Aminosugar Modified Lipid A und Analogues

Lipopolysaccharide (LPS) is a primary constituent of the outer leaflet of the outer membrane of Gram-negative bacteria. The lipid A domain anchors the molecule in the outer membrane contributing to the maintenance of its integrity and represents a key factor in immune stimulation via its detection by the host TLR4. The biosynthesis of lipid A is a highly conserved process, however, a number of latent enzymes modify the canonical lipid A structure in the late biosynthetic steps, e.g., by addition of phosphoethanolamine or aminosugars ß-L-4-aminoarabinose (ß- L-Ara4N) and a-D-galactosamine (a-D-Gal2N) to the lipid A phosphate residues. This is considered to be a bacterial strategy to promote survival by providing resistance to the components of innate immune system (e.g. cationic antimicrobial peptides) and modulation of host immune response to infection. Lipid A preparations purified from bacterial cultures often suffer from the lack of consistency due to heterogeneity, protein contaminations and loss of an intrinsically labile glycosidic phosphate substitution. Immunological, biochemical, and biophysical experiments to probe the function of Gal2N and Ara4N modifications of the lipid A phosphates require an access to chemically defined homogenous synthetic lipid A and epitopes. The project intends first chemical synthesis of lipid A variants and neoglycoconjugates modified at the glycosidic phosphate with a-D-Gal2N and ß-L-Ara4N, corresponding to the native LPS structures of Francisella tularensis and Burkholderia /Pseudomonas, respectively. The synthesis entails elaboration of an efficient chemical approach towards stereocontrolled instalment of phosphodiester linkage connecting glycosidic centres of reducing glucosamine unit of lipid A carbohydrate backbone and a second aminosugar (a-D-Gal2N and ß-L-Ara4N). In support of ongoing vaccine studies the target synthetic F. tularensis lipid A-based carbohydrate antigens containing a-D-Gal2N at the reducing phosphate will be converted to neoglycoconjugates and used for immunisation of mice against F. tularensis in an effort to produce an antibody response that could be cross-reactive with virulent Francisella strains. (S.Vogel, University of Maryland, Baltimore, USA). Synthetic, divergently acylated ß-L-Ara4N-modified Lipid A analogues will be tested for endotoxic activity in TLR4/MD-2 transfected cells (R. Jerala, National Institute of Chemistry, Slovenia), the influence of ß-L-Ara4N substitution on the recognition of the modified lipid A by TLR4/MD-2 receptor complexes and on the specific binding to recombinant human and murine MD-2 will be assessed. This knowledge is crucial for the deciphering of the pathogenic molecular mechanisms leading to poor clinical outcome of airway infections caused by Burkholderia cepacia complex and Pseudomonas CF strains. Furthermore, this data will contribute to better understanding of the relationship between chemical structure of lipid A and its TLR4-regulated endotoxic activity, which could impart new insights into external manipulation of immune activation and design of new antiseptic drugs and adjuvants. Affinities of synthetic aminosugar-modified lipid A variants to antimicrobial peptides will be assessed as well, which might provide a potential basis for Lipid A-targeted design of selective peptide-like antibiotics.

Endotoxins or lipopolysaccharides, which are situated in the outer membrane of the cell wall of Gram-negative bacteria, represent pathogen associated molecular patterns (PAMPs) that could be detected by mammalian innate immune system. Immune cells such as macrophages or dendritic cells, as well as other cell types, possess specific receptor proteins (Toll-like Receptors) which are responsible for the recognition of PAMPs and the ensuing initiation of the innate immune response. Very minor concentrations of Endotoxins are sensed by the Toll-like Receptor 4 (TLR4) which, upon activation by lipopolysaccharide, triggers a cascade of immune reactions resulting in the release of pro-inflammatory mediators (cytokines). The latter contributes to the establishment of an immediate immune response to infection and eradication of invading pathogens. TLR4 can recognize and bind only a small terminal part of Endotoxin which is called Lipid A and consists of a carbohydrate di-glucosamine backbone that is substituted by the two anionic phosphate groups and by up to six long-chain acyl lipid tails. This amphiphilic compound was nominated to endotoxic principle of lipopolysaccharide since the activation of TLR4 depends solely on the chemical structure of Lipid A. Some bacteria (Escerihia coli, Salmonella etc.) possess endotoxically active hexaacylated Lipid A, other bacteria modify their Lipid A in such a way that it cannot be recognised by TLR4 and, therefore, is unable to activate the innate immune defence mechanisms. For instance, a gram-negative pathogen Burkholderia cenocepacia, which readily colonises the respiratory tract of immunocompromised and cystic fibrosis (CF) patients, modifies the negatively charged phosphate groups of its Lipid A by a positively charged aminosugar 4-amino-4-deoxy-arabinose (Ara4N). This modification confers the resistance to antibiotics and the hosts endogenous defence mechanisms which results in extreme high mortality of Burkholderia infection in CF patients. The phosphate group of Lipid A of another infectious pathogen, Francisella tularensis, is decorated by aminosugar galactosamine (GalN). To unveil the influence of these unique Lipid A modifications on the activation of the innate and adaptive immune responses we have synthetically prepared Ara4N modified Burkholderia Lipid A as well as neoglycoconjugates containing carbohydrate subunits of Ara4N- and GalN modified Lipid A. Synthetic epitope containing components of GalN modified Francisella Lipid A, which is conserved in all virulent Francisella strains, might be utilized in diagnostic immunoassays as capture antigen. The synthetic neoglycoconjugates could be of considerable use for the generation of specific antibodies having potential application in the therapy of relevant infections. The central results of the project have led to an extended understanding of the interaction of Ara4N-modified Burkholderia Lipid A with its recognition receptor TLR4, which could be established in the framework of cooperations with research groups in Germany, Switzerland and Belgium. We have shown that Ara4N modification of the Lipid A phosphate group enables the activation of TLR4 by Ara4N-modified Lipid A from Burkholderia, in contrast to its unmodified counterpart which is largely endotoxically inactive. Our data contribute to better comprehending of the structure activity relationships between Lipid A and its TLR4-regulated endotoxic activity, which imparts new insights on the design of TLR4-dependent immuno-therapeutics for the treatment of relevant acute infectious and chronic diseases.