The Beauty of Asymmetric Membranes: Reconstitution of the Outer Membrane of Gram-Negative Bacteria.

The architecture of the lipid matrix of the outer membrane of Gram-negative bacteria is extremely asymmetric: Whereas the inner leaflet is composed of a phospholipid mixture, the outer leaflet is built up by glycolipids. For most Gram-negative species, these glycolipids are lipopolysaccharides (LPS), for a few species, however, glycosphingolipids. We demonstrate experimental approaches for the reconstitution of these asymmetric membranes as (i) solid supported membranes prepared by the Langmuir-Blodgett technique, (ii) planar lipid bilayers prepared by the Montal-Mueller technique, and (iii) giant unilamellar vesicles (GUVs) prepared by the phase transfer method. The asymmetric GUVs (aGUVs) composed of LPS on one leaflet are shown for the first time. They are characterized with respect to their phase behavior, flip-flop of lipids and their usability to investigate the interaction with membrane active peptides or proteins. For the antimicrobial peptide LL-32 and for the bacterial porin OmpF the specificity of the interaction with asymmetric membranes is shown. The three reconstitution systems are compared with respect to their usability to investigate domain formation and interactions with peptides and proteins.

The mode of action of the lantibiotic lacticin 3147--a complex mechanism involving specific interaction of two peptides and the cell wall precursor lipid II.

Lacticin 3147 is a two-peptide lantibiotic produced by Lactococcus lactis in which both peptides, LtnA1 and LtnA2, interact synergistically to produce antibiotic activities in the nanomolar concentration range; the individual peptides possess marginal (LtnA1) or no activity (LtnA2). We analysed the molecular basis for the synergism and found the cell wall precursor lipid II to play a crucial role as a target molecule. Tryptophan fluorescence measurements identified LtnA1, which is structurally similar to the lantibiotic mersacidin, as the lipid II binding component. However, LtnA1 on its own was not able to substantially inhibit cell wall biosynthesis in vitro; for full inhibition, LtnA2 was necessary. Both peptides together caused rapid K(+) leakage from intact cells; in model membranes supplemented with lipid II, the formation of defined pores with a diameter of 0.6 nm was observed. We propose a mode of action model in which LtnA1 first interacts specifically with lipid II in the outer leaflet of the bacterial cytoplasmic membrane. The resulting lipid II:LtnA1 complex is then able to recruit LtnA2 which leads to a high-affinity, three-component complex and subsequently inhibition of cell wall biosynthesis combined with pore formation.

Calcium adsorption and displacement: characterization of lipid monolayers and their interaction with membrane-active peptides/proteins.

BACKGROUND: The first target of antimicrobial peptides (AMPs) is the bacterial membrane. In the case of Gram-negative bacteria this is the outer membrane (OM), the lipid composition of which is extremely asymmetric: Whereas the inner leaflet is composed of a phospholipid mixture, the outer leaflet is made up solely from lipopolysaccharides (LPSs). LPS, therefore, represents the first target of AMPs. The binding and intercalation of polycationic AMPs is driven by the number and position of negatively charged groups of the LPS. Also, proteins other than cationic AMPs can interact with LPS, e.g. leading eventually to a neutralization of the endotoxic effects of LPS. We compared different biophysical techniques to gain insight into the properties of the electrical surface potentials of lipid monolayers and aggregates composed of LPSs and various phospholipids and their interaction with peptides and proteins. RESULTS: The net negative charge calculated from the chemical structure of the phospholipid and LPS molecules is linearly correlated with the adsorption of calcium to two-dimensional lipid monolayers composed of the respective lipids. However, the zeta-potentials determined by the electrophoretic mobility of LPS aggregates can only be interpreted by assuming a dependence of the plane of shear on the number of saccharides and charged groups. Various peptides and proteins were able to displace calcium adsorbed to monolayers. CONCLUSION: To characterize the electrical properties of negatively charged phospholipids and LPSs and their electrostatic interaction with various polycationic peptides/proteins, the adsorption of calcium to and displacement from lipid monolayers is a suitable parameter. Using the calcium displacement method, the binding of peptides to monolayers can be determined even if they do not intercalate. The interpretation of zeta-potential data is difficulty for LPS aggregates, because of the complex three-dimensional structure of the LPS molecules. However, the influence of peptides/proteins on the zeta-potential can be used to characterize the underlying interaction mechanisms.

Lipid-specific membrane activity of human beta-defensin-3.

