Covalent Lectin Inhibition and Application in Bacterial Biofilm Imaging

Abstract

Biofilm formation by pathogenic bacteria is a hallmark of chronic infections. In many cases, lectins play key roles in establishing biofilms. The pathogen Pseudomonas aeruginosa often exhibiting various drug resistances employs its lectins LecA and LecB as virulence factors and biofilm building blocks. Therefore, inhibition of the function of these proteins is thought to have potential in developing "pathoblockers" preventing biofilm formation and virulence. A covalent lectin inhibitor specific to a carbohydrate binding site is described for the first time. Its application in the LecA-specific in vitro imaging of biofilms formed by P. aeruginosa is also reported.

Keywords: Pseudomonas aeruginosa; biofilms; carbohydrates; glycomimetics; lectins.

Figure 1

A) Galactoside recognition by the bacterial lectin LecA (pdb: 3ZYF^[15]); B) Electrophilic epoxide derivatives 2 and 3 for the targeting of Cys62. The distances from Cys62‐S to C6 and O6 of 1 are between 4.1 and 4.3 Å. (sc=side chain, bb=backbone).

Scheme 1

Synthesis of LecA‐directed epoxides and competitive binding to LecA. i) acetone, ZnCl₂, H₂SO₄; ii) (COCl)₂, DMSO, NEt₃, CH₂Cl₂, −78 °C to 0 °C; iii) PPh₃*MeI, NaH, DMSO; iv) 70 % HOAc aq.; v) Ac₂O, pyridine; vi) PhOH, BF₃*Et₂O, CH₂Cl₂, −20 °C–r.t.; vii) NaOMe, MeOH, r.t.; viii) mCPBA, NaHCO₃, CH₂Cl₂/MeOH;.

Figure 2

Covalent binding mode of 3 with LecA established by mass spectrometry. Deconvoluted intact protein MS spectra of LecA: A) without inhibitor, and B) with inhibitor 3; C) MALDI‐ISD experiments with c‐ion series annotated in a range from 5000 to 8000 m/z.

Figure 3

Crystal structure of epoxide 3 in complex with LecA at 1.80 Å resolution in the non‐covalent binding mode obtained at pH 4.6 (pdb code 5MIH). A) Electron density displayed at 1σ for ligand and Cys62 side chain. B) Interaction of the ligand with LecA: the epoxy oxygen atom accepts hydrogen bonds from His50 and one protein‐bound water molecule. Furthermore, His50 established a CH–π interaction with the phenyl agylycon. In the crystal structure, the sulfur atom of Cys62 is 3.3 Å away from C7 of ligand 3.

Scheme 2

Synthesis of LecA‐directed propargylated epoxides with LecA inhibition data and synthesis of fluorescent derivative 17. i) hydoquinone monopropargyl ether, BF₃*Et₂O, CH₂Cl₂, −20 °C–r.t.; ii) mCPBA, NaHCO₃, CH₂Cl₂, 0 °C–r.t.; iii) NaOMe, MeOH, 0 °C; iv) CuSO₄, sodium ascorbate, H₂O, DMF, r.t.

Figure 4

Covalent binding of 17 to LecA established by SDS‐PAGE. A) Fluorescence imaging; B) Coomassie staining; M* BenchMark fluorescent protein standard (Thermo), M^# protein marker III (Applichem).

Figure 5

Galleries of LecA‐dependent staining of P. aeruginosa biofilms with 17. A) P. aeruginosa PAO1 wt or B) the lecA knockout (ΔlecA) mutant, both expressing mCherry from pMP7605, were incubated at 37 °C for 24 h with agitation (180 rpm). Biofilms were stained with the covalent LecA ligand fused to fluorescein (17) for 10–30 min. Z‐stacks (232×232 μm) were recorded every 2 μm at 561 nm for mCherry (red, A and B, upper panels) and 488 nm for fluorescein (green, A and B middle panels). The galleries show every 4th z‐stack recorded. Lower panels show merged images of both channels (488 nm and 561 nm).

Figure 6

Three‐dimensional imaging of LecA‐dependent staining of P. aeruginosa PAO1 biofilms with 17. A) P. aeruginosa PAO1 wt or B) the lecA knockout (ΔlecA) mutant expressing mCherry from pMP7605 were incubated at 37 °C for 24 h with agitation (180 rpm). Biofilms were stained with 17 for 10–30 min. Z‐stacks (232×232 μm) were recorded every 2 μm at 561 nm for mCherry (red) and 488 nm for fluorescein (green). The 3D images show merged images of both channels (488 nm and 561 nm) from top and side views.