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

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 (Lewis^a, Lewis^b, Lewis^x, siayl-Lewis^x 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.

Keywords: Pseudomonas aeruginosa; anti-bacterial; glycocluster; glycoconjugate; oligosaccharide.

Figure 1

Structure of the fucosyl cluster F1 used for the first screening.

Figure 2

Examples of results obtained for five natural oligosaccharides. The reference fucocluster was F1. On the y-axis, the fluorescent signal recorded after incubation of F1 with LecB and 0.1 µM of fucose was set at 100%. All other fluorescent signals were then normalized to that reference. (Le^a: Lewis^a tetraose, Galβ1-3(Fucα1-4)GlcNAcβ1-3Gal; 3FL: 3-Fucosyllactose, Galβ1-4(Fucα1-3)Glc; Le^x: Lewis^x triose, Galβ1-4( Fucα1-3)GlcNAcβ1-3Gal; Blood group A: A trisaccharide antigen, GalNAcα1-3(Fucα1-2)Gal; Galili antigen: Galili pentasaccharide, Galα1-3(Galβ1-4GlcNAcβ1-3)₂Galβ1-4Glc); see supporting information S1).

Figure 3

Overview of the screening of the 156 glycans. 36 competed for the interaction of LecB with the immobilized fucocluster F1.

Figure 4

(a) Lewis^a interaction with Lectin LecB tetramer (calcium ions in green). 2D diagram (Biovia Discovery Studio 2016) of the interaction of LecB tetramer with (b) Lewis^a tetrasaccharide, (c) Lewis^x tetrasaccharide and (d) 3-Fucosyllactose (3FL).

Figure 5

Structure of azide solid support 157 N³-propargyl furanoside uracyl 158–160 and propargyl pyranoside 161–163 scaffolds.

Scheme 1

Synthesis of tosyl di- and tetra-ethyleneglycyl (2-cyanoethyl) N,N-diisopropyl phosphoramidites 165a–b. Cne: 2-cyano-ethyl.

Scheme 2

Synthesis of propargylated N-acetyl oligosaccharides 166–170. Le^a: Lewis^a tetrasaccharide, Le^b: Lewis^b tetrasaccharide, 3FL: 3-Fucosyllactose, Le^x: Lewis^x tetrasaccharide and sLe^x: sialyl-Lewis^x pentasaccharide.

Scheme 3

Synthesis of uracylfuranoside-centered oligo-saccharide cluster oligonucleotide conjugates (G1 to G6 series) SPOS Solid-phase oligonucleotide synthesis, Cne: Cyanoethyl, grey ball represents CPG: Controlled pore glass.

Scheme 4

Synthesis of pyranoside-centered oligosaccharide cluster oligonucleotide conjugates (G7 to G12 series). SPOS Solid-phase oligonucleotide synthesis, Cne: Cyanoethyl, grey ball represents CPG: Controlled pore glass.

Scheme 5

Synthesis of oligosaccharide-oligonucleotide conjugates (M-series).

Figure 6

The fluorescent signal of Alexa 647 LecB conjugate was recorded as a function of the oligoglycoclusters. NC and PC stand for negative and positive control. Structures of NC_2, PC_1 and PC_2 are displayed Figure 7. NC_1 is a single stranded DNA. G1 and G2 are arabinose-centered clusters with an EG₂ or EG₄ linker respectively. In a similar manner, G3 and G4 are xylose-centered, G5 and G6 ribose-centered, G7 and G8 glucose-centered, G9 and G10 galactose-centered, and finally G11 and G12 mannose-centered. Average and error bars were calculated from 32 repetition spots from one experiment.

Figure 7

Structure of the two positive controls PC_1 and PC_2 [27] and the negative control NC_2. [42].

Figure 8

(top) Modelling of the interaction of G9-Lewis^a with LecB. The two loops, V69-P73 and D96-D99, are in green and purple respectively. (bottom) Details of the interaction of G9-Lewis^a with LecB.