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

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.