Antibacterial mechanism of a multitarget antibacterial peptide CpAtt against Staphylococcus aureus

Abstract 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.