides novel insights into the regulatory mechanism underlying biofilm formation and highlights potential targets for the development of future antibacterial strategies. SIGNIFICANCE STATEMENT: This study elucidates the critical role of the eamB gene in Aeromonas hydrophila, a significant aquatic pathogen, by demonstrating its impact on biofilm formation and physiological traits. Through comparative proteomic analysis, we identified 616 differentially abundant proteins in the ΔeamB mutant, revealing its involvement in key metabolic pathways such as amino acid metabolism, flagellar assembly, and fatty acid degradation. Notably, eamB deletion enhanced biofilm formation, while exogenous amino acids like cysteine and O-acetylserine obviously increased biofilm formation in the ΔeamB strain. These findings highlight EamB as a regulator of biofilm formation, offering novel molecular insights into bacterial pathogenicity. This research advances our understanding of biofilm-associated antibiotic resistance and provides potential targets for developing strategies to mitigate infections caused by A. hydrophila in aquaculture and public health.