Andre Wiese

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.

Dual Role of Lipopolysaccharide (LPS)-Binding Protein in Neutralization of LPS and Enhancement of LPS-Induced Activation of Mononuclear Cells

ABSTRACT The lipopolysaccharide (LPS)-binding protein (LBP) has a concentration-dependent dual role in the pathogenesis of gram-negative sepsis: low concentrations of LBP enhance the LPS-induced activation of mononuclear cells (MNC), whereas the acute-phase rise in LBP concentrations inhibits LPS-induced cellular stimulation. In stimulation experiments, we have found that LBP mediates the LPS-induced cytokine release from MNC even under serum-free conditions. In biophysical experiments we demonstrated that LBP binds and intercalates into lipid membranes, amplified by negative charges of the latter, and that intercalated LBP can mediate the CD14-independent intercalation of LPS into membranes in a lipid-specific and temperature-dependent manner. In contrast, prior complexation of LBP and LPS inhibited binding of these complexes to membranes due to different binding of LBP to LPS or phospholipids. This results in a neutralization of LPS and, therefore, to a reduced production of tumor necrosis factor by MNC. We propose that LBP is not only present as a soluble protein in the serum but may also be incorporated as a transmembrane protein in the cytoplasmic membrane of MNC and that the interaction of LPS with membrane-associated LBP may be an important step in LBP-mediated activation of MNC, whereas LBP-LPS complexation in the serum leads to a neutralization of LPS.

Biochemical and biophysical characterization of in vitro folded outer membrane porin PorA of Neisseria meningitidis

Two subtypes of the outer membrane porin PorA of Neisseria meningitidis, P1.6 and P1.7,16, were folded in vitro after overexpression in, and isolation from Escherichia coli. The PorA porins could be folded efficiently by quick dilution in an appropriate buffer containing the detergent n-dodecyl-N, N-dimethyl-1-ammonio-3-propanesulphonate. Although the two PorA porins are highly homologous, they required different acidities for optimal folding, that is, a pH above the pI was needed for efficient folding. Furthermore, whereas trimers of PorA P1.7,16 were almost completely stable in 2% sodium dodecyl sulphate (SDS), those of P1.6 dissociated in the presence of SDS. The higher electrophoretic mobility of the in vitro folded porins could be explained by the stable association of the RmpM protein to the porins in vivo. This association of RmpM contributes to the stability of the porins. The P1.6 pores were moderately cation-selective and displayed a single-channel conductance of 2.8 nS in 1 M KCl. The PorA P1.6 pores, but not the PorA P1.7,16 pores, showed an unusual non-linear dependence of the single-channel conductance on the salt concentration of the subphase. We hypothesize that a cluster of three negatively charged residues in L5 of P1.6 is responsible for the higher conductance at low salt concentrations.

Interaction of CAP18-Derived Peptides with Membranes Made from Endotoxins or Phospholipids

Antimicrobial peptides with alpha-helical structures and positive net charges are in the focus of interest with regard to the development of new antibiotic agents, in particular against Gram-negative bacteria. Interaction between seven polycationic alpha-helical CAP18-derived peptides and different types of artificial membranes composed of phosphatidylcholine or lipopolysaccharide of the Gram-negative bacterium Escherichia coli were investigated using different biophysical techniques. Results obtained from fluorescence energy transfer spectroscopy with liposomes, monolayer measurements on a Langmuir trough, and electrophysiological measurements on planar reconstituted asymmetric bilayer membranes including the lipid matrix of the outer membrane of E. coli were correlated, and these data were, furthermore, correlated with structural parameters of the peptides (net charge, alpha-helical content, hydrophobic moment, and hydrophobicity). All peptides induced current fluctuations in planar membranes due to the formation of transient lesions above a peptide- and lipid-specific minimal clamp voltage. Antibacterial activity was exhibited only by those peptides that induced lesion formation in the reconstituted outer membrane at clamp voltages below the transmembrane potential of the natural membrane. Thus, we propose that the physicochemical properties of both the peptides as well as of the target membranes are important for antibacterial activity.

The Dual Role of Lipopolysaccharide as Effector and Target Molecule

Abstract Lipopolysaccharides (LPS) are major integral components of the outer membrane of Gram-negative bacteria being exclusively located in its outer leaflet facing the bacterial environment. Chemically they consist in different bacterial strains of a highly variable O-specific chain, a less variable core oligosaccharide, and a lipid component, termed lipid A, with low structural variability. LPS participate in the physiological membrane functions and are, therefore, essential for bacterial growth and viability. They contribute to the low membrane permeability and increase the resistance towards hydrophobic agents. They are also the primary target for the attack of antibacterial drugs and proteins such as components of the hosts immune response. When set free LPS elicit, in higher organisms, a broad spectrum of biological activities. They play an important role in the manifestation of Gram-negative infection and are therefore termed endotoxins. Physico-chemical parameters such as the molecular conformation and the charges of the lipid A portion, which is responsible for endotoxin-typical biological activities and is therefore termed the ‘endotoxic principle’ of LPS, are correlated with the biological activity of chemically different LPS.

Planar asymmetric lipid bilayers of glycosphingolipid or lipopolysaccharide on one side and phospholipids on the other: membrane potential, porin function, and complement activation

We have determined some physicochemical properties of the monosaccharide-type fraction (GSL-1) of glycosphingolipids, the major glycolipid components of the outer leaflet of the Gram-negative species Sphingomonas paucimobilis. These properties included the state of order of the hydrocarbon moiety, the effective molecular area, surface charge density, and intrinsic transmembrane potential profile of reconstituted planar asymmetric GSL-1/phospholipid bilayer membranes. We have, furthermore, investigated the insertion into and the function of porin channels in the reconstituted bilayers and the complement-activating capability of GSL-1 surfaces. All results were compared with respective data for deep rough mutant lipopolysaccharide of Salmonella minnesota R595. We found a remarkable agreement in most functional properties of the two glycolipids.

Influence of the lipid matrix on incorporation and function of LPS-free porin from Paracoccus denitrificans

We have studied the role of lipopolysaccharide (LPS) for the insertion of LPS-free porin from Paracoccus denitrificans into planar lipid bilayers and its function therein. For this, we reconstituted the porin into different asymmetric planar lipid bilayers with or without LPS and into symmetric phospholipid bilayers. LPS-free porin added to the various bilayer systems was found to induce a step-wise increase in membrane conductance with different incorporation rates, depending on the presence of LPS in the bilayer leaflet opposite to porin addition. The incorporation rate into asymmetric LPS/phospholipid membranes from the phospholipid side was more than 10-fold higher than that observed for pure phospholipid membranes. The porin formed general diffusion pores without any salt specificity. The mean single-channel conductance did not depend on the presence of LPS and was about 4.2 nS for a subphase containing 1 M KCl in all systems tested. At certain applied transmembrane voltages, which depended on membrane composition and were approximately greater than 100 mV for the LPS/phospholipid system, single-channel closing in three steps was observed. Differences in the voltage dependence of porin-channel closing could be correlated with the surface charge of the bilayer. From the voltage-dependent gating behaviour proof for an oriented incorporation of the porin molecules, depending on the side of porin addition, and evidence for their orientation could be derived. Measurements at temperatures above and below the beta<==>alpha phase transition temperature of LPS gave evidence for the influence of membrane rigidity on the gating behaviour.

Interaction between lipopolysaccharide (LPS), LPS-binding protein (LBP), and planar membranes.

Abstract The mechanism of interaction of the lipopolysaccharide (LPS)binding protein, LBP, with differently composed symmetric and asymmetric planar lipid bilayers was investigated in electrical measurements (membrane current, potential, capacitance). From a change of the inner membrane potential difference, binding of LBP to membranes was deduced. After addition of LBP to one side of the membrane, binding of antiLBP antibodies and LPS to LBP on both sides of the bilayer was observed. Effects resulting from an interaction of antiLBP antiserum with membranebound LBP depend on the side of addition of the antiserum, indicating a directed intercalation of LBP into the membrane. Addition of LPS to the same side as LBP may induce a change of the conformation of LBP or its orientation in the membrane. Based on these observations, we propose that LBP intercalates in a directed orientation into negativelycharged membranes and assumes a transmembrane configuration. Moreover, preincubated complexes of LPS and LBP do not interact with membranes. These experiments show that reconstituted planar membranes are a suitable tool for investigations of the interaction of non poreforming proteins that are involved in signal transduction.

Investigation into the interaction of the bacterial protease OmpT with outer membrane lipids and biological activity of OmpT:lipopolysaccharide complexes

Outer-membrane proteases T (OmpT) are important defence molecules of Gram-negative bacteria such as Escherichia coli found in particular in clinical isolates. We studied the interaction of OmpT with the membrane-forming lipids phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) from the inner leaflet and lipopolysaccharide (LPS) from the outer leaflet of the outer membrane. These investigations comprise functional aspects of the protein–lipid interaction mimicking the outer-membrane system as well as the bioactivity of LPS:OmpT complexes in the infected host after release from the bacterial surface. The molecular interaction of the lipids PE, PG, and LPS with OmpT was investigated by analysing molecular groups in the lipids originating from the apolar region (methylene groups), the interface region (ester), and the polar region (phosphates), and by analysing the acyl-chain melting-phase behaviour of the lipids. The activity of OmpT and LPS:OmpT complexes was investigated in biological test systems (human mononuclear cells and Limulus amoebocyte lysate assay) and with phospholipid model membranes. The results show a strong influence of OmpT on the mobility of the lipids leading to a considerable fluidization of the acyl chains of the phospholipids as well as LPS, and a rigidification of the phospholipid, but not LPS head groups. From this, a dominant role of the protein on the function of the outer membrane can be deduced. OmpT released from the outer membrane still contains slight contaminations of LPS, but its strong cytokine-inducing ability in mononuclear cells, which does not depend on the Toll-like receptors 2 and 4, indicates an LPS-independent mechanism of cell activation. This might be of general importance for infections induced by Gram-negative bacteria.

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

BackgroundThe 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.ResultsThe 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 ζ-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.ConclusionTo 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 ζ-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 ζ-potential can be used to characterize the underlying interaction mechanisms.