A multifunctional antimicrobial peptide with cell-penetrating and β-lactam-potentiating activities for combating methicillin-resistant Staphylococcus aureus and carbapenem-resistant Enterobacteriaceae infections.

AIMS: The global health crisis posed by New Delhi metallo-β-lactamase-producing carbapenem-resistant Enterobacteriaceae (NDM-CRE) and methicillin-resistant Staphylococcus aureus (MRSA) underscores the urgent need for novel antimicrobial agents. Natural antimicrobial peptides (AMPs) often suffer from limited modes of action, narrow antimicrobial spectra, and poor cellular permeability. To address these challenges, we engineered X1-3, a rationally designed AMP that exhibits direct bactericidal activity, efficient cell penetration and synergistic potentiation of β-lactams.

METHODS

X1-3 was generated via structure-activity relationship-guided optimization and machine learning-assisted screening, followed by characterization of its physicochemical properties and safety profile. Its antimicrobial and β-lactam synergistic activities were evaluated against clinical NDM-CRE and MRSA isolates using checkerboard assays, time-kill kinetics, and confocal microscopy. Mechanistic studies combined transcriptomic profiling with flow cytometry, gel retardation, and enzymatic assays to elucidate multimodal actions involving membrane disruption, DNA binding, and specific inhibition of NDM-5 and PBP2a. Intracellular penetration and in vivo therapeutic potential were assessed in RAW264.7 macrophage infection model and murine systemic infection model, respectively.

RESULTS

X1-3 exhibits high stability and robust activity against NDM-CRE, MRSA, and vancomycin-resistant Enterococcus, with low resistance induction. Additionally, X1-3 shows a robust anti-virulence profile by inhibiting biofilm formation and eradicating mature biofilms in both NDM-CRE and MRSA. Mechanistic studies revealed that X1-3 induces bacterial death primarily through membrane disruption and multiple synergistic mechanisms (DNA binding and metabolic perturbations). As a β-lactam adjuvant, X1-3 enhances antibiotic efficacy by disrupting proton motive force (PMF) to suppress efflux pumps and directly targeting NDM-CRE- and MRSA-associated resistance enzymes, including NDM and penicillin-binding protein 2a (PBP2a). Furthermore, it penetrates host cells via lipid raft-mediated internalization to eradicate intracellular pathogens and attenuate inflammation. In vivo, the combination of X1-3 with β-lactam antibiotics showed promising therapeutic potential with no observable systemic toxicity. Notably, the treatment effectively alleviated the MRSA-infected wound.

CONCLUSIONS

Rational structural optimization enables X1-3 to function as both a direct bactericide and a β-lactam adjuvant. This dual strategy significantly improves efficacy against clinical NDM-CRE and MRSA, offering a novel therapeutic paradigm.