Modulation of Elastic Properties through Unsaturated Lipid Content as a Mechanism for Inducing Resistance to Amphiphilic Antimicrobial Peptides

Abstract

We explore whether changes in fatty acid composition in bacterial membrane models lead to inhibition of amphiphilic antimicrobial peptide-(AMPs) through the modulation of the membrane elastic properties. Specifically, we examined whether changes in the unsaturated/saturated fatty acid ratio, which is controlled by bacteria, inhibit AMP function. In phosphatidylglycerol (PG) lipid bilayers we observe a 10-fold increase in resistance to Magainin-2 (MAG) in POPG (16:0/18:1) vs. DPPG (16:0/16:0) bilayers and a 20-fold increase in resistance in DOPG (18:1/18:1) vs. DPPG bilayers, as measured by calcein release at 45 C (where lipids are in the liquid-crystalline phase). By analyzing the leakage kinetics, we find that the activation energy for pore formation in DPPG/POPG mixtures increases linearly with POPG content, confirming that unsaturation is energetically unfavorable for pore formation. Laurdan polarization (GP) measurements correlate well with MAG resistance, suggesting that head group spacing is an indicator of resistance (increased head-group spacing could allow for more peptides to partition into the head group region, instead of becoming inserted into the bilayer as bilayer-spanning monomers). The changes in potency cannot be explained solely by the increased head group spacing, however. We therefore performed leakage experiments using known modifiers of membrane elastic properties (Triton-X, capsaicin) that both reduce bilayer stiffness but cause opposite changes in curvature. Bilayer softening plays a role in decreasing MAG's potency, whereas the curvature alters only the leakage kinetics, not potency. Based on previous studies, we propose that membrane softening somehow makes it more difficult for MAG to reach a critical concentration necessary to produce pore formation⎯maybe because the energetics of lateral association among bilayer-spanning MAG monomers favors the monomers in softer bilayers. Unsaturated lipids may therefore influence MAG potency by modulating membrane stiffness.