Membrane interaction of antimicrobial peptides using E. coli lipid extract as model bacterial cell membranes and SFG spectroscopy

Abstract

Supported lipid bilayers are used as a convenient model cell membrane system to study biologically important molecule-lipid interactions in situ. However, the lipid bilayer models are often simple and the acquired results with these models may not provide all pertinent information related to a real cell membrane. In this work, we use sum frequency generation (SFG) vibrational spectroscopy to study molecular-level interactions between the antimicrobial peptides (AMPs) MSI-594, ovispirin-1 G18, magainin 2 and a simple 1,2-dipalmitoyl-d62-sn-glycero-3-phosphoglycerol (dDPPG)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) bilayer. We compared such interactions to those between the AMPs and a more complex dDPPG/Escherichia coli (E. coli) polar lipid extract bilayer. We show that to fully understand more complex aspects of peptide-bilayer interaction, such as interaction kinetics, a heterogeneous lipid composition is required, such as the E. coli polar lipid extract. The discrepancy in peptide-bilayer interaction is likely due in part to the difference in bilayer charge between the two systems since highly negative charged lipids can promote more favorable electrostatic interactions between the peptide and lipid bilayer. Results presented in this paper indicate that more complex model bilayers are needed to accurately analyze peptide-cell membrane interactions and demonstrates the importance of using an appropriate lipid composition to study AMP interaction properties.

Keywords: Antimicrobial peptide; Bacterial cell; Membrane mimetics; Peptide–lipid interactions; Sum frequency generation; Vibrational spectroscopy.

Fig. 1

SFG spectra collected before (black) and after (red) the addition of MSI-594 to the subphase of (a) dDPPG/POPG bilayer in the C-D stretching frequency range; (b) dDPPG/E. coli polar extract bilayer in the C-D stretching frequency range; (c) dDPPG/ POPG bilayer in the C-H stretching frequency range; (d) dDPPG/E. coli polar extract bilayer in the C-H stretching frequency range.

Fig. 2

Time-dependent SFG signal observed from the dDPPG/POPG bilayer (top) and the dDPPG/E. coli polar extract bilayer (bottom). The arrow in the insert shows the time when the MSI-594 was added to the lipid bilayer subphase.

Fig. 3

SFG spectra of Amide I signal from the MSI-594 associated with (top) dDPPG/POPG bilayer and (bottom) dDPPG/E. coli polar extract bilayer.

Fig. 4

SFG spectra collected before (black) and after (red) the addition of ovispirin-1 G18 to the subphase of (a) dDPPG/POPG bilayer in the C-D stretching frequency range; (b) dDPPG/E. coli polar extract bilayer in the C-D stretching frequency range; (c) dDPPG/ POPG in the C-H stretching frequency range; (d) dDPPG/E. coli polar extract bilayer in the C-H stretching frequency range.

Fig. 5

SFG time-dependent signal detected from (a) dDPPG/POPG bilayer, inset is focused on the time period directly before and after the injection of the peptide to the subphase; (b) dDPPG/E. coli polar extract bilayer; (c) dDPPG/E. coli polar extract bilayer zoomed in before and after the injection of ovispirin-1 G18 to the subphase. Peptide injection is indicated by arrow.

Fig. 6

SFG spectra of Amide I signal from the ovispirin-1 associated with (top) dDPPG/POPG bilayer and (bottom) dDPPG/E. coli polar extract bilayer.

Fig. 7

SFG spectra collected before (black) and after (red) the addition of magainin 2 to the subphase of (a) dDPPG/POPG bilayer in the C-D stretching frequency range; (b) dDPPG/E. coli polar extract bilayer in the C-D stretching frequency range; (c) dDPPG/ POPG in the C-H stretching frequency range; (d) dDPPG/E. coli polar extract bilayer in the C-H stretching frequency range. The magainin subphase concentration is 800 nM.

Fig. 8

SFG time-dependent signal detected from (a) dDPPG/POPG bilayer, (b) dDPPG/POPG bilayer in the first 800 seconds, (c) dDPPG/E. coli polar extract bilayer; (c) dDPPG/E. coli polar extract bilayer zoomed in the first 1000 seconds, before and after the injection of magainin 2 to the subphase to reach 800 nM. Peptide injection is indicated by arrow.

Fig. 9

SFG spectra of Amide I signal from the magainin 2 associated with (top) dDPPG/POPG bilayer and (bottom) dDPPG/E. coli polar extract bilayer. The magainin 2 subphase concentration is 800 nM.

Fig. 10

SFG spectra collected before (black) and after (red) the addition of magainin 2 to the subphase of (a) dDPPG/POPG bilayer in the C-D stretching frequency range; (b) dDPPG/E. coli polar extract bilayer in the C-D stretching frequency range; (c) dDPPG/ POPG in the C-H stretching frequency range; (d) dDPPG/E. coli polar extract bilayer in the C-H stretching frequency range. The magainin subphase concentration is 2.0 μM.

Fig. 11

SFG time-dependent signal detected from (a) dDPPG/POPG bilayer, (b) dDPPG/POPG bilayer in the first 600 seconds, (c) dDPPG/E. coli polar extract bilayer; (c) dDPPG/E. coli polar extract bilayer zoomed in the first 800 seconds, before and after the injection of magainin 2 to the subphase to reach 2.0 μM. Peptide injection is indicated by arrow.

Fig. 12

SFG spectra of Amide I signal from the magainin 2 associated with (top) dDPPG/POPG bilayer and (bottom) dDPPG/E. coli polar extract bilayer. The magainin 2 subphase concentration is 2.0 μM.