How do Antimicrobial Peptides Interact with the Outer Membrane of Gram-Negative Bacteria? Role of Lipopolysaccharides in Peptide Binding, Anchoring, and Penetration.

Gram-negative bacteria possess a complex structural cell envelope that constitutes a barrier for antimicrobial peptides that neutralize the microbes by disrupting their cell membranes. Computational and experimental approaches were used to study a model outer membrane interaction with an antimicrobial peptide, melittin. The investigated membrane included di[3-deoxy-d-manno-octulosonyl]-lipid A (KLA) in the outer leaflet and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) in the inner leaflet. Molecular dynamics simulations revealed that the positively charged helical C-terminus of melittin anchors rapidly into the hydrophilic headgroup region of KLA, while the flexible N-terminus makes contacts with the phosphate groups of KLA, supporting melittin penetration into the boundary between the hydrophilic and hydrophobic regions of the lipids. Electrochemical techniques confirmed the binding of melittin to the model membrane. To probe the peptide conformation and orientation during interaction with the membrane, polarization modulation infrared reflection absorption spectroscopy was used. The measurements revealed conformational changes in the peptide, accompanied by reorientation and translocation of the peptide at the membrane surface. The study suggests that melittin insertion into the outer membrane affects its permeability and capacitance but does not disturb the membrane's bilayer structure, indicating a distinct mechanism of the peptide action on the outer membrane of Gram-negative bacteria.

Membrane Potentials Trigger Molecular-Scale Rearrangements in the Outer Membrane of Gram-Negative Bacteria.

The structural complexity of the cell envelope of Gram-negative bacteria limits the fabrication of realistic models of bacterial cell membranes. A vertical Langmuir-Blodgett withdrawing was used to deposit a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) monolayer on the Au(111) surface. The second leaflet composed of di[3-deoxy-D-manno-octulosonyl]-lipid A (KLA) was deposited using Langmuir-Schaefer transfer. The use of an electrode material as a support for the POPE-KLA bilayer allowed electrochemical control of the membrane's stability, compactness, and structure. Capacitance-potential curves showed a typical pattern for the supported lipid bilayers electrochemical characteristic. The minimum membrane capacitance was ∼4 µF cm-2 and did not change in the following desorption-adsorption cycles, indicating the presence of a stable bilayer structure with an asymmetric composition of both leaflets. However, at a molecular scale, as elucidated in spectroelectrochemical experiments, large differences in the response of both leaflets to electric potentials were observed. The acyl chains in POPE and KLA existed in a liquid state. The quantitative analysis of the CH stretching modes indicated potential-driven reorientations in the hydrophobic fragment of the bilayer, already in the adsorbed state. To assign observed rearrangements to POPE and KLA lipids in both leaflets, per-deuterated d31-POPE was transferred into the inner leaflet. Since no potential-dependent changes of the CD2 stretching modes in the d31-POPE-KLA bilayer were observed, reorientations in the acyl chain region were assigned to the KLA molecules. Mg2+ ions were bound to the polar head groups of KLA. The strength of electrostatic interactions in the polar head group region of KLA was dependent on the direction of the electric field. At negative electric potentials, the binding of divalent cations weakened, which gave the KLA molecules increased orientational flexibility. This behavior in electric fields is peculiar for the outer membrane and indicates that the microbial cell membranes have different electrochemical properties than phospholipid bilayers.

Structural changes in the model of the outer cell membrane of Gram-negative bacteria interacting with melittin: an in situ spectroelectrochemical study.

The cell membrane of Gram-negative bacteria interacting with an antimicrobial peptide presents a complex supramolecular assembly. Fabrication of models of bacterial cell membranes remains a large experimental challenge. Langmuir-Blodgett and Langmuir-Schaefer (LS-LB) transfer makes possible the deposition of multicomponent asymmetric lipid bilayers onto a gold surface. Two lipids: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and di[3-deoxy-D-manno-octulosonyl]-lipid A (KLA) were used to deposit a model of the outer membrane of Gram-negative bacteria on the Au(111) substrate. The use of gold as the solid substrate enables control of the membrane potential. Molecular scale changes in the model membrane exposed to physiological electric fields and interacting with melittin antimicrobial peptide are discussed in this paper. The interaction of the outer membrane with melittin leads to an increase in the membrane capacitance and permeability to ions and water. The stability of the outer membrane with bound melittin decreases at positive membrane potentials. In situ polarization modulation infrared reflection absorption spectroscopy is used to investigate membrane potential-dependent changes in the structure of the outer membrane interacting with melittin. The hydration of the ester carbonyl groups is not affected by the interaction with melittin. However, the orientation and hydrogen bond network with the carboxylate groups in KLA changes drastically after POPE-KLA bilayer interacts with melittin. We propose that the positively charged groups in the amino acids present at the C-terminus of the peptide interact directly with the polar head group of KLA. Simultaneously, the packing order in hydrocarbon chains in the membrane with bound melittin increases. A hydrophobic match between the chains in the lipids and the peptide, which spans the membrane, seems to be responsible for the ordering of the hydrocarbon chains region of the bilayer. The N-terminus enters into the hydrophobic region of the membrane and forms a channel to the hydrophilic head groups in POPE.

The shape of lipid molecules affects potential-driven molecular-scale rearrangements in model cell membranes on electrodes.

Planar asymmetric lipid bilayers composed of phosphatidylethanolamine and phosphatidylglycerol lipids are transferred onto a gold electrode surface. Lipids containing two saturated, one monounsaturated and two monounsaturated hydrocarbon chains compose the model membranes. Results of electrochemically controlled polarization modulation infrared reflection absorption spectroscopy and quartz crystal microbalance with energy dissipation studies reveal two different types of electric potential-dependent structural rearrangements in the bilayers. They are correlated with the geometry of the lipid molecule. Packing parameter correlates the cross-section area of the hydrophobic and hydrophilic parts of amphiphilic molecules. In bilayers composed of lipids with the packing parameter <1, the hydrocarbon chains are tilted with respect to the bilayer plane and the polar head groups are well hydrated. At a threshold potential an abrupt flow of water through the bilayer is connected with membrane dehydration and upward orientation of the chains. In bilayers composed of lipids with packing parameter ≥1, electric potentials have negligible effect on the membrane structure. A simple rule correlating the packing parameter with molecular scale changes occurring at electrified membranes has a large diagnostic implication for biomimetic studies and our understanding of molecular processes occurring in biological cell membranes.