Almuhanna, Yasir Saleh I
(2021)
Role of lectin receptors in recognition of Pseudomonas aeruginosa biofilms.
PhD thesis, University of