Mechanism of Action of Surface Immobilized Antimicrobial Peptides Against Pseudomonas aeruginosa

Abstract

Bacterial colonization and biofilm development on medical devices can lead to infection. Antimicrobial peptide-coated surfaces may prevent such infections. Melimine and Mel4 are chimeric cationic peptides showing broad-spectrum antimicrobial activity once attached to biomaterials and are highly biocompatible in animal models and have been tested in Phase I and II/III human clinical trials. These peptides were covalently attached to glass using an azidobenzoic acid linker. Peptide attachment was confirmed using X-ray photoelectron spectroscopy and amino acid analysis. Mel4 when bound to glass was able to adopt a more ordered structure in the presence of bacterial membrane mimetic lipids. The ability of surface bound peptides to neutralize endotoxin was measured along with their interactions with the bacterial cytoplasmic membrane which were analyzed using DiSC(3)-5 and Sytox green, Syto-9, and PI dyes with fluorescence microscopy. Leakage of ATP and nucleic acids from cells were determined by analyzing the surrounding fluid. Attachment of the peptides resulted in increases in the percentage of nitrogen by 3.0% and 2.4%, and amino acid concentrations to 0.237 nmole and 0.298 nmole per coverslip on melimine and Mel4 coated surfaces, respectively. The immobilized peptides bound lipopolysaccharide and disrupted the cytoplasmic membrane potential of Pseudomonas aeruginosa within 15 min. Membrane depolarization was associated with a reduction in bacterial viability by 82% and 63% for coatings melimine and Mel4, respectively (p < 0.001). Disruption of membrane potential was followed by leakage of ATP from melimine (1.5 ± 0.4 nM) or Mel4 (1.3 ± 0.2 nM) coated surfaces compared to uncoated glass after 2 h (p < 0.001). Sytox green influx started after 3 h incubation with either peptide. Melimine coatings yielded 59% and Mel4 gave 36% PI stained cells after 4 h. Release of the larger molecules (DNA/RNA) commenced after 4 h for melimine (1.8 ± 0.9 times more than control; p = 0.008) and after 6 h with Mel4 (2.1 ± 0.2 times more than control; p < 0.001). The mechanism of action of surface bound melimine and Mel4 was similar to that of the peptides in solution, however, their immobilization resulted in much slower (approximately 30 times) kinetics.

Activity of Antimicrobial Peptides and Ciprofloxacin against Pseudomonas aeruginosa Biofilms

“…Application of either AMP also reduced the biofilm mass compared to buffer treated controls. Previously, immobilized melimine and Mel4 killed adherent P. aeruginosa cells and reduced total adhered cells [ 39 ].…”

A New Era of Antibiotics: The Clinical Potential of Antimicrobial Peptides

“…The covalent attachment of an antimicrobial molecule removes the need for recurrent dosing and avoids non-compliance of individual dosing regimens. A phase III clinical trial of covalently attached Mel4 contact lenses found that, with extended wear (14 days), the incidence of corneal infiltration was reduced by 50% [ 95 ]. No cytotoxicity or corneal irritation was observed.…”

An Update on Antimicrobial Peptides (AMPs) and Their Delivery Strategies for Wound Infections

“…This is attributed to the relatively low specificity of AMP mechanisms of action, as well as to the variety of these mechanisms implied in bacterial killing. In general, AMPs are quite resistant to the development of bacterial resistance [69].…”

“…The only way to eradicate bacteria in this situation is implant removal, which implies surgery and close follow-up of the patients for their complete recovery. In this context, it is worth noticing that AMPs, such as AG-30 [36], AG-30/5C [37], WRL3 [41], melimine and Mel4 [69], 73c, and D-73 [94] are very potent against biofilm formation.…”

“…In general, the bacterial colonization of medical devices favors biofilm development, which in turn will lead to device failure due to peri-implantitis [119]. For instance, P. aeruginosa colonization of catheters and other medical devices accounts for 10-20% of all related infections [69]. Therefore, coating medical devices with a layer of antibacterial membrane or covalently attaching different In general, the bacterial colonization of medical devices favors biofilm development, which in turn will lead to device failure due to peri-implantitis [119].…”