Staphylococcus aureus (S. aureus) poses a serious threat to food safety and global health, driving the demand for natural and safe antimicrobials. CpAtt, an attacin-like antimicrobial peptide identified previously in our laboratory, demonstrated potent antibacterial activity against S. aureus. This study systematically elucidated the multi-target antibacterial mechanisms of CpAtt against S. aureus. The bacteriostatic curve confirmed that 1 MIC CpAtt effectively inhibited the S. aureus proliferation within 12 h. CpAtt disrupted cell wall and membrane integrity in S. aureus, as evidenced by blurred ultrastructure features, and significant leakage of nucleic acids, proteins, alkaline phosphatase, and β-galactosidase. Increased propidium iodide uptake increase further indicated that CpAtt increased cell membrane permeability. Transcriptomic analysis revealed that CpAtt disrupts multiple physiological processes. Specifically, these genes involved in the peptidoglycan biosynthesis pathway, the accessory gene regulator system, the TCA cycle, and succinate dehydrogenase were downregulated, whereas genes encoding for F-type ATPase, Cytochrome c oxidase, and Cytochrome bd complex were upregulated. Consequently, intracellular ATP levels dropped significantly, while reactive oxygen species levels decreased, suggesting that CpAtt impaired the energy metabolismwithout inducing the oxidative damage. In vitro binding assays and molecular docking analyses indicated that CpAtt interacts with genomic DNA primarily through electrostatic interactions between its positively charged residues and the anionic phosphate backbone. Notably, at sub-inhibitory concentrations, CpAtt modulated the transcription of adhesion-related genes in S. aureus, thereby suppressing biofilm formation. Consistently, in vivo experiments preliminarily demonstrated that CpAtt promotes skin wound healing in mice while conferring protection against infection when administered at appropriate doses. Collectively, these findings indicated that the antibacterial activity of CpAtt on S. aureus involves multiple targets, offering a strategic basis for the development of novel preservatives and antibacterial agents.

To explore the antibacterial effect and mechanism of antimicrobial peptide LL-1 against Salmonella, the minimum inhibitory concentration (MIC) was determined by the doubling dilution method, and the antibacterial effect was evaluated by the growth inhibition curve. Then, the bacterial morphology was observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The effect of LL-1 on the cell wall and cell membrane of Salmonella was evaluated by detecting the leakage of intracellular nucleic acids, proteins and alkaline phosphatase (ALP) as well as by conducting propidium iodide (PI) staining experiments. The binding of LL-1 to Salmonella DNA was detected by nucleic acid gel electrophoresis. Finally, the effect of LL-1 on the energy metabolism of Salmonella was evaluated by measuring the activities of intracellular succinate dehydrogenase (SDH), NADP-malate dehydrogenase (NADP-MDH) and ATP levels. The results showed that the MIC was 6.25 μg/mL, and LL-1 had a good antibacterial effect in dose- and time-dependent manners. Salmonella treated with LL-1 showed morphological changes such as cell shrinkage, cell membrane dissolution, and plasmolysis. LL-1 resulted in the leakage of intracellular nucleic acids, proteins and ALP and an increase in the fluorescence intensity of PI-stained bacterial cells. Additionally, LL-1 could bind to bacterial DNA. Increasing LL-1 concentration resulted in a decrease in the intracellular ATP content, SDH and NADP-MDH activities. In conclusion, LL-1 could exert its antibacterial activity against Salmonella by increasing the permeability of the cell membrane and cell wall, thereby causing the leakage of intracellular contents, binding to DNA, and affecting bacterial energy metabolism. This study lays the foundation for further research on the antibacterial mechanism and application of LL-1.