Ulrich Seydel

Aggregates Are the Biologically Active Units of Endotoxin

For the elucidation of the very early steps of immune cell activation by endotoxins (lipopolysaccharide, LPS) leading to the production and release of proinflammatory cytokines the question concerning the biologically active unit of endotoxins has to be addressed: are monomeric endotoxin molecules able to activate cells or is the active unit represented by larger endotoxin aggregates? This question has been answered controversially in the past. Inspired by the observation that natural isolates of lipid A, the lipid moiety of LPS harboring its endotoxic principle, from Escherichia coli express a higher endotoxic activity than the same amounts of the synthetic E. coli-like hexaacylated lipid A (compound 506), we looked closer at the chemical composition of natural isolates. We found in these isolates that the largest fraction was hexaacylated, but also significant amounts of penta- and tetraacylated molecules were present that, when administered to human mononuclear cells, may antagonize the induction of cytokines by biologically active hexaacylated endotoxins. We prepared separate aggregates of either compound 506 or 406 (tetraacylated precursor IVa), mixed at different molar ratios, and mixed aggregates containing both compounds in the same ratios. Surprisingly, the latter mixtures showed higher endotoxic activity than that of the pure compound 506 up to an admixture of 20% of compound 406. Similar results were obtained when using various phospholipids instead of compound 406. These observations can only be understood by assuming that the active unit of endotoxins is the aggregate. We further confirmed this result by preparing monomeric lipid A and LPS by a dialysis procedure and found that, at the same concentrations, only the aggregates were biologically active, whereas the monomers showed no activity.

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.

Chemical structure of glycosphingolipids isolated from Sphingomonas paucimobilis

Two novel glycosphingolipids were isolated fromSphingomonas paucimobilis and their structures were completely elucidated. The glycosyl portion of the glycosphingolipid consists of an α‐D‐Manp‐[1→2)‐α‐D‐Galp‐(1→6)‐α‐D‐GlcpN‐(1→4)‐α‐D‐GlcpA‐R tetrasaccharide. The hydrophobic residue R was found to be heterogeneous with respect to the dihydrosphingosine residue.Erythro‐1,3‐dihydroxy‐2‐amino‐octadecane anderythro‐1,3‐dihydroxy‐2‐amino‐cis‐13,14‐methyleneoctadecane were identified in comparable amounts. Both dihydrosphingosine derivatives were quantitatively substituted by an (S)‐2‐hydroxymyristic acid in amide linkage.

New Insights Into Endotoxin-Induced Activation of Macrophages: Involvement of a K+ Channel in Transmembrane Signaling

LPS (endotoxins) activate cells of the human immune system, among which are monocytes and macrophages, to produce endogenous mediators. These regulate the immune response, but may also cause severe harm leading to septic shock. The activation of monocytes/macrophages by LPS is mediated by a membrane-bound LPS receptor, mCD14. As mCD14 lacks a transmembrane domain, a further protein is required for the signal transducing step to the cell interior. Here we show, using excised outside-out membrane patches, that activation of a high-conductance Ca2+- and voltage-dependent potassium channel is an early step in the transmembrane signal transduction in macrophages. The channel is activated by endotoxically active LPS in a dose-dependent manner. Channel activation can be completely inhibited by LPS antagonists and by anti-CD14 Abs. Activation of the channel is essential for LPS-induced cytokine production as shown by its inhibition by selective K+ channel blockers.

Lipopolysaccharide‐binding protein mediates CD14‐independent intercalation of lipopolysaccharide into phospholipid membranes

Lipopolysaccharides (LPS, endotoxin) stimulate mononuclear cells to release cytokines which initiate endotoxic effects. Interaction of LPS at low concentrations with target cells is CD14‐independent whereas at high LPS concentrations it is CD14‐independent. Here, we demonstrate by resonance energy transfer (RET) technique that nonspecific, CD14‐independent intercalation of LPS into membrane systems can be mediated by lipopolysaccharide‐binding protein (LBP). It is proposed that in this pathway, LBP breaks down LPS aggregates, transports the smaller units to and inserts them into the phospholipid cell matrix. We furthermore show that LBP also mediates the intercalation of other negatively charged amphiphilic molecules. We propose a model explaining CD14‐independent cell activation at high endotoxin concentrations.

