Abstract
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.
Highlights
The emergence of antibiotic-resistant bacterial strains calls for new anti-bacterial strategies
We first prepared the different building blocks corresponding to an azide solid support 157 [40], trihydroxylated alkyne scaffolds prepared from arabino, ribo- and xylofuranosyl uracils (158–160) [28], tetrahydroxylated scaffolds corresponding to propargyl-galactoside, glucoside and mannoside (161–163) [27] (Figure 5), two different linkers corresponding to tosyl-di- or tetra-ethyleneglycol phosphoramidites (165a–b)
LecB, as previously observed with fucoclusters clusters. This result shows that the topology seems to be more important than the valence on the Concerning influence of the length of the linker, our study showed that a shorter linker (EG2)
Summary
The emergence of antibiotic-resistant bacterial strains calls for new anti-bacterial strategies. One of them consists of inhibiting virulent factors in particular lectins involved in bacterial adhesion and/or biofilm formation using monovalent glycomimetics [1] or multivalent glycoclusters. The soluble homotetrameric fucophilic lectin LecB of PA is classified as a virulent factor and is involved in biofilm formation, host/bacteria and bacteria/bacteria interaction, cytotoxicity and inhibition of ciliated removal [10]. It consists of four subunits of 11.7 kDa (114 amino-acids). With the aim to find oligosaccharide of high affinity to LecB and to synthesize oligosaccharide clusters of higher affinity to LecB that could compete with the natural endogenous ligands of the lectin, we developed a double screening strategy. The resulting glycocluster glycocluster arraytowas usedthe to assess of the oligoglycoclusters by a readout direct readout array was used assess affinitythe ofaffinity the oligoglycoclusters to LecB to byLecB a direct of the of the fluorescence in comparison to the corresponding monovalent fluorescence signal signal in comparison to the corresponding monovalent ligand.ligand
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