Interaction of the surface bound antimicrobial peptides melimine and Mel4 with Staphylococcus aureus

Abstract

Melimine and Mel4 are cationic antimicrobial peptides which can resist biofilm development once bound to biomaterials. The aim of the current study was to determine the mode of action of bound melimine and Mel4 against S. aureus. The peptides were covalently attached to glass using an azidobenzoic acid linker. The amount of attached peptides was confirmed by XPS and amino acid analysis and their covalent attachment by SDS extraction. The release of autolysins after interaction of S. aureus with immobilized peptides was determined in cell free supernatants. The interaction of immobilized peptides with lipoteichoic acid was confirmed by ELISA. Membrane damage by surface bound peptides was assessed using DiSC(3)-5 (membrane potential sensitive), Syto-9 (membrane permeable) and PI (membrane impermeable) dyes with fluorescence microscopy. Release of ATP and nucleic acids (DNA/RNA) was measured in the surrounding fluid. Attachment of the peptides resulted in increased N% for melimine (5.4 ± 1.8%) and for Mel4 (4.8 ± 1.8%). The concentrations of immobilised amino acids were 0.297 nmole for melimine and 0.358 nmole for Mel4. SDS extraction released < 15% of peptides from the glass. The immobilized peptides bound ≥ 4 times more LTA than control surfaces. More autolysins (8 ± 2%; p = 0.026) were released from Mel4 than melimine or control surfaces. Membrane depolarization occurred at 15 min and was associated with a reduction in bacterial viability ≥ 37% for both peptides (p < 0.001). Disruption of the membrane potential resulted in loss of ATP from melimine (0.9 ± 0.4 nM) or Mel4 (0.6 ± 0.3 nM) coated surfaces compared to control (p < 0.001). Melimine coatings yielded 27 ± 11% (p = 0.026) and Mel4 gave 17 ± 12% (p = 0.150) PI stained cells after 4 h. DNA/RNA was released only by melimine coatings (2.1 ± 0.1 times; p = 0.011) compared to process control at 6 h. Both bound peptides resulted in the release of ATP, but only melimine released DNA/RNA while Mel4-coating resulted in the release of autolysins. Since the mode of action of melimine and Mel4 relate to the cell surface, they have potential for the development of infection-resistant implants.