Effects of Polyunsaturated Fatty Acids and Metallation on the Antimicrobial Activity and Membrane-Disruptive Properties of Host-Defense Metallopeptide PISCIDIN 1

Abstract

Piscidins are host-defense peptides that exhibit properties critical for the development of novel therapeutics. While the capability of piscidins to physically disrupt membranes is well accepted, their ability to bind Cu2+ and chemically damage membranes is less understood. Metallation lowers their charge, which could affect membrane binding. It also provides a redox center for the formation of radicals that can convert unsaturated fatty acids (UFAs) into membrane-destabilizing oxidized phospholipids (OxPLs). Such mechanistic possibility is important to examine since bacteria such as Vibrio species incorporate exogenous poly-UFA (PUFA) in their membranes to affect membrane properties and virulence. Here, we investigated how metallation and the UFA-content of membranes influence the mechanism of action and antimicrobial potency of P1, a particularly membrane-active piscidin. First, using dye leakage experiments with model membranes constituted of phosphoglycerol (PG) and phosphatidylcholine (PC), we quantified the permeabilization of P1/P1-Cu on membranes containing 10% of either 18:2 PUFA or Aldo-PC, its oxidized version. We found that the combined effect of metallating P1 and making Aldo-PC available yielded a ten-fold increase in membranolysis. Second, we used 31P solid-state NMR to characterize the partitioning behaviors of P1/P1-Cu in these membranes. Both peptides demonstrated stronger affinity for PG than PC. This effect was more pronounced for P1-Cu than P1 in the Aldo-PC-containing bilayers. Hence, differences in the membrane partitioning of P1 and P1-Cu in OxPL-containing membranes underlie their contrasted permeabilization effects. Third, we probed how the changing PUFA-content of bacterial cell membranes affects virulence upon P1/P1-Cu exposure. When V. cholerae bacteria were fed specific PUFAs (18:2; 20:4; 20:5; 22:6) and either peptide, peptide activity was enhanced with all FAs, except the 20:4. This demonstrates the importance of considering exogenous UFAs in antimicrobial treatments.