Antibiofilm activity of a recombinant mussel-derived antimicrobial protein through surface charge-mediated hydration layer formation.

Abstract

Biofilm formation on biomedical devices remains a major cause of persistent infections and antibiotic resistance. In this study, we developed a charge-mediated, anti-adhesive platform by immobilizing recombinant mussel adhesive protein-antimicrobial peptide fusions (MAP-AMPs) on solid substrates via a simple EDC/NHS covalent coupling reaction. Using recombinant MAP-foot protein-151 (MAP-fp-151) as a versatile scaffold, immobilized MAP-AMPs were characterized by attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy, scanning electron microscopy (SEM), atomic force microscopy (AFM), and Coomassie brilliant blue staining. While soluble MAP-AMPs exhibited potent bactericidal activity against both reference and antibiotic-resistance clinical E. coli strains, their immobilized forms primarily inhibited biofilm formation rather than exhibiting direct bactericidal activity. Computational simulations and experimental surface analyses revealed that the cationic MAP-AMPs formed island-like clusters that elevated surface positive potential and organized a dense, long-range hydration layer extending beyond the clusters into the bare surface regions. This mechanism was experimentally validated using Kelvin probe force microscopy (KPFM) and contact angle analysis, which showed elevated surface potential, increased water retention, and reduced water contact angles. Bacterial adhesion on the MAP-AMP2-immobilized surface was significantly suppressed, with adhesion rates reduced to 27.62-45.16% across all tested strains. These results indicate that MAP-AMP-immobilized surfaces provide a versatile, biocompatible, and resistance-independent strategy for preventing biofilm formation through charge-mediated hydration, offering broad potential for biomedical device coatings and antifouling applications.