Lipid tails modulate antimicrobial peptide membrane incorporation and activity

Abstract

Membrane disrupting antimicrobial peptides (AMPs) are often amphipathic peptides that interact directly with lipid bilayers. AMPs are generally thought to interact mostly with lipid head groups, but it is less clear how the lipid alkyl chain length and saturation modulate interactions with membranes. Here, we used native mass spectrometry to measure the stoichiometry of three different AMPs-LL-37, indolicidin, and magainin-2-in lipid nanodiscs. We also measured the activity of these AMPs in unilamellar vesicle leakage assays. We found that LL-37 formed specific hexamer complexes but with different intermediates and affinities that depended on the bilayer thickness. LL-37 was also most active in lipid bilayers containing longer, unsaturated lipids. In contrast, indolicidin incorporated to a higher degree into more fluid lipid bilayers but was more active with bilayers with thinner, less fluid lipids. Finally, magainin-2 incorporated to a higher degree into bilayers with longer, unsaturated alkyl chains and showed more activity in these same conditions. Together, these data show that higher amounts of peptide incorporation generally led to higher activity and that AMPs tend to incorporate more into longer unsaturated lipid bilayers. However, the activity of AMPs was not always directly related to amount of peptide incorporated.

Keywords: Antimicrobial peptides; LL-37; Membrane interactions; Nanodiscs; Native mass spectrometry.

Figure 1.

(A) Schematic of addition of LL-37 to nanodiscs. (B–E) Relative amounts of incorporation of different LL-37 stoichiometries in nanodiscs containing DLPG (B), DMPG (C), DPPG (D), or SOPG (E), at 3:1 (purple), 6:1 (blue), 9:1 (green), or 18:1 (grey) molar ratios of LL-37:nanodisc. Data for DMPG are adapted from Walker et al.[7] (C) Adapted with permission from ref. . Note: hypothetical structures are meant to illustrate the method and not propose a particular structure.

Figure 2.

Average incorporation of peptides in nanodiscs containing different lipids at 3:1 (A, C, E) and 9:1 (B, D, F) ratios of peptide:nanodisc for LL-37 (A, B), indolicidin (C, D), and magainin-2 (E, F). Connected lines above bars indicate significance as determined by a T-test at the 95% confidence level. Solid, dashed, and dotted lines are used only to help differentiate between overlapping lines. Data for DMPG are from Walker et al.[7]

Figure 3.

ULV Leakage Assay with DMPG (A) or SOPG (B) ULVs with addition of 3:1, 9:1, or 18:1 ratios of LL-37:nanodisc. Fluorescence of carboxyfluorescein encapsulated LUVs was measured for 5 minutes. At 5 minutes, LL-37 was added at the molar ratios indicated. At 25 minutes, 0.1% Triton was added to determine maximum fluorescence. Results were normalized to highest and lowest fluorescence values, and the mean of the normalized fluorescence from 3 different ULV preparations was calculated. The shaded areas indicate the standard error of the mean for each stoichiometry.