Study of the interaction of lactoferricin B with phospholipid monolayers and bilayers.

Bovine lactoferricin (LfcinB) is an antimicrobial peptide obtained from the pepsin cleavage of lactoferrin. The activity of LfcinB has been extensively studied on diverse pathogens, but its mechanism of action still has to be elucidated. Because of its nonspecificity, its mode of action is assumed to be related to interactions with membranes. In this study, the interaction of LfcinB with a negatively charged monolayer of dipalmitoylphosphatidylglycerol has been investigated as a function of the surface pressure of the lipid film using in situ Brewster angle and polarization modulation infrared reflection absorption spectroscopy and on transferred monolayers by atomic force microscopy and polarized attenuated total reflection infrared spectroscopy. The data show clearly that LfcinB forms stable films at the air-water interface. They also reveal that the interaction of LfcinB with the lipid monolayer is modulated by the surface pressure. At low surface pressure, LfcinB inserts within the lipid film with its long molecular axis oriented mainly parallel to the acyl chains, while at high surface pressure, LfcinB is adsorbed under the lipid film, the hairpin being preferentially aligned parallel to the plane of the interface. The threshold for which the behavior changes is 20 mN/m. At this critical surface pressure, LfcinB interacts with the monolayer to form discoidal lipid-peptide assemblies. This structure may actually represent the mechanism of action of this peptide. The results obtained on monolayers are correlated by fluorescent probe release measurements of dye-containing vesicles made of lipids in different phases and support the important role of the lipid fluidity and packing on the activity of LfcinB.

Biophysical studies of the interactions between the phage varphiKZ gp144 lytic transglycosylase and model membranes.

The use of naturally occurring lytic bacteriophage proteins as specific antibacterial agents is a promising way to treat bacterial infections caused by antibiotic-resistant pathogens. The opportunity to develop bacterial resistance to these agents is minimized by their broad mechanism of action on bacterial membranes and peptidoglycan integrity. In the present study, we have investigated lipid interactions of the gp144 lytic transglycosylase from the Pseudomonas aeruginosa phage varphiKZ. Interactions with zwitterionic lipids characteristic of eukaryotic cells and with anionic lipids characteristic of bacterial cells were studied using fluorescence, solid-state nuclear magnetic resonance, Fourier transform infrared, circular dichroism, Langmuir monolayers, and Brewster angle microscopy (BAM). Gp144 interacted preferentially with anionic lipids, and the presence of gp144 in anionic model systems induced membrane disruption and lysis. Lipid domain formation in anionic membranes was observed by BAM. Gp144 did not induce disruption of zwitterionic membranes but caused an increase in rigidity of the lipid polar head group. However, gp144 interacted with zwitterionic and anionic lipids in a model membrane system containing both lipids. Finally, the gp144 secondary structure was not significantly modified upon lipid binding.

Cholesterol sulfate and Ca(2+) modulate the mixing properties of lipids in stratum corneum model mixtures.

The influence of cholesterol sulfate (CS) and calcium on the phase behavior of lipid mixtures mimicking the stratum corneum (SC) lipids was examined using vibrational spectroscopy. Raman microspectrocopy showed that equimolar mixtures of ceramide, palmitic acid, and cholesterol underwent a phase transition in which, at low temperatures, lipids formed mainly a mosaic of microcrystalline phase-separated domains, and above 45 degrees C, a more fluid and disordered phase in which the three lipid species were more miscible. In the presence of Ca(2+), there was the formation of fatty acid-Ca(2+) complexes that led to domains stable on heating. Consequently, these lipid mixtures remained heterogeneous, and the fatty acid molecules were not extensively involved in the formation of the fluid lipid phase, which included mainly ceramide and cholesterol. However, the presence of CS displaced the association site of Ca(2+) ions and inhibited the formation of domains formed by the fatty acid molecules complexed with Ca(2+) ions. This work reveals that CS and Ca(2+) modulate the lipid mixing properties and the lipid order in SC lipid models. The balance in the equilibria involving Ca(2+), CS, and fatty acids is proposed to have an impact on the organization and the function of the epidermis.

Isothermal titration calorimetric study of calcium association to lipid bilayers: influence of the vesicle preparation and composition.

The association of Ca2+ ions with phospholipid bilayers was investigated using isothermal titration calorimetry. The study reveals that the binding enthalpy of these cations to bilayers formed with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) depends strongly on the method of preparation of the unilamellar vesicles. Extruded vesicles lead to an exothermic association, whereas sonicated ones lead to an endothermic association. In the later case, the calorimetric signal is sensitive to the length of the sonication period. It is proposed that a reorganization of the lipid bilayers under stress, obtained with sonicated small unilamellar vesicles, contributes to the calorimetric signal upon the titration with Ca2+. The analysis of the titrations indicates that, as expected, the nature of the association of Ca2+ with negatively charged phospholipid bilayers is essentially of electrostatic nature. Using a Scatchard approach, it is found that bilayers become saturated in Ca2+ approximately when the electroneutrality of the bilayer interface is reached. Moreover, the affinity constant was reduced by the increase of the ionic strength of the aqueous buffer. It was found that the intrinsic binding constant of Ca2+ to membranes containing 30 and 50 mol% of POPG was about 11 mM-1, in a MES buffer containing 10 mM NaCl, at pH 5.6.