Anti-antimicrobial peptides: folding-mediated host defense antagonists

Abstract

Antimicrobial or host defense peptides are innate immune regulators found in all multicellular organisms. Many of them fold into membrane-bound α-helices and function by causing cell wall disruption in microorganisms. Herein we probe the possibility and functional implications of antimicrobial antagonism mediated by complementary coiled-coil interactions between antimicrobial peptides and de novo designed antagonists: anti-antimicrobial peptides. Using sequences from native helical families such as cathelicidins, cecropins, and magainins we demonstrate that designed antagonists can co-fold with antimicrobial peptides into functionally inert helical oligomers. The properties and function of the resulting assemblies were studied in solution, membrane environments, and in bacterial culture by a combination of chiroptical and solid-state NMR spectroscopies, microscopy, bioassays, and molecular dynamics simulations. The findings offer a molecular rationale for anti-antimicrobial responses with potential implications for antimicrobial resistance.

Keywords: Antimicrobial Peptides; Membrane Biophysics; Microscopy; Molecular Dynamics; Nuclear Magnetic Resonance; Protein Design; Protein Folding.

FIGURE 1.

Peptide design. A, cathelicidin type: bovine myeloid antimicrobial peptide-27 (b27) and anti-b27. B, cecropin type: native cecropin B (cB), cecropin B template (cBt), and anti-cecropin B template (anti-cBt). Linear sequences (top) and configured onto helical wheels (bottom) are monomeric with 3.6 residues (left) and coiled-coil with 3.5 residues (right) per turn. Antimicrobial peptides and antagonists are shown in blue and red, respectively. Mutations in cBt are in black, and key residues in helix-disrupting motifs are underlined. Three overlapping G(X)_nG motifs are highlighted by horizontal brackets. Double-headed arrows denote electrostatic e-g′ interactions.

FIGURE 2.

Peptide folding probed by CD spectroscopy. A, CD spectra for b27 (red line), anti-b27 (blue line), and anti-b27:b27 (black line) in 10 mm phosphate buffer. B, CD spectra for b27 (red line) and anti-b27 (blue line) in anionic membranes. C and D, thermal unfolding curves (C) and their first derivatives for anti-b27:b27 (D) as a function of temperature at 222 nm. E, CD spectra for cB (green line), cBt (red line), anti-cBt (blue line), anti-cBt:cB (magenta line), and anti-cBt:cBt (black line) in 10 mm phosphate buffer. F, CD spectra for cB (green line), cBt (red line), and anti-cBt (blue line) in anionic membranes. G and H, thermal unfolding curves (G) and their first derivatives (H) for anti-cBt:cBt as a function of temperature at 222 nm. CD spectra are for 30 μm concentration of each peptide, pH 7.4, room temperature. Thermal unfolding curves are for 15 μm (red line), 30 μm (black line), and 50 μm (blue line) concentration of each peptide. Lipid-peptide ratio for CD spectra in anionic membranes was 100:1.

FIGURE 3.

Stoichiometry of anti-cBt:cBt interactions. A, CD points recorded for 222 nm by titrating anti-cBt into cBt (30 μm). The intersection of the lines fitted on the titration curve indicates a 1:1 binding stoichiometry. B, sedimentation equilibrium analysis. Experimental data (open circles) were collected at 37,000 rpm for a 100 μm sample at 20 °C. The line is a calculated curve for an ideal dimer. C, isothermal titration calorimetry of the interactions. Heat absorbed (μcal/s) for each isotherm is plotted versus titration time (min) and shows endothermic binding (top panel). Integrated heats (kcal/mol) are plotted versus anti-cBt:cBt molar ratios (bottom panel).

FIGURE 4.

Molecular dynamics simulations of cecropin assembly. Secondary structure visualization after 50 ns for cBt (A) and anti-cBt (B), and for anti-cBt:cBt (C) at the initial configuration (0 ns) and after 100 ns (D).

FIGURE 5.

Cecropin folding in anionic membranes. A, LD spectra for cBt (dotted line), anti-cBt (bold line), cBt added to anti-cBt (dashed line) and pre-formed anti-cBt:cBt (dot-dashed line). Lipid-peptide ratio was 100:1 (20 μm peptide), pH 7.4, room temperature. Arrows point to π-π* and n-π* electronic transition bands. B, solid-state NMR spectra for blank (no peptide, black), cBt (red), and anti-cBt:cBt (blue). Lipid-peptide ratio was 25:1, pH 7.4, room temperature. Vertical dashed lines are to assist comparison between the innermost and outermost splittings of the three spectra.

FIGURE 6.

Stain-dead S. aureus cells. A, percentage of stain-dead cells as a function of time for cB (100 μm, red squares), cBt (25 μm, green triangles), anti-cBt (250 μm, purple crosses), and anti-cBt:cBt (125:25 μm, blue diamonds). B, confocal microscopy images of cells stained with propidium iodide after incubation with corresponding peptide. C, absorbances measured overnight at 600 nm for the bacterium incubated with anti-cBt:cBt at different ratios (black pillars), for the bacterium without peptide and for the culture medium only (gray pillars). Error bars, S.D.

FIGURE 7.

A, 100× light micrographs of Gram-stained E. coli and P. aeruginosa after overnight incubations with peptide. B, averaged total of Gram-stained cells. Shown is anti-cBt:cBt at (100:10 μm).