Limiting an antimicrobial peptide to the lipid-water interface enhances its bacterial membrane selectivity: a case study of MSI-367

Abstract

In a minimalist design approach, a synthetic peptide MSI-367 [(KFAKKFA)(3)-NH(2)] was designed and synthesized with the objective of generating cell-selective nonlytic peptides, which have a significant bearing on cell targeting. The peptide exhibited potent activity against both bacteria and fungi, but no toxicity to human cells at micromolar concentrations. Bacterial versus human cell membrane selectivity of the peptide was determined via membrane permeabilization assays. Circular dichroism investigations revealed the intrinsic helix propensity of the peptide, β-turn structure in aqueous buffer and extended and turn conformations upon binding to lipid vesicles. Differential scanning calorimetry experiments with 1,2-dipalmitoleoyl-sn-glycero-3-phosphatidylethanolamine bilayers indicated the induction of positive curvature strain and repression of the fluid lamellar to inverted hexagonal phase transition by MSI-367. Results of isothermal titration calorimetry (ITC) experiments suggested the possibility of formation of specific lipid-peptide complexes leading to aggregation. (2)H nuclear magnetic resonance (NMR) of deuterated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) multilamellar vesicles confirmed the limited effect of the membrane-embedded peptide at the lipid-water interface. (31)P NMR data indicated changes in the lipid headgroup orientation of POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine lipid bilayers upon peptide binding. Membrane-embedded and membrane-inserted states of the peptide were observed via sum frequency generation vibrational spectroscopy. Circular dichroism, ITC, and (31)P NMR data for Escherichia coli lipids agree with the hypothesis that strong electrostatic lipid-peptide interactions embrace the peptide at the lipid-water interface and provide the basis for bacterial cell selectivity.

Figure 1

(A) Hemolysis profile of MSI-367 in six independent experiments at indicated concentrations (μg/mL). Maximum lysis (100%) was observed with 2% Triton 100. (B) MSI-367 induced ANS uptake into E. coli membrane. Fluorescence spectrum of ANS equilibrated with E. coli cells (1), and in the presence of 0.67 μM (2), 1.33 μM (3), 2.0 μM (4) and 2.67 μM (5) quantities of MSI-367. The E. coli cell density, as measured by the OD_600, was 1.20. The blue shift in the fluorescence emission maximum indicates the extent of penetration of ANS into E. coli membrane as a function of peptide concentration.

Figure 2

(A) Helix wheel representation of MSI-367 and CD spectra of MSI-367 in different media: phosphate buffered saline pH 7.4 (1), SUVs of POPC (2), SUVs of E. coli total lipids (3), and TFE (4). Peptide concentration was 30 μM. The concentration of POPC was 550 μM, and that of E. coli lipids was 840 μM. When all the residues are assumed to be part of the helix, the polar angle subtended by the helix is 154°. (B) ¹H-NMR spectra of MSI-367: 2.53 mM peptide in 20 mM sodium phosphate buffer (pH 6.0) (traces (b) and (d)) obtained from a 900 MHz Bruker spectrometer (East Lansing, MI); 1.5 mM peptide and 180 mM SDS-d23 in 10 mM sodium phosphate buffer (pH 6.0) (traces (a) and (c)).

Figure 2

(A) Helix wheel representation of MSI-367 and CD spectra of MSI-367 in different media: phosphate buffered saline pH 7.4 (1), SUVs of POPC (2), SUVs of E. coli total lipids (3), and TFE (4). Peptide concentration was 30 μM. The concentration of POPC was 550 μM, and that of E. coli lipids was 840 μM. When all the residues are assumed to be part of the helix, the polar angle subtended by the helix is 154°. (B) ¹H-NMR spectra of MSI-367: 2.53 mM peptide in 20 mM sodium phosphate buffer (pH 6.0) (traces (b) and (d)) obtained from a 900 MHz Bruker spectrometer (East Lansing, MI); 1.5 mM peptide and 180 mM SDS-d23 in 10 mM sodium phosphate buffer (pH 6.0) (traces (a) and (c)).

Figure 3

DSC thermograms of DiPoPE incorporated with different concentrations of MSI-367. The heating scan rate was 1 °C/min. The vertical dotted line shows the phase transition temperature (T_m=43 °C) of pure DiPoPE. The phase transition thermograms of DiPoPE containing 0.2 and 0.4 mole% of peptide are also shown.

Figure 4

(A) de-Paked ²H quadrupole coupling spectra of POPC-d₃₁ MLVs and POPC-d₃₁ MLVs incorporated with 3 and 5 mole% of MSI-367. (B) Plot of order parameter versus lipid acyl chain carbon number: POPC-d₃₁ (empty triangles), POPC-d₃₁/3 mol%MSI-367 (filled circles), POPC-d₃₁/5 mol%MSI-367 (empty circles).

Figure 5

³¹P chemical shift spectra of aligned POPC (A), POPE (B) and POPG (C) lipid bilayers in the presence and absence of MSI-367. The peptide concentrations are indicated in the figure. Each spectrum was obtained from 4 mg lipids and required 100–1000 transients.

Figure 6

³¹P chemical shift spectra of aligned E. coli lipid bilayers in the presence and absence of MSI-367 (A) and MSI-78 (B). The peptide concentrations are indicated in the figure. Each spectrum was obtained from 4 mg lipids and required 100–1000 transients.

Figure 7

Titration calorimetry thermograms of POPC (A) and E. coli (B) lipid into peptide titrations. Aliquots of a 10 μL lipid solution (~20 mM) were added to the peptide solution (5 μM) in the reaction cell (V = 1.0 mL). Top panel shows the calorimeter trace. The enthalpy of reaction, which is calculated by integration of the calorimeter traces, is given in bottom panel.

Figure 8

SFG-Vibrational spectra: Amide I signals obtained from the single POPG bilayer in contact with 0.8 μM MSI-78 (A), and 0.8 μM (B), 1.2 μM (C) and 2.0 μM (D) MSI-367 solutions. Open squares: ppp spectra; filled squares: ssp spectra. While there is no signal observed from 0.8 μM MSI-367, a low-intensity ppp signal was observed for 1.2 μM concentration of the peptide.

Figure 9

The solid line represents the relationship between the SFG ppp/ssp signal strength ratio and the tilt angle (θ) of a helical peptide by assuming an identical orientation relative to the lipid bilayer-normal. Experimentally measured data points are shown for 0.8 μM MSI-78 and 1.2 μM MSI-367.