Nanoscale imaging reveals laterally expanding antimicrobial pores in lipid bilayers

Abstract

Antimicrobial peptides are postulated to disrupt microbial phospholipid membranes. The prevailing molecular model is based on the formation of stable or transient pores although the direct observation of the fundamental processes is lacking. By combining rational peptide design with topographical (atomic force microscopy) and chemical (nanoscale secondary ion mass spectrometry) imaging on the same samples, we show that pores formed by antimicrobial peptides in supported lipid bilayers are not necessarily limited to a particular diameter, nor they are transient, but can expand laterally at the nano-to-micrometer scale to the point of complete membrane disintegration. The results offer a mechanistic basis for membrane poration as a generic physicochemical process of cooperative and continuous peptide recruitment in the available phospholipid matrix.

Keywords: antibiotics; de novo protein design; innate host defense; nanometrology; nanoscopy.

Fig. 1.

Peptide design and folding. (A) Amhelin sequence, linear and on a helical wheel, and as an amphipathic helix spanning ∼3.15 nm (in blue, 2ZTA PDB entry rendered with PyMoL). (B) CD spectra of amhelin (20 μM) in 10 mM phosphate buffer (red line), ZUVs (blue line), and AUVs (green line). (C) LD spectra of amhelin (solid line) and the non-AMP (dashed line) (both at 20 μM) in AUVs. (D) ³¹P MAS ssNMR spectra of AUVs mixed with amhelin at different lipid–peptide ratios, −0.9 ppm (large peak) and 0.2 ppm (small peak) resonances arise from the PC and PG headgroups, respectively. (E) The rmsd for the molecular dynamics simulation of a model octameric amhelin pore (initial configuration in Inset) in an AUV bilayer. (F) Initial (Left) and later stage (Right) configurations of a model hexameric amhelin pore in the bilayer.

Fig. 2.

SIMS analysis of amhelin-treated supported lipid bilayers. (A) SIMS images of ¹²C¹⁴N^–, ¹²C¹⁵N^–, and ¹²C¹⁵N^–/¹²C¹⁴N^– signals from the supported lipid bilayers treated with the isotopically labeled peptide. (B) ¹²C¹⁵N^–/¹²C¹⁴N^– ratio expressed as HSI images. The rainbow scale changes from blue (natural abundance ratio of 0.37%) to red (40%, >100 times the natural ratio). This image is the sum of several sequential images to enhance the statistical significance of the measured ratios. (C) SIMS images of ¹²C¹⁴N^–, ¹²C¹⁵N^–, and ¹²C¹⁵N^–/¹²C¹⁴N^– signals from the supported lipid bilayers with no peptide. Incubation conditions: 10 μM, pH 7.4, 20 °C.

Fig. 3.

Amhelin-treated supported lipid bilayers. (A) In-air AFM topographic images with a cross-section along the highlight line. (B) Schematic representation of pore edges showing the thickness of the SLB (3.2 nm), the maximum observed height (4 nm), and the difference between the two (0.5–0.8 nm) accounted for by possible protrusion variants, three shown. For clarity, only one peptide (blue cylinder) and one phospholipid per layer are shown (aliphatic chains in gray, headgroups in pink). Incubation conditions: 10 μM, pH 7.4, 20 °C.

Fig. 4.

In-water AFM imaging of amhelin-treated supported lipid bilayers. (A) Topography of supported lipid bilayers during incubation with amhelin. Color scale (see Inset, 0 min): 3 nm (0–20 min); 9 nm (30–120 min). (B) Topography image after 40 min incubation with cross-sections along the highlighted lines. Incubation conditions: 0.5 μM, pH 7.4, 20 °C.

Fig. 5.

Proposed pore expansion mechanism for amphipathic antimicrobial peptides. Antimicrobial peptides (blue cylinders) bind to the surface of the membrane (S-state), insert into lipid bilayers forming pores (I-state), which can then expand indefinitely (E-state).