Disruption of Asymmetric Lipid Bilayer Models Mimicking the Outer Membrane of Gram-Negative Bacteria by an Active Plasticin.

The outer membrane (OM) of Gram-negative bacteria is a complex and asymmetric bilayer that antimicrobial peptides must disrupt in order to provoke the cell lysis. The inner and external leaflets of the OM are mainly composed of phospholipids (PL), and lipopolysaccharide (LPS), respectively. Supported lipid bilayers are interesting model systems to mimic the lipid asymmetric scaffold of the OM and determine the quantitative and mechanistic effect of antimicrobial agents, using complementary physicochemical techniques. We report the formation of asymmetric PL/LPS bilayers using the Langmuir-Blodgett/Langmuir-Schaefer technique on two different surfaces (sapphire and mica) with synthetic phospholipids constituting the inner leaflet and bacteria-extracted mutant LPS making up the outer one. The combination of neutron reflectometry and atomic force microscopy techniques allowed the examination of the asymmetric scaffold structure along the normal to the interface and its surface morphology in buffer conditions. Our results allow discrimination of two structurally related peptides, one neutral and inactive, and the other cationic and active. The active cationic plasticin PTCDA1-KF disrupts the asymmetric OM at relevant concentrations through a carpeting scenario characterized by a dramatic removal of lipid molecules from the surface.

Influence of myelin proteins on the structure and dynamics of a model membrane with emphasis on the low temperature regime.

Myelin is an insulating, multi-lamellar membrane structure wrapped around selected nerve axons. Increasing the speed of nerve impulses, it is crucial for the proper functioning of the vertebrate nervous system. Human neurodegenerative diseases, such as multiple sclerosis, are linked to damage to the myelin sheath through demyelination. Myelin exhibits a well defined subset of myelin-specific proteins, whose influence on membrane dynamics, i.e., myelin flexibility and stability, has not yet been explored in detail. In a first paper [W. Knoll, J. Peters, P. Kursula, Y. Gerelli, J. Ollivier, B. Demé, M. Telling, E. Kemner, and F. Natali, Soft Matter 10, 519 (2014)] we were able to spotlight, through neutron scattering experiments, the role of peripheral nervous system myelin proteins on membrane stability at room temperature. In particular, the myelin basic protein and peripheral myelin protein 2 were found to synergistically influence the membrane structure while keeping almost unchanged the membrane mobility. Further insight is provided by this work, in which we particularly address the investigation of the membrane flexibility in the low temperature regime. We evidence a different behavior suggesting that the proton dynamics is reduced by the addition of the myelin basic protein accompanied by negligible membrane structural changes. Moreover, we address the importance of correct sample preparation and characterization for the success of the experiment and for the reliability of the obtained results.

Membrane thickness and the mechanism of action of the short peptaibol trichogin GA IV.

Trichogin GA IV (GAIV) is an antimicrobial peptide of the peptaibol family, like the extensively studied alamethicin (Alm). GAIV acts by perturbing membrane permeability. Previous data have shown that pore formation is related to GAIV aggregation and insertion in the hydrophobic core of the membrane. This behavior is similar to that of Alm and in agreement with a barrel-stave mechanism, in which transmembrane oriented peptides aggregate to form a channel. However, while the 19-amino acid long Alm has a length comparable to the membrane thickness, GAIV comprises only 10 amino acids, and its helix is about half the normal bilayer thickness. Here, we report the results of neutron reflectivity measurements, showing that GAIV inserts in the hydrophobic region of the membrane, causing a significant thinning of the bilayer. Molecular dynamics simulations of GAIV/membrane systems were also performed. For these studies we developed a novel approach for constructing the initial configuration, by embedding the short peptide in the hydrophobic core of the bilayer. These calculations indicated that in the transmembrane orientation GAIV interacts strongly with the polar phospholipid headgroups, drawing them towards its N- and C-termini, inducing membrane thinning and becoming able to span the bilayer. Finally, vesicle leakage experiments demonstrated that GAIV activity is significantly higher with thinner membranes, becoming similar to that of Alm when the bilayer thickness is comparable to its size. Overall, these data indicate that a barrel-stave mechanism of pore formation might be possible for GAIV and for similarly short peptaibols despite their relatively small size.

Structure of self-organized multilayer nanoparticles for drug delivery.

The combined use of cryo-TEM, dynamic light scattering, and small-angle X-ray and neutron scattering techniques allows a detailed structural model of complex pharmaceutical preparations of soybean lecithin/chitosan nanoparticles used as drug vectors to be worked out. Charge-driven self-organization of the lipid(-)/polysaccharide(+) vesicles occurs during rapid injection, under mechanical stirring, of an ethanol solution of soybean lecithin into a chitosan aqueous solution. We conclude that beyond the charge inversion region of the phase diagram, i.e., entering the redissolution region, the initial stages of particle formation are likely to be affected by a re-entrant condensation effect at the nanoscale. This behavior resembles that at the mesoscale which is well-known for polyion/amphiphile systems. Close to the boundary of the charge inversion region, nanoparticle formation occurs under a maximum condensation condition at the nanoscale and the complexation-aggregation process is driven toward a maximum multilamellarity. Interestingly, the formulation that maximizes vesicle multilamellarity corresponds to that displaying the highest drug loading efficiency.