In the present study, we characterized CF-14, a novel antimicrobial peptide derived from the catfish skin mucus. The objective of this study was to explore the antimicrobial mechanism of CF-14 against Escherichia coli. The agar-diffusion assay and the microdilution method were used to evaluate the antimicrobial activity and the minimum inhibitory concentration (MIC) of CF-14 against E. coli, respectively. In addition, the absorbance of the bacterial suspension filtrate at 260 nm was measured to quantify the leakage of bacterial cytoplasmic components. The bacterial morphological changes were observed by scanning electron microscopy, while confocal microscopy was used to investigate the localization site of CF-14 in E.coli. The DNA binding ability of CF-14 was evaluated using gel retardation assay and the binding of CF-14 to DnaK was evaluated using Discovery Studio. The results demonstrated that CF-14 exhibited strong antimicrobial activity against E.coli with an MIC of 31.3 μg/mL. Unlike common cationic anti-microbial peptides (AMPs) that target the cellmembrane, CF-14 penetrated the E.coli cell membrane and induced only minormembrane perturbations. Furthermore, the antimicrobial mechanism of CF-14 against E.coli involved DNA binding and competitive inhibition of bacterial DnaK. Finally, by deleting or replacing the amino acid sequence, the antibacterial activity of CF-14 was affected, which helped the optimization of amino acid sequence. Therefore, CF-14 can be a potential antimicrobial peptide.
Keywords: Antibacterial mechanism; Antibiotics; CF-14; DnaK; Escherichia coli.
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