The Pseudomonas aeruginosa lectin LecB binds to the exopolysaccharide Psl and stabilizes the biofilm matrix

Abstract

Pseudomonas aeruginosa biofilms are composed of exopolysaccharides (EPS), exogenous DNA, and proteins that hold these communities together. P. aeruginosa produces lectins LecA and LecB, which possess affinities towards sugars found in matrix EPS and mediate adherence of P. aeruginosa to target host cells. Here, we demonstrate that LecB binds to Psl, a key matrix EPS, and this leads to increased retention of both cells and EPS in a growing biofilm. This interaction is predicted to occur between the lectin and the branched side chains present on Psl. Finally, we show that LecB coordinates Psl localization in the biofilm. This constitutes a unique function for LecB and identifies it as a matrix protein that contributes to biofilm structure through EPS interactions.

Fig. 1

LecB binds to the exopolysaccharide Psl. a FLLA assay shows that  LecB is able to bind to fucose/mannose-containing polysaccharides immobilized on the wells of microtiter plates. FITC-conjugated LecB does not bind to uncoated or glucose-rich polysaccharide alginate, and Pel-rich materials (ΔwspF Δpsl). However, it binds to mannan, fucose-rich proteins (mucin), Psl-rich materials (ΔwspF and ΔwspF Δpel), and to purified Psl. ***p < 0.0001, t-test, n > 3. b LecB western blot showing coimmunopreciptation of LecB and Psl. Lane 1 was loaded with purified LecB. PtnG Dynabeads coated with anti-Psl antibodies were incubated with LecB (lane 2) or purified Psl and LecB (lane 3). Eluted material was immunoblotted using anti-LecB antibodies. c Psl blot showing coimmunopreciptation of LecB and Psl. Protein G Dynabeads coated with anti-LecB antibodies were incubated with purified LecB (lane 1) or with LecB and Psl (lane 2). After wash steps, eluted material was immunoblotted using anti-Psl antibodies. Lane 3 was loaded with purified Psl

Fig. 2

LecB binds α-mannobiose, but not β-mannobiose. Titration of a α-1,2′ mannobiose (Psl side chain) into purified LecB, b α-1,3′ mannobiose into purified LecB (not present in Psl), and c β-1,3′ mannobiose (Psl linear chain) into purified LecB. d Chemical structure of the Psl displaying the α-1,2′ mannobiose side chain and β-1,3′ mannobiose in linear chain

Fig. 3

Modeling of LecB–Psl interactions favors binding to the side chain of Psl. a Overall conformation of the docked Psl pentasaccharide is represented as α-D-mannopyranose in cyan, β-D-mannopyranose in gray, α-L-rhamnopyranose in orange, and β-D-glucopyranose in yellow. Oxygen atoms are labeled in red. b Structural model of Psl docking in LecB binding site is favored to occur in the α-1,2′ mannobiose. LecB is depicted in white, water molecules are the red spheres, the green ones are Ca⁺² ions, and Psl follows the same color code from (a)

Fig. 4

LecB binds mature biofilms in Psl-rich regions. Exogenously added FITC-conjugated LecB binds to biofilms containing Psl. a PAO1 4-day-old biofilm stained with Syto 62 (magenta), HHA-TRITC (red), and LecB-FITC (green) showed that LecB binds to similar regions as HHA. b Although PAO1 Δpsl produces very few aggregates relative to PAO1, those that were found were negative for HHA and LecB staining. c The same was observed for a PA14 biofilm, which cannot produce Psl, but still makes Pel. Scale bars = 25 µm

Fig. 5

LecB mutants display a defect on mature aggregate formation under flow conditions. a PAO1 4-day-old biofilm displays a complex mature structure in NB media while b ΔlecB 4-day-old biofilm do not fully develop into mature structures. c Complemented strain ΔlecB pJNLecB 0.05% arabinose 4-day-old biofilm develop structures that are very similar to PAO1. Biofilms are stained for biomass with Syto 62 at 2.5 µM. Scale bars = 25 µm

Fig. 6

LecB coordinates Psl positioning in the matrix. a Syto 62 (magenta) and HHA-TRITC (red) stained biofilms of ΔlecB pJNLecB grown in NB without arabinose forms a flat biofilm with b Psl distribution being fairly homogenous throughout the biomass (the x-axis, RFU, corresponds to the intensity of PSL staining.). Psl intensity profiles were generated from an average of nine independent micrographs. Inset is a representative Psl staining micrograph that was used to generate the profile. c Biofilms of ΔlecB pJNLecB grown in NB with the addition of 0.05% arabinose present a similar structure and d Psl distribution to the ones formed by PAO1. e Syto 62 (magenta) and HHA-TRITC (red) stained biofilms of ΔcdrA ΔlecB pJNLecB grown in NB without arabinose display a loose carpet of cells with f no particular Psl localization pattern. g ΔcdrA ΔlecB pJNLecB grown in NB with the addition of 0.05% arabinose rescues PAO1 phenotype and h usual Psl placement. i Biofilms of ΔcdrA ΔlecB pBADCdrAB grown in NB with the addition of 0.05% arabinose results in the formation of an undifferentiated thick layer of cells with j no particular Psl organization. Psl distribution measurements were performed in nine images from three different experiments per condition. Scale bars = 25 µm

Fig. 7

LecB retains cells and matrix material in adherent biomass. a Schematics of procedures performed in the samples from the tube biofilm experiment. Tubes are represented containing biomass (green) and media (blue). b Quantification of non-adherent and adherent ΔlecB pJNLecB cells without or with 0.2% arabinose. Absence of LecB results in increased number of non-adherent cells compared with when LecB is present. Expression of lecB increases the number of cells adhered to the tube surface compared with when LecB is not present. ***p < 0.0001, *p < 0.005 t-test, n > 3. c Dot blot of non-adherent fractions derived from ΔlecB pJNLecB without or with 0.2% arabinose 6-day-old tube biofilms. Immunodetection was performed using anti-Psl antibodies. Expression of lecB leads to decreased levels of released Psl when compared with the uninduced condition