Antimicrobial activity and membrane selective interactions of a synthetic lipopeptide MSI-843

Abstract

Lipopeptide MSI-843 consisting of the nonstandard amino acid ornithine (Oct-OOLLOOLOOL-NH2) was designed with an objective towards generating non-lytic short antimicrobial peptides, which can have significant pharmaceutical applications. Octanoic acid was coupled to the N-terminus of the peptide to increase the overall hydrophobicity of the peptide. MSI-843 shows activity against bacteria and fungi at micromolar concentrations. It permeabilizes the outer membrane of Gram-negative bacterium and a model membrane mimicking bacterial inner membrane. Circular dichroism investigations demonstrate that the peptide adopts alpha-helical conformation upon binding to lipid membranes. Isothermal titration calorimetry studies suggest that the peptide binding to membranes results in exothermic heat of reaction, which arises from helix formation and membrane insertion of the peptide. 2H NMR of deuterated-POPC multilamellar vesicles shows the peptide-induced disorder in the hydrophobic core of bilayers. 31P NMR data indicate changes in the lipid head group orientation of POPC, POPG and Escherichia colitotal lipid bilayers upon peptide binding. Results from 31P NMR and dye leakage experiments suggest that the peptide selectively interacts with anionic bilayers at low concentrations (up to 5 mol%). Differential scanning calorimetry experiments on DiPOPE bilayers and 31P NMR data from E.coli total lipid multilamellar vesicles indicate that MSI-843 increases the fluid lamellar to inverted hexagonal phase transition temperature of bilayers by inducing positive curvature strain. Combination of all these data suggests the formation of a lipid-peptide complex resulting in a transient pore as a plausible mechanism for the membrane permeabilization and antimicrobial activity of the lipopeptide MSI-843.

Fig. 1

MSI-843 induced ANS uptake into E. coli membrane. Fluorescence spectra of ANS equilibrated with E. coli cells (a), and in the presence of 0.67 (b), 1.33 (c), 2.0 (d), 2.67 (e) and 3.35 μM (f) concentrations of MSI-843. The E. coli cell density, as measured at OD₆₀₀, was 0.065.

Fig. 2

Extent of carboxyfluoresceine dye leakage as a function of time and peptide concentration from POPC/POPG (3 : 1) SUVs. Lipid concentration was 26 μM.

Fig. 3

(A) CD spectra of MSI-843 in (a) Tris buffer (10 mM Tris–HCl, 150 mM NaCl, 2 mM EDTA, pH = 7.4) and (b) 2 mM POPC in Tris buffer. Peptide concentration was 20 μM and P / L was 1 : 100. (B) Helical wheel projection of MSI-843.

Fig. 4

Titration calorimetry thermograms of POPC and POPC/POPG (3 : 1) SUVs (20 mM) titrated against a solution of MSI-843 (660 μM) at 25 °C. Aliquots of a 10 μL peptide solution were added to the vesicle suspension in the reaction cell (V = 1.280 mL). Panel A shows the calorimeter trace for POPC SUVs. The enthalpy of reaction, which was calculated by integrating the calorimeter traces, is given in panel B (POPC SUVs filled circles; POPC/POPG (3 : 1) SUVs filled squares).

Fig. 5

(A) de-Paked ²H quadrupole coupling spectra of POPC-d₃₁ MLVs with and without the lipopeptide MSI-843. (B) ²H order parameter profiles for POPC-d₃₁ bilayers containing 0 mol% (filled circles), 3 mol% (empty squares) and 5 mol% (empty circles) MSI-843.

Fig. 6.

³¹P spectra of oriented bilayers containing MSI-843. The lipid bilayer normal was parallel to B_o. Spectra of (A) POPC, (B) POPG, and (C) E. coli lipid bilayers were obtained at 25 °C with the indicated MSI-843 concentrations (mol%).

Fig. 7

³¹P spectra of oriented pure E. coli total lipid membrane bilayers at 35 (A) and 50 °C (B).³¹P spectra of oriented E. coli total lipid membrane bilayers containing 3 mol% of MSI-843 at (C) 70, (D) 75 and (E) 80 °C.

Fig. 8

DSC thermograms of DiPoPE MLVs containing (A) 0, (B) 0.2, and (C) 0.4 mol% MSI-843. The rates of heating and cooling were 1 °C/min.