Biophysical Investigations Elucidating the Mechanisms of Action of Antimicrobial Peptides and Their Synergism

Abstract

Biophysical and structural investigations are presented with a focus on the membrane lipid interactions of cationic linear antibiotic peptides such as magainin, PGLa, LL37, and melittin. Observations made with these peptides are distinct as seen from data obtained with the hydrophobic peptide alamethicin. The cationic amphipathic peptides predominantly adopt membrane alignments parallel to the bilayer surface; thus the distribution of polar and non-polar side chains of the amphipathic helices mirror the environmental changes at the membrane interface. Such a membrane partitioning of an amphipathic helix has been shown to cause considerable disruptions in the lipid packing arrangements, transient openings at low peptide concentration, and membrane disintegration at higher peptide-to-lipid ratios. The manifold supramolecular arrangements adopted by lipids and peptides are represented by the 'soft membranes adapt and respond, also transiently' (SMART) model. Whereas molecular dynamics simulations provide atomistic views on lipid membranes in the presence of antimicrobial peptides, the biophysical investigations reveal interesting details on a molecular and supramolecular level, and recent microscopic imaging experiments delineate interesting sequences of events when bacterial cells are exposed to such peptides. Finally, biophysical studies that aim to reveal the mechanisms of synergistic interactions of magainin 2 and PGLa are presented, including unpublished isothermal titration calorimetry (ITC), circular dichroism (CD) and dynamic light scattering (DLS) measurements that suggest that the peptides are involved in liposome agglutination by mediating intermembrane interactions. A number of structural events are presented in schematic models that relate to the antimicrobial and synergistic mechanism of amphipathic peptides when they are aligned parallel to the membrane surface.

Keywords: carpet model; cecropin; local disorder; magainin; membrane macroscopic phase; membrane pore; membrane topology; peptide-lipid interactions; soft membrane adapt and respond, also transiently (SMART) model; toroidal pore.

Figure 1

Schematic models illustrating how antimicrobial peptides work and interact with membranes (A–D), and how two peptides can interact synergistically in a membrane environment (E–G). (A) Peptides such as magainin partition into the membrane interface and cause disordering of the lipid packing. (B) Bilayer openings form stochastically when the peptide concentration increases locally, or when the membrane disrupts at high peptide-to-lipid ratios [72]. Along the openings, the peptides can insert and cross in in-planar or at tilted alignments. (C) In molecular dynamics calculations schematic, amphipathic helices have been simulated to form double belts [73], an arrangement which also agrees with the in-planar alignment of the peptide helices observed by solid-state nuclear magnetic resonance NMR spectroscopy [38]. (D) Fluorescence quenching experiments suggest mesophase structures formed by in-plane oriented helices [74]. (E) The membrane disruptive properties of one peptide (yellow) help the insertion of another one (blue), which by itself is less likely to partition into membranes of high negative curvature [75]. (F) Peptide-peptide contacts result in the agglutination of liposomes (Figure 3) [76], and could be responsible for synergistic enhancement of activities. (G) A more densely packed mesophase arrangement forms in the presence of two peptides with complementary charge distribution such as magainin 2 and PGLa [77]. Notably, multiple mechanisms, such as a combination of E, F, and G may apply. Panels A, B, E, and F show side views, panels C, D, and G show top views of the lipid bilayer.

Figure 2

Isothermal Titration Calorimetry measurements of large unilamellar vesicles (LUVs) made of palmitoyl-oleoyl-phosphatidylethanolamine/ palmitoyl-oleoyl-phosphatidylglycerol POPE/POPG (3/1) at 440 µM total lipid concentration, into which solutions of 85 µM magainin 2 (A), 140 µM PGLa (B), and the equimolar mixture of PGLa and magainin 2 at 105 µM total peptide concentration (C) have been injected successively. Buffer: 10 mM Tris-HCl, pH 7, 100 mM NaCl. The inserts show close-ups of the regions t > 2215 s. The fittings of the data with a single binding site model are displayed as solid lines in panels D–F. The open circles in panel F show the difference of the heat of reactions when comparing the mixture of peptides with the sums obtained from the magainin 2 and PGLa titrations (i.e., half the intensities of the fitted curves shown in panels D and E, dashed line). The values of the enthalpies (ΔH), entropies (ΔS), binding constant (K), and apparent stoichiometry (N) are reported in the corresponding graphs.

Figure 3

Dynamic light s cattering (DLS) size measurement of LUVs made of 440 µM POPE/POPG (3/1) and incubated with magainin 2 (long dashed line), PGLa (short dashed line) or an equimolar mixture of PGLa and magainin 2 (continuous line) in 10 mM Tris-HCl buffer pH 7. The vesicles were made by mechanical extrusion through a 100 nm pore filter and the peptide to lipid ratio was kept constant at P/L = 3.6% for all tree measurements (this corresponds to the same condition for peptide injection at t ≈ 2030 s in Figure 2C). The averaged hydrodynamic radius < R_H > is indicated in the upper right of the graphs.