Synthesis of Acinetobacter LPS ligands of collectins

Lectins, which constitute important components of the innate mammalian immune system, bind to manifold carbohydrate ligands exposed on the cell surface of pathogenic fungi, bacteria and viruses. The group of C-reactive lectins requires the presence of calcium ions for the binding to the hydroxyl groups of carbohydrates. Prominent examples of these lectins are the mannose-binding protein (MBL) and the lung surfactant proteins SP-A and SP-D, which induce immediate immune reactions upon contact with microbes in the serum and at the surface-layer of lung tissue. The weak individual sugar-lectin binding interactions are strongly enhanced due to multivalent presentation of the carbohydrate ligands at the cell surface. Previous studies have shown, that surface structures of Acinetobacter bbind with unusual high affinity to the murine mannose-binding protein MBL-A. Since the binding region is not known in molecular detail and carbohydrate fractions from bacterial lipopolysaccharide (LPS) may only be isolated in small amounts and insufficient purity, chemical synthesis of defined subunits will be performed. The synthesis is based on the core region of bacterial lipopolysaccharide from Acinetobacter haemolyticus, a member of a bacterial genus causing severe nosocomial infections and being notoriously resistant to antibiotic therapies. The proposal aims at the preparation of oligosaccharides up to the pentasaccharide level containing a specific phosphate groups as well octulosonic sugar acids (Kdo, Ko), improving synthetic strategies for the synthesis of Ko and Ko glycosyl donors and investigating glycoside coupling reactions under batch and microfluidic conditions. The compounds have been designed as monovalent glycosides, as multivalent assemblies and as neoglycoproteins. The products will then be used for binding studies with MBL-A, SP-A and SP-D in order to define the topology of the carbohydrate recognition on a molecular basis. The molecular details will be elucidated by binding and crystallographic studies within established long-standing cooperations with groups in Germany, Canada and the US and will include lectin-binding assays tests, surface-plasmon resonance studies and isothermal microcalorimetric measurements. As complementary techniques, molecular modeling and NMR spectroscopic techniques will be used. The outcome of the project should contribute to a better understanding of bacterial carbohydrate-lectin interaction with charged sugar ligands and translate into novel approaches towards the development of anti- inflammatory agents to be used in anti-inflammatory and antiallergic therapies.

New methods for the chemical synthesis of bacterial cell-surface carbohydrates have been developed providing a basis for the development of potential vaccines and diagnostic tools in the context of clinically relevant bacterial infections. This topic is of significant medical and socio-political global relevance due to the increase of bacterial resistance against antibiotics and the occurrence of multi-drug-resistant bacterial strains. In particular, infections caused by Acinetobacter are notoriously difficult to treat and eradicate and have led to the closure of intensive care units in hospitals. The target structures addressed within the project are located in the outer membrane of the Gram-negative bacterial cell wall and comprise lipid-bound carbohydrates (lipopolysaccharides) involved in a multitude of interactions with components of the innate and adaptive immune system. The project was specifically focused on a structurally conserved part of Acinetobacter lipopolysaccharide containing higher-carbon sugars of seven and eight carbon atoms. The cell-wall carbohydrates of Acinetobacter are characterized by unique structures of the conserved bacterial 8-carbon sugar acid Kdo (3-deoxy-octulosonic acid) and the occurrence of an acid-stable Kdo variant, termed Ko. In the framework of the project, these unique Acinetobacter carbohydrate units were synthesized for the first time with significant improvements in yields and selectivity. This way, altogether a compound library of 20 oligosaccharides was prepared. The synthetic products and cell wall fragments isolated from bacteria were then subjected to detailed binding studies with antibodies and lectins in cooperation with groups from the research Centre Borstel (Germany) and the University of Victoria (Canada). Binding experiments with mannose-binding lectins (MBL-A, MBL-C) revealed enhanced binding of a few synthetic products, which, however, were still of lower potency compared to the unusual high reactivity of the bacterial cell-wall fragments. The data obtained thus fas would indicate that the region which is more close to the surface of the bacterial membrane could be responsible for this pronounced activity. Selected synthetic modified Kdo antigens were also subjected to crystallographic studies with Kdo-specific monoclonal antibodies and provided the first evidence to support the hypothesis of a selection of already preformed antibodies by the antigen. This feature leads to significant efficiency of the innate immune response, since the recognition of these bacteria-specific higher-carbon sugars is genetically determined in the germline sequence. The synthetic methods developed in the framework of the project will be applicable for the generation of Acinetobacter-specific antibodies.