Single-Molecule Resolution of Antimicrobial Peptide Interactions with Supported Lipid A Bilayers

Abstract

The molecular interactions between antimicrobial peptides (AMPs) and lipid A-containing supported lipid bilayers were probed using single-molecule total internal reflection fluorescence microscopy. Hybrid supported lipid bilayers with lipid A outer leaflets and phospholipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)) inner leaflets were prepared and characterized, and the spatiotemporal trajectories of individual fluorescently labeled LL37 and Melittin AMPs were determined as they interacted with the bilayer surfaces comprising either monophosphoryl or diphosphoryl lipid A (from Escherichia coli) to determine the impact of electrostatic interactions. Large numbers of trajectories were obtained and analyzed to obtain the distributions of surface residence times and the statistics of the spatial trajectories. Interestingly, the AMP species were sensitive to subtle differences in the charge of the lipid, with both peptides diffusing more slowly and residing longer on the diphosphoryl lipid A. Furthermore, the single-molecule dynamics indicated a qualitative difference between the behavior of AMPs on hybrid Lipid A bilayers and on those composed entirely of DOPE. Whereas AMPs interacting with a DOPE bilayer exhibited two-dimensional Brownian diffusion with a diffusion coefficient of ∼1.7 μm²/s, AMPs adsorbed to the lipid A surface exhibited much slower apparent diffusion (on the order of ∼0.1 μm²/s) and executed intermittent trajectories that alternated between two-dimensional Brownian diffusion and desorption-mediated three-dimensional flights. Overall, these findings suggested that bilayers with lipid A in the outer leaflet, as it is in bacterial outer membranes, are valuable model systems for the study of the initial stage of AMP-bacterium interactions. Furthermore, single-molecule dynamics was sensitive to subtle differences in electrostatic interactions between cationic AMPs and monovalent or divalent anionic lipid A moieties.

Figure 1

Schematic of Langmuir-Blodgett/Langmuir-Schaefer deposition, as described in the text. To see this figure in color, go online.

Figure 2

Complementary cumulative probability distributions of residence times for AMPs on lipid A bilayers. The lines represent the best fits to the sum of two exponentials. To see this figure in color, go online.

Figure 3

Average residence times for fluorescently labeled LL37 and melittin on diphosphoryl E. coli lipid A/DOPE asymmetric bilayers (blue) and monophosphoryl asymmetric bilayers (orange). The error bars represent the SD for three separate experimental trials. To see this figure in color, go online.

Figure 4

Average diffusion coefficients determined from single-molecule tracking for fluorescent DOPE inserted into the outer leaflet and peptides adsorbed to diphosphoryl lipid A/DOPE asymmetric bilayers (blue), monophosphoryl asymmetric bilayers (orange), and DOPE symmetric bilayers (green). The error bars represent the SD for three separate experimental trials. To see this figure in color, go online.

Figure 5

(A) Probability density distributions of diffusive steps along the x axis for melittin on DOPE (magenta triangles) and monophosphoryl lipid A (black squares). The lines represent fits of the central peaks to a single Gaussian distribution. (B) Representative trajectories for melittin on DOPE (magenta) and monophosphoryl lipid A (black) are shown. To see this figure in color, go online.

Figure 6

(A) Waiting-time probability distribution and (B) characteristic timescale for AMPs on asymmetric lipid A SLBs. The waiting-time probabilities are offset by factors of 10 for clarity, with the LL37-diphosphoryl lipid A series having no offset. The lines are best fits of a stretched exponential to the data. To see this figure in color, go online.