Defensins represent a major component of innate host defense against bacteria, fungi, and enveloped viruses. One potent defensin found, e.g., in epithelia, is the polycationic human beta-defensin-3 (hBD3). We investigated the role of the lipid matrix composition, and in particular the presence of negatively charged lipopolysaccharides (LPS) from sensitive (Escherichia coli, Salmonella enterica serovar Minnesota) or resistant (Proteus mirabilis) Gram-negative bacteria or of the zwitterionic phospholipids of human cells, in determining the action of polycationic hBD3 on the different membranes, and related to their biological activity. The main focus was directed on data derived from electrical measurements on a reconstitution system of the OM as a planar asymmetric bilayer composed on one side of LPS and on the other of a phospholipid mixture. Our results demonstrate that the antimicrobial activity and the absence of cytotoxicity can be explained by the lipid-specificity of the peptide. A clear correlation between these aspects of the biological activity of hBD3 and its interaction with lipid matrices could be found. In particular, hBD3 could only induce lesions in those membranes resembling the lipid composition of the OM of sensitive bacterial strains. The permeation through the membrane is a decisive first step for the biological activity of many antimicrobial peptides. Therefore, we propose that the lipid-specificity of hBD3 as well as some other membrane-active antimicrobial peptides is important for their activity against bacteria or mammalian cells.

Lipid-mediated resistance of Gram-negative bacteria against various pore-forming antimicrobial peptides.

Lipopolysaccharides (LPSs) play a dual role as target and as effector molecules. The knowledge of the LPS-induced activation of human immune cells is increasing; however, surprisingly, much less effort seems to be directed towards the understanding of the mechanisms leading to the killing of the bacterial organisms, which eventually results in the release of LPS from the bacterial surface into the blood circulation. We demonstrate mechanisms of interaction of peptides of the innate immune system (e.g. defensins and cathelicidins) as well as of externally administered antibiotics (e.g. Polymyxin B) with Gram-negative bacteria. The main focus is directed on data derived from electrical measurements on a reconstitution system of the outer membrane as an asymmetric bilayer composed on one side of LPS and on the other of phospholipids. All these antimicrobial peptides (AMPs) are membrane-active and induce the permeabilization of the reconstituted membranes by the formation of lesions. We found that differences in the activity of the AMPs against various sensitive and resistant Gram-negative bacteria can be explained solely by variations in the chemical structure of LPS, e.g. in the composition of the sugar head group. A reduction of the net negative charge of LPS is responsible for a reduced interaction with the polycationic AMPs and thus for resistance. A most important side effect of positively charged AMPs is the neutralization of the negatively charged LPS released from the bacterial surface as a consequence of AMP-induced killing.

Inner field compensation as a tool for the characterization of asymmetric membranes and Peptide-membrane interactions.

Symmetric and asymmetric planar lipid bilayers prepared according to the Montal-Mueller method are a powerful tool to characterize peptide-membrane interactions. Several electrical properties of lipid bilayers such as membrane current, membrane capacitance, and the inner membrane potential differences and their changes can be deduced. The time-resolved determination of peptide-induced changes in membrane capacitance and inner membrane potential difference are of high importance for the characterization of peptide-membrane interactions. Intercalation and accumulation of peptides lead to changes in membrane capacitance, and membrane interaction of charged peptides induces changes in the charge distribution within the membrane and with that to changes in the membrane potential profile. In this study, we establish time-resolved measurements of the capacitance minimization potential DeltaPsi on various asymmetric planar lipid bilayers using the inner field compensation method. The results are compared to the respective ones of inner membrane potential differences DeltaPhi determined from ion carrier transport measurements. Finally, the time courses of membrane capacitances and of DeltaPsi have been used to characterize the interaction of cathelicidins with reconstituted lipid matrices of various Gram-negative bacteria.

Pore formation and function of phosphoporin PhoE of Escherichia coli are determined by the core sugar moiety of lipopolysaccharide.

The lipid matrix of the outer membrane of Gram-negative bacteria is an asymmetric bilayer composed of a phospholipid inner leaflet and a lipopolysaccharide outer leaflet. Incorporated into this lipid matrix are, among other macromolecules, the porins, which have a sieve-like function for the transport or exclusion of hydrophilic substances. It is known that a reduced amount of porins is found in the outer membrane of rough mutants as compared with wild-type bacteria. This observation was discussed to be caused by a reduced number of insertion sites in the former. We performed electrical measurements on reconstituted planar bilayers composed of lipopolysaccharide on one side and a phospholipid mixture on the other side using lipopolysaccharide from various rough mutant strains of Salmonella enterica serovar Minnesota. We found that pore formation by PhoE trimers that were added to the phospholipid side of the bilayers increased with the increasing length of the lipopolysaccharide core sugar moiety. These results allow us to conclude that the length of the sugar moiety of lipopolysaccharide is the parameter governing pore formation and that no particular insertion sites are required. Furthermore, we found that the voltage gating of the porin channels is strongly dependent on the composition of the lipid matrix.