Strong Membrane Permeabilization Activity Can Reduce Selectivity of Cyclic Antimicrobial Peptides

Abstract

Selectivity is a key requirement for membrane-active antimicrobials to be viable in therapeutic contexts. Therefore, the rational design or suitable selection of new compounds requires adequate mechanistic understanding of peptide selectivity. In this study, we compare two similar cyclic peptides that differ only in the arrangement of their three hydrophobic tryptophan (W) and three positively charged arginine (R) residues, yet exhibit different selectivities. This family of peptides has previously been shown to target the cytoplasmic membrane of bacteria, but not to act directly by membrane permeabilization. We have systematically studied and compared the interactions of the two peptides with zwitterionic phosphatidylcholine (PC) and negatively charged phosphatidylglycerol/phosphatidylethanolamine (PG/PE) model membranes using various biophysical methods to elucidate the mechanism of the selectivity. Like many antimicrobial peptides, the cyclic, cationic hexapeptides investigated here bind more efficiently to negatively charged membranes than to zwitterionic ones. Consequently, the two peptides induce vesicle leakage, changes in lipid packing, vesicle aggregation, and vesicle fusion predominantly in binary, negatively charged PG/PE membranes. The peptide with the larger hydrophobic molecular surface (three adjacent W residues) causes all these investigated effects more efficiently. In particular, it induces leakage by asymmetry stress and/or leaky fusion in zwitterionic and charged membranes, which may contribute to high activity but reduces selectivity. The unselective type of leakage appears to be driven by the more pronounced insertion into the lipid layer, facilitated by the larger hydrophobic surface of the peptide. Therefore, avoiding local accumulation of hydrophobic residues might improve the selectivity of future membrane-active compounds.

Figure 1

Absorption measurements characterize the adsorption and insertion of antimicrobial peptides into lipid monolayers. Changes in the surface pressure, Δπ, of a lipid monolayer 35 min after adding the peptide solution to the subphase (final concentration 900 nM) as a function of the initial surface pressure π₀. (Dark green squares) c(RW)₃ adsorption to DMPC monolayers, (light green squares) c(RW)₃ adsorption to DMPG/DMPE (1:1) monolayers, (dark red dots) cR₃W₃ adsorption so DMPC monolayers, (light red dots) cR₃W₃ adsorption to DMPG/DMPE (1:1) monolayers. (10 mM TRIS; 110 mM NaCl; 0.5 mM EDTA; pH 7.4; 20 °C) The corresponding measured surface pressures π over time are provided in Figure SI1.

Figure 2

Isothermal titration calorimetry analysis of the interaction of antimicrobial peptides with model membranes. (A) 20 mM POPC liposome suspension was titrated into 0.02 mM c(RW)₃ solution, (B) 10 mM POPG/POPE (1:1) liposome suspension was titrated into 0.1 mM c(RW)₃ solution, (C) 20 mM POPC liposome suspension was titrated into 0.1 mM cR₃W₃ solution, (D) 5 mM POPG/POPE (1:1) liposome suspension was titrated into 0.1 mM cR₃W₃ solution. Dots and squares represent the integrated heat per injection, lines the fit curve. The corresponding fitting parameters are summarized in Table 1. (10 mM TRIS; 110 mM NaCl; 0.5 mM EDTA; pH 7.4; 25 °C) The depicted data is representative of several experiments with slightly varied initial concentrations. Data in panel D has been published before and is reproduced here from ref (46) with permission from the Royal Society of Chemistry. Raw thermograms are provided in Figure SI2.

Figure 3

Laurdan fluorescence spectroscopy provides insight into lipid headgroup packing and membrane fluidity. This is expressed as the general polarization GP, which is calculated from the emission spectra provided in Figure SI3. Changes in membrane order, ΔGP, of 0.3 mM liposome suspensions over 2 h are shown as function of peptide concentration. (A) c(RW)₃ was added to POPC and POPG/POPE (1:1) liposomes, (B) cR₃W₃ was added to POPC and POPG/POPE (1:1) liposomes. (10 mM TRIS; 110 mM NaCl; 0.5 mM EDTA; pH 7.4; 25 °C).

Figure 4

Dynamic light scattering (DLS) analysis reveal the particle size (bars) and size distribution PDI (dots) of 30 μM liposome suspension after incubation with various concentrations of antimicrobial peptides for 24 h. Asterisks mark a particle sizes >1000 nm. (A) POPC liposomes and (B) POPG/POPE (1:1) liposomes were incubated with c(RW)₃. (C) POPC liposomes and (D) POPG/POPE (1:1) liposomes were incubated with cR₃W₃. (10 mM TRIS; 110 mM NaCl; 0.5 mM EDTA; pH 7.4; 25 °C) Data in panel D has been published before and is reproduced here from ref (46) with permission from the Royal Society of Chemistry.

Figure 5

Lipid mixing efficiency LME of 30 μM liposome suspension after incubation with various concentrations of antimicrobial peptides. (A) POPG/POPE (1:1) liposomes (light green) were incubated with c(RW)₃. (B) POPC liposomes (dark red) and POPG/POPE (1:1) liposomes (light red) were incubated with cR₃W₃. Data with poor intensity and therefore of questionable reliability are depicted in gray. (10 mM TRIS; 110 mM NaCl; 0.5 mM EDTA; pH 7.4; 25 °C) POPG/POPE data in panel B has been published before and is reproduced here from ref (46) with permission from the Royal Society of Chemistry. The corresponding emission spectra are provided in Figure SI4.

Figure 6

Calcein leakage reveals the permeabilization behavior of antimicrobial peptides. The total vesicle leakage L_total of 30 μM liposome suspensions is shown as a function of peptide concentration at increasing incubation times. (A) POPC liposomes were incubated with c(RW)₃, (B) POPG/POPE (1:1) liposomes were incubated with c(RW)₃, (C) POPC liposomes were incubated with cR₃W₃, (D) POPG/POPE (1:1) liposomes were incubated with cR₃W₃. As illustrated in Figure SI6, data with a decrease in Sum of B of more than 20% is depicted in gray. (10 mM TRIS; 110 mM NaCl; 0.5 mM EDTA; pH 7.4; 25 °C) Data in panel D has been published before and is reproduced here from ref (46) with permission from the Royal Society of Chemistry.