CONFORMATIONAL STUDIES OF SYNTHETIC LIPID A ANALOGUES AND PARTIAL STRUCTURES BY INFRARED SPECTROSCOPY

Synthetic lipid A analogues and partial structures were analyzed and compared with natural hexaacyl lipid A from E. coli applying Fourier transform infrared spectroscopy. The investigations comprised (i) the measurement of the beta <=> alpha phase transition of the acyl chains via monitoring of the symmetric stretching vibration of the methylene groups, (ii) an estimation of the supramolecular aggregate structures evaluating vibrations from the interface like ester carbonyl and applying theoretical calculations (iii) a determination of the inter- and intramolecular conformations monitoring functional groups from the interface and the diglucosamine backbone (ester carbonyl, phosphate). The phase transition temperature Tc was found to be nearly a linear function of the number of acyl chains for most bisphosphoryl compounds indicating comparable packing density, whereas the deviating behaviour of some samples indicated a higher packing density. From the determination of the supramolecular aggregate structures (cubic, HII) of natural hexaacyl lipid A by X-ray small-angle diffraction, the existence of the same aggregate structures also for the synthetic hexaacyl lipid A was deduced from the nearly identical thermotropism of the ester carbonyl band. From this, a good approximation of the supramolecular structures of all synthetic samples was possible on the basis of the theory of Israelachvili. The analysis of the main phosphate band, together with that of the Tc data and former colorimetric results, allowed the establishment of a model of the intermolecular conformations of neighbouring lipid A/LPS molecules. The biological relevance of the findings is discussed in terms of the strongly varying biological activity (between high and no activity) of the samples.

Molecular mechanisms of polymyxin B-membrane interactions: direct correlation between surface charge density and self-promoted transport.

Abstract. We have studied the interaction of the polycationic peptide antibiotic polymyxin B (PMB) with asymmetric planar bilayer membranes via electrical measurements. The bilayers were of different compositions, including those of the lipid matrices of the outer membranes of various species of Gram-negative bacteria. One leaflet, representing the bacterial inner leaflet, consisted of a phospholipid mixture (PL; phosphatidylethanolamine, -glycerol, and diphosphatidylglycerol in a molar ratio of 81:17:2). The other (outer) leaflet consisted either of lipopolysaccharide (LPS) from deep rough mutants of PMB-sensitive (Escherichia coli F515) or -resistant strains (Proteus mirabilis R45), glycosphingolipid (GSL-1) from Sphingomonas paucimobilis IAM 12576, or phospholipids (phosphatidylglycerol, diphytanoylphosphatidylcholine). In all membrane systems, the addition of PMB to the outer leaflet led to the induction of current fluctuations due to transient membrane lesions. The minimal PMB concentration required for the induction of the lesions and their size correlated with the charge of the lipid molecules. In the membrane system resembling the lipid matrix of a PMB-sensitive strain (F515 LPS/PL), the diameters of the lesions were large enough (d= 2.4 nm ± 8%) to allow PMB molecules to permeate (self-promoted transport), but in all other systems they were too small. A comparison of these phenomena with membrane effects induced by detergents (dodecyltriphenylphosphonium bromide, dodecyltrimethylammonium bromide, sodiumdodecylsulfate) revealed a detergent-like mechanism of the PMB-membrane interaction.

Physical aspects of structure and function of membranes made from lipopolysaccharides and free lipid A

Abstract The physical structure of the lipid component of the outer membrane of Gram-negative bacteria, here mutants of Salmonella minnesota , was studied with different optical and calorimetric techniques on the bilayer and with film balance measurements on the monolayer system. Special emphasis was laid on the elucidation of the phase behavior of lipopolysaccharides and its isolated lipid component, free lipid A, differing in the length of the polysaccharide moiety. All samples exhibit the phase transition gel-liquid crystalline of the hydrocarbon chains with T c for free lipid A lying well above the growth temperature of the bacteria (37°C)m while the mutant lipopolysaccharides show values in the range 3-–37°C. The state of order of the hydrocarbon chains at 37°C is lowest for the deep rough mutant lipopolysaccharides. Thus, an explanation of the high sensitivity of the mutants Re and Rd against hydrophobic drugs is possible. The behaviour of several physical quantities, e.g. enthalpy and cooperativity of the phase transition indicates that free lipid A and, to a lower extent, also the deep rough mutant lipopolysaccharide (mR595) form inverted structures. From this a model is derived for the mechanism of incorporation of lipopolysaccharide into the outer leaflet of the outer membrane after its lipid and saccharide parts have been translocated separately from the cytoplasmic membrane.

The generalized endotoxic principle

Bacterial lipopolysaccharides (endotoxins, LPS) belong to the most potent immunostimulators in mammals. The endotoxic principle of LPS is located in its lipid A moiety, which for Escherichia coli‐type LPS consists of a hexaacylated diphosphoryl diglucosamine backbone. This lipid A adopts a cubic inverted aggregate structure from which a conical shape of the molecule can be deduced, whereas the tetraacyl lipid A precursor IVa adopts a cylindrical shape and is endotoxically inactive, but antagonizes active LPS. We hypothesize that non‐lipid A amphiphiles with similar physicochemical properties of amphiphilicity, charge, and shape, might mimic the respective lipid A. To test this hypothesis, phospholipid‐like amphiphiles with six acyl chains attached to a bisphosphorylated serine‐like backbone of varying length replacing the diglucosamine backbone were synthesized. The compound with a short backbone fulfills all criteria of an endotoxic agonist, and that with longbackbone fulfills those of an antagonist. This holds true for the human as well as for the murine system. Interestingly, these compounds are inactive in the Limulus amebocyte lysate test which is specific for LPS diglucosamine backbone. These results define a general endotoxic principle and, furthermore, provide new insights into an understanding of early steps of endotoxin action.