Non‐Membrane Active Peptide Resensitizes MRSA to β‐Lactam Antibiotics and Inhibits S. aureus Virulence

Abstract Methicillin‐resistant Staphylococcus aureus (MRSA) is a serious global health threat due to its high morbidity and mortality rates, creating a dire need for novel therapeutic strategies. Antimicrobial peptides (AMPs), with broad‐spectrum activity and low propensity for resistance development, show promise as effective antibiotic adjuvants to reverse multidrug‐resistance in bacteria. Herein, it is uncovered that a potent and non‐toxic AMP termed GN1 substantially resensitizes MRSA to multiple β ‐lactam antibiotics at low concentrations. Mechanistic studies indicate that GN1 functions by suppressing both the production and enzymatic activity of MRSA‐associated resistance determinants, including penicillin‐binding protein 2a (PBP2a) and β ‐lactamase. Additionally, GN1 exhibits a robust anti‐virulence profile by inhibiting MRSA biofilm formation and staphyloxanthin production. Furthermore, GN1 induces bacterial metabolic perturbation, resulting in glutamate accumulation and oxidative damage. Importantly, the combination of GN1 with β ‐lactam antibiotics effectively mitigates MRSA‐induced infections in the animal infection models. Collectively, these findings suggest that GN1 represents a potent β ‐lactam adjuvant and anti‐virulence agent, offering a safe and versatile solution to combat MRSA infections.