Screening of a Library of Oligosaccharides Targeting Lectin LecB of Pseudomonas Aeruginosa and Synthesis of High Affinity Oligoglycoclusters.

The Gram negative bacterium Pseudomonas aeruginosa (PA) is an opportunistic bacterium that causes severe and chronic infection of immune-depressed patients. It has the ability to form a biofilm that gives a selective advantage to the bacteria with respect to antibiotherapy and host defenses. Herein, we have focused on the tetrameric soluble lectin which is involved in bacterium adherence to host cells, biofilm formation, and cytotoxicity. It binds to l-fucose, d-mannose and glycan exposing terminal fucose or mannose. Using a competitive assay on microarray, 156 oligosaccharides and polysaccharides issued from fermentation or from the biomass were screened toward their affinity to LecB. Next, the five best ligands (Lewisa, Lewisb, Lewisx, siayl-Lewisx and 3-fucosyllactose) were derivatized with a propargyl aglycon allowing the synthesis of 25 trivalent, 25 tetravalent and 5 monovalent constructions thanks to copper catalyzed azide alkyne cycloaddition. The 55 clusters were immobilized by DNA Directed immobilization leading to the fabrication of a glycocluster microarray. Their binding to LecB was studied. Multivalency improved the binding to LecB. The binding structure relationship of the clusters is mainly influenced by the carbohydrate residues. Molecular simulations indicated that the simultaneous contact of both binding sites of monomer A and D seems to be energetically possible.

Synthesis of α-galactosyl epitopes by metabolically engineered Escherichia coli.

The α-Gal epitope is a carbohydrate structure, Galα-3Galß-4GlcNAc-R, expressed on glycoconjuguates in many mammals, but not in humans. Species that do not express this epitope have present in their serum large amounts of natural anti-Gal antibodies, which contribute to organ hyperacute rejection during xenotransplantation. We first describe the efficient conversion of lactose into isoglobotriaose (Galα-3Galß-4Glc) using high cell density cultures of a genetically engineered Escherichia coli strain expressing the bovine gene for α-1,3-galactosyltransferase. Attempts to produce the Galili pentasaccharide (Galα-3Galß-4GlcNAcß-3Galß-4Glc) by additionally expressing the Neisseria meningitis lgtA gene for ß-1,3-N-acetylglucosaminyltransferase and the Helicobacter pylori gene for ß-1,4-galactosyltransferase were unsuccessful and led to the formation of a series of long chain oligosaccharides formed by the repeated addition of the trisaccharide motif [Galß-4GlcNAcß-3Galα-3] onto a lacto-N-neotetraose primer. The replacement of LgtA by a more specific ß-1,3-N-acetylglucosaminyltransferase from H. pylori, which was unable to glycosylate α-galactosides, prevented the formation of these unwanted compounds and allowed the successful formation of the Galili pentasaccharide and longer α-Gal epitopes.