Effect of Lipid Bilayer Composition on the Membrane-Binding Ability and Depth of Insertion of Antimicrobial Piscidin 1: A Solid-State Deuterium NMR Study

Abstract

Piscidins constitute a family of amphipathic, cationic, antimicrobial peptides (AMPs) found in the skin, gills and gastrointestinal tracts of hybrid striped bass. Unstructured in water, they become alpha-helical in the presence of phospholipids. It is believed that their ability to disrupt phospholipid bilayers is related to their antimicrobial effects on yeasts and bacteria. Interestingly, the minimum inhibitory concentrations of piscidin tend to be higher for yeasts than most bacteria. Here, we used Circular Dichroism (CD) and deuterium solid-state Nuclear Magnetic Resonance (ssNMR) to investigate the membrane-binding ability and depth of insertion of piscidin 1 in different membrane mimetic systems. Aligned lipid bilayer preparations were made using 3:1 POPC/POPG-d31 (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol-d31) and 3:1 POPC/POPS-d31 (1-palmitoyl 2-oleoyl phosphatidylserine-d31) to mimic bacterial and yeast cell membranes, respectively. To test the effects of ergosterol, a sterol found in yeasts, we added 20% ergosterol (molar ratio) to the lipid mixtures. We hypothesized that both PS and ergosterol would reduce the ability of piscidin to bind and disrupt bilayers.Deuterium quadrupolar splittings, which were obtained for deuterons in the methylene and terminal methyl groups of the phospholipid acyl chains, were used to assess the disordering effect of piscidin on the acyl chains. Our results suggest that the type of anionic lipid component (e.g., PG versus PS) does not affect acyl chain disordering by piscidin while ergosterol appears to decrease its disordering effect. The data also give evidence that membrane-bound piscidin is located close to the phospholipid headgroups. Overall, these studies help us gain some insight into specific physicochemical features of lipid membranes that affect piscidin-lipid interactions. The implications in terms of piscidin's mode of action will be discussed.