Multiple peptide resistance factor (MprF)-mediated Resistance of Staphylococcus aureus against antimicrobial peptides coincides with a modulated peptide interaction with artificial membranes comprising lysyl-phosphatidylglycerol

Abstract

Modification of the membrane lipid phosphatidylglycerol (PG) of Staphylococcus aureus by enzymatic transfer of a l-lysine residue leading to lysyl-PG converts the net charge of PG from -1 to +1 and is thought to confer resistance to cationic antimicrobial peptides (AMPs). Lysyl-PG synthesis and translocation to the outer leaflet of the bacterial membrane are achieved by the membrane protein MprF. Consequently, mutants lacking a functional mprF gene are in particular vulnerable to the action of AMPs. Hence, we aim at elucidating whether and to which extent lysyl-PG modulates membrane binding, insertion, and permeabilization by various AMPs. Lysyl-PG was incorporated into artificial lipid bilayers, mimicking the cytoplasmic membrane of S. aureus. Moreover, we determined the activity of the peptides against a clinical isolate of S. aureus strain SA113 and two mutants lacking a functional mprF gene and visualized peptide-induced ultrastructural changes of bacteria by transmission electron microscopy. The studied peptides were: (i) NK-2, an α-helical fragment of mammalian NK-lysin, (ii) arenicin-1, a lugworm β-sheet peptide, and (iii) bee venom melittin. Biophysical data obtained by FRET spectroscopy, Fourier transform infrared spectroscopy, and electrical measurements with planar lipid bilayers were correlated with the biological activities of the peptides. They strongly support the hypothesis that peptide-membrane interactions are a prerequisite for eradication of S. aureus. However, degree and mode of modulation of membrane properties such as fluidity, capacitance, and conductivity were unique for each of the peptides. Altogether, our data support and underline the significance of lysyl-PG for S. aureus resistance to AMPs.

FIGURE 1.

Transmission electron microscopy images of peptide-treated S. aureus (clinical isolate). A, no peptide. B, NK-2 (20 μm). C, C7S (20 μm). D, NK11 (200 μm). E, melittin (12 μm). F, Ar-1 (20 μm). G, C/S-Ar-1 (20 μm). H, R/K-Ar-1 (20 μm). Each bar represents 0.5 μm.

FIGURE 2.

Peptide-membrane intercalation monitored by FRET spectroscopy. Peptides were added at 50 s. An increase of the I_Donor/I_Acceptor ratio corresponded to a reduced FRET efficiency and indicated insertion of peptides into the membranes. Experiments were done in 20 mm Hepes, 150 mm NaCl, pH 7.4, at 37 °C. A and B, dose-dependent intercalation of NK-2 at the indicated concentrations with vesicles composed of DOPG (A) and DOPG:lysyl-DOPG (B). C and D, intercalation of 0.4 μm NK-2 (solid black line), NK11 (dotted black line), melittin (solid gray line), Ar-1 (dotted gray line), and control (solid light gray line) into liposomes made of DOPG (C) and DOPG:lysyl-DOPG (D) (50:50, by mol). For better visualization of NK11 data, the control curve has been omitted in D.

FIGURE 3.

Influence of peptides on the phase transition behavior of lipids. The temperature dependence of the wave number of the symmetric methylene stretching vibration of the lipid acyl chains was determined by FTIR for pure lipids (A), for DPPG in combination with peptides (B), and for an equimolar mixture of DPPG:lysyl-DPPG in combination with peptides (C). The lipid-to-peptide molar ratio was 1:0.3. The second heating scan is shown. Lipids are as follows: DPPG (black circles), lysyl-DPPG (open circles), and DPPG:lysyl-DPPG (gray circles). Lipid:peptide combinations are as follows: NK-2 (filled diamonds), C7S (open diamonds), NK11 (open triangles), melittin (filled triangles), Ar-1 (filled squares), and C/S-Ar-1 (open squares).

FIGURE 4.

Peptide-induced changes in membrane capacitance and current traces of planar lipid bilayers. Peptides were added at 0 min to the cis side of lipid bilayers mimicking S. aureus ΔmprF mutant (PG:PE, left column), as well as wild type (PG:lysyl-PG:PE; right column) cytoplasmic membranes. First row (A and B), 0.5 μm NK-2. Second row (C and D), 0.1 μm melittin. Third row (E and F), 0.5 μm Ar-1. Transmembrane current and membrane capacitance (inset, capacitance is given as C/C₀) were monitored over time. The trans leaflet of each bilayer was formed of PG:PE. An applied positive voltage (dotted line) reflects the situation at the biological membrane (inside negative) as the trans side of the planar bilayer was grounded.

FIGURE 5.

Formation of membrane pores with defined conductivity levels by Ar-1. An asymmetric lipid bilayer consisting of PG:lysyl-PG:PE (25:25:50) at the cis side and PG:PE (50:50) at the trans side was formed after Ar-1 (0.5 μm) had been added to the bathing solution. After setting a clamp voltage (100 mV), we observed defined conductivity levels. An applied positive voltage reflects the situation at the biological membrane (inside negative) as the trans side of the planar bilayer was grounded. nS, nanosiemens.