Tuneable poration: host defense peptides as sequence probes for antimicrobial mechanisms

Abstract

The spread of antimicrobial resistance stimulates discovery strategies that place emphasis on mechanisms circumventing the drawbacks of traditional antibiotics and on agents that hit multiple targets. Host defense peptides (HDPs) are promising candidates in this regard. Here we demonstrate that a given HDP sequence intrinsically encodes for tuneable mechanisms of membrane disruption. Using an archetypal HDP (cecropin B) we show that subtle structural alterations convert antimicrobial mechanisms from native carpet-like scenarios to poration and non-porating membrane exfoliation. Such distinct mechanisms, studied using low- and high-resolution spectroscopy, nanoscale imaging and molecular dynamics simulations, all maintain strong antimicrobial effects, albeit with diminished activity against pathogens resistant to HDPs. The strategy offers an effective search paradigm for the sequence probing of discrete antimicrobial mechanisms within a single HDP.

Figure 1

Cecropin sequence probes. Peptide sequences, linear (a) and configured on helical wheels (b). Blue and grey cylinders denote helical N-terminal (blue) and C-terminal (grey) domains. Lysines are in blue, arginines and neutral polar residues are in light blue, relative identities of hydrophobic residues are in yellow. Glycine residues are in red and glycine zippers are highlighted by overarching horizontal brown brackets. Glycine residues in ChoC and their replacements in ChoM are in orange in the helical wheels. Green lines in the helical wheels of ChoC and ChoM indicate polar angles. The AGPA hinge and the W2 residue are underlined. A coiled-coil designation, gabcdef, is shown along the CecM sequence. Heptad repeats are shown underneath. Only two i, i + 7 pairs are given for clarity. (c) A schematic representation of a carpet-like mechanism by CecB oriented flat on a phospholipid bilayer. For clarity, only one phospholipid per leaflet is shown (aliphatic chains in grey, headgroups in pink).

Figure 2

Membrane disruption mechanisms in DLPC/DLPG (3:1, molar ratio) supported lipid bilayers. (a) Schematic representations of relative orientations of cecropin peptides in lipid bilayers: (from top down) CecB, CecM, Choc and ChoM. Designations are as in Fig. 1. (b) Topography of SLBs treated with cecropin peptides (left) and cross-sections along the highlighted lines (right). The images were taken at 90 min (CecB), 15 min (CecM), 10 min (ChoC) and 6 min (ChoM) of incubation. Color scale is 6 nm.

Figure 3

Peptide orientations in membranes. (a) 0.5 µs snapshots of molecular dynamics simulations for individual cecropin peptides in AUVs. (b) A representative simulation for ChoM following an equilibration phase (0 ns). Key: peptide helices are in magenda, lipid aliphatic chains are in cyan, phospholipid headgroups are green spheres (see also Movies S1–S4).

Figure 4

ChoM orientation in AUVs. (a) Oriented ²H NMR quadrupolar splittings in AUVs (POPC/POPG, 3:1 molar ratio) at L/P ratio 25 for four ChoM mutants each with a single deuterated alanine (Ala-d₃) mutation at positions 4, 8, 12 and 20, respectively. (b) A GALA-derived helix model showing relative positions of labelled Ala-d₃ (brown).