Carbohydrates from Pseudomonas aeruginosa biofilms interact with immune C-type lectins and interfere with their receptor function

Sonali Singh,Farah Hussain,Miguel Cámara,Ana C Da Silva,David L Williams,Daniel A Mitchell Daniel A Mitchell,Yasir Almuhanna,Peter J Rice,Janice Koranteng,Paul Williams,Darryl Jackson,Zuchao Ma,Gökhan Yilmaz ,Kelly C Lee ,Sirina Muntaka,Douglas W Lowman,Yasuhiko Irie,Mohammad Y Alshahrani,Francesca Mastrotto,Christopher Gell ,Bridget M Graves ,Rucha Juarez,Luisa Martı́nez-Pomares

doi:10.1038/s41522-021-00257-w

Abstract

Bacterial biofilms represent a challenge to the healthcare system because of their resilience against antimicrobials and immune attack. Biofilms consist of bacterial aggregates embedded in an extracellular polymeric substance (EPS) composed of polysaccharides, nucleic acids and proteins. We hypothesised that carbohydrates could contribute to immune recognition of Pseudomonas aeruginosa biofilms by engaging C-type lectins. Here we show binding of Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN, CD209), mannose receptor (MR, CD206) and Dectin-2 to P. aeruginosa biofilms. We also demonstrate that DC-SIGN, unlike MR and Dectin-2, recognises planktonic P. aeruginosa cultures and this interaction depends on the presence of the common polysaccharide antigen. Within biofilms DC-SIGN, Dectin-2 and MR ligands appear as discrete clusters with dispersed DC-SIGN ligands also found among bacterial aggregates. DC-SIGN, MR and Dectin-2 bind to carbohydrates purified from P. aeruginosa biofilms, particularly the high molecular weight fraction (HMW; >132,000 Da), with KDs in the nM range. These HMW carbohydrates contain 74.9–80.9% mannose, display α-mannan segments, interfere with the endocytic activity of cell-associated DC-SIGN and MR and inhibit Dectin-2-mediated cellular activation. In addition, biofilm carbohydrates reduce the association of the DC-SIGN ligand Lewisx, but not fucose, to human monocyte-derived dendritic cells (moDCs), and alter moDC morphology without affecting early cytokine production in response to lipopolysaccharide or P. aeruginosa cultures. This work identifies the presence of ligands for three important C-type lectins within P. aeruginosa biofilm structures and purified biofilm carbohydrates and highlights the potential for these receptors to impact immunity to P. aeruginosa infection.

Highlights

Pseudomonas aeruginosa is a versatile opportunistic pathogen that causes acute infection after invasive procedures and burns, and chronic infections in patients with persistent lung disease and compromised immunity[1]
To determine the location of DC-SIGN, MR and Dectin-2 ligands within P. aeruginosa biofilms, we analysed the binding of DC-SIGN, MR and Dectin-2 Fc proteins to biofilms generated under flow conditions in XVivo-15 medium using confocal microscopy
These differences could be caused by the use on an alternative detection system or changes in carbohydrate structure when biofilms are formed under flow conditions. Together these results support the display of DC-SIGN, MR and Dectin-2 ligands by P. aeruginosa biofilms, with each receptor exhibiting different binding profiles

Summary

Introduction

Pseudomonas aeruginosa is a versatile opportunistic pathogen that causes acute infection after invasive procedures and burns, and chronic infections in patients with persistent lung disease and compromised immunity[1]. The use of indwelling catheters and implants during hospital procedures, as well as use of contact lenses create niches that are effectively colonised by P. aeruginosa which exploits an armoury of cell-associated and secreted virulence determinants that facilitate invasion and establishment of infection[4]. Biofilms contribute to P. aeruginosa persistence by increasing tolerance to antimicrobial agents and immune defences[1]. Within these bacterial communities, cells are embedded in an extracellular polymeric substance (EPS) matrix mainly composed of polysaccharides, nucleic acids and proteins[6,7]. Pel is cationic and largely composed of N-acetylgalactosamine and N-acetyl-glucosamine[10]

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Conclusion