Role of biofilm carbohydrates during chronic infection

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 P. aeruginosa biofilms by engaging C-type lectins such as Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN, CD209), mannose receptor (MR, CD206) and Dectin-2. We have shown robust binding of DC-SIGN and weak binding of MR 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 concentrate into discrete clusters with dispersed DC-SIGN ligands also found among bacteria aggregates. We purified carbohydrates from P. aeruginosa biofilms and demonstrate binding of DC-SIGN, MR and Dectin-2 to these preparations, preferentially the high molecular weight fraction (HMW; >132,000Da) with KDs in the nM range. These HMW carbohydrates contain 74.9-80.9% mannose, display -mannan segments, interfere with the endocytic activity of cell-associated DC-SIGN and MR and, unexpectedly, inhibit Dectin-2-mediated cellular activation. We hypothesis that these important C-type lectins will impact immunity to P. aeruginosa infection. The first aim of this project is to establish the contribution of DC-SIGN, MR and Dectin-2 to the recognition of P. aeruginosa biofilms using in vitro cell cultures with primary human macrophages, dendritic cells and monocytes.

For instance, data to date indicate that biofilm carbohydrates induce phenotypical changes in monocyte-derived dendritic cells, but we still do not know the biological consequences of these changes. We will explore this aspect by looking at dendritic cell-T cell co-cultures and dendritic cell migration. Analysis of macrophage and monocyte behaviour will include cytokine production, changes in gene expression and microbicidal activity against P. aeruginosa. A second aspect of the project will explore the potential role of biofilm carbohydrates in modulating the response of macrophages, dendritic cells and monocytes to the fungal pathogen C. albicans.