Biophysical implications for MP1-lipid membrane interactions

We have shown a synergistic enhancement of the rate and extent of membrane permeabilization by MP1 peptides when PE and PS lipids are present in the lipid membrane. This picture is confirmed and corroborated by complementary experiments using three different model membrane systems: LUVs, GUVs, and planar-supported bilayers. We consider the perturbation of the membrane by MP1 peptides in two steps: 1) binding of the peptides to the membrane, and 2) perturbation of the bilayer structure by bound peptides to induce leakage.

Binding isotherms ( Fig. 2 ) reveal that PS lipids cause a 7–8-fold increase in peptide bound to the membrane. This strongly outweighs the small ∼10% reduction in bound peptide concentration caused by the PE lipids. Therefore, we find that the dominant role of PS lipids’ contribution to the membrane disruption by MP1 is a large increase in peptide binding to the membrane.

The role of PE lipids in MP1-induced membrane disruption is twofold: 1) PE increases the susceptibility of the membrane to permeabilization by bound peptides, and 2) PE facilitates the formation of larger transmembrane pores. First, when the extent of GUV leakage is normalized to bound peptide concentration in the dose-response curves in Fig. 3 , c and d, it can be seen that 4–5 times lower bound peptide concentration is required to induce a similar leakage response compared to comparable GUVs without PE lipids. Second, GUV and AFM experiments corroborate the effect of larger pores forming in the presence of PE. Quantitative analysis of GUV leakage profiles in Fig. 6 and Table 2 reveal that the presence of PE increases the permeability of membranes by an order of magnitude compared to membranes lacking in PE. Furthermore, once pores formed in GUVs, they quickly (within seconds) grew large enough in size to allow larger macromolecules (3 and 10 kDa) to permeate the membrane ( Table 1 ); this compared to several tens of seconds for larger pores to form in GUVs lacking PE. Crucially, the formation of larger pores for PE-containing membranes is directly visualized by AFM ( Fig. 8 ), where the observed pore diameters are ∼5 times larger in the presence of PE (and hence ∼20–30 times larger in average pore area, consistent with the order-of-magnitude increase in permeability reported for the GUVs).

The formation of transmembrane pores was confirmed by complementary experimental systems and techniques. Rapid translocation of membrane-impermeable leakage markers across GUV membranes, an all-or-none LUV fluorescence leakage assay, and direct visualization of transmembrane defects by AFM imaging of planar bilayers, all confirm this to be true. While these pores are fairly long lived, the membranes were sometimes observed to temporarily reseal, regaining their barrier properties. This can clearly be seen in the leakage profiles of individual GUVs in Figs. 5 a, S4 , and S5 . GUV and planar bilayer imaging experiments also strongly suggest differences in the mechanism of pore formation depending on whether PE lipids are present. Images of GUVs that did not contain PE lipids often exhibited bright spots of increased local lipid concentrations on the membrane, which we interpret to be local aggregation of peptides and lipid ( Fig. 7 ). Similarly, AFM images showed locally raised regions of lipid scattered across the membrane for these lipid compositions ( Fig. S9 ) before the formation of pores ( Fig. S10 ). This contrasted to the pore-formation mechanism observed in the presence of PE, where local aggregates were not directly observed on the GUV surface and time-resolved AFM imaging showed pore growth to occur by the stepwise micellization and loss of lipid from the edge of the pores ( Fig. S8 ).

Besides the increased binding due to PS and the increased membrane susceptibility and pore size due to PE, the synergistic enhancement of membrane disruption facilitated by these lipids can be observed in the kinetics of initial permeabilization events. GUV experiments showed that PC/PE/PS GUVs leaked a factor-of-two quicker than other membrane compositions ( Table 1 ). This is again corroborated by the AFM studies where defects were observed in PC/PE/PS membranes almost immediately after peptide addition, whereas perturbations of other membrane compositions took a few 10 s of minutes to evolve. The complementary pore-promoting effects of PS on bound peptide concentrations and PE on membrane susceptibility far outweigh their slight inhibitory effects on each other’s roles (PE causes a slight reduction in binding affinity ( Fig. 2 ) and PS causes a decrease in the membrane susceptibility to bound peptide ( Fig. 3 , c and d)). This is apparent from the effects of MP1 on GUVs, where PC/PE/PS membranes experience the greatest membrane perturbation for any given total peptide concentration ( Fig. 3 , a and b) and the larger number of pores observed on the membrane surface by AFM ( Fig. 8 ). Therefore, our combined results provide a detailed mechanistic picture of the importance of increased PS and PE lipid concentrations in synergistically enhancing the membrane’s propensity for significant disruption of its barrier properties by MP1 peptides.

Variations in lipid composition are responsible for differences in membrane properties such as charge, fluidity, lateral pressure profiles, and mechanical moduli. Changes in these fundamental membrane properties directly affect their interactions with extraneous compounds, such as antimicrobial peptides. The cationic nature of the MP1 peptide is likely an important component in the initial step of peptide action, in which the peptide recognizes the target membrane due to electrostatic interactions and binds to it in a structured form, most of the time as a helix. Therefore, the inclusion of anionic PS lipids in these membranes increases these electrostatic interactions with the MP1 peptide (net charge = +2e). However, MP1-membrane interactions cannot be solely driven by electrostatics as these peptides were also found to disrupt zwitterionic PC and PC/PE membranes, likely through secondary hydrophobic initial binding interactions that lead to a significantly lower bound concentration of peptide compared to the anionic membranes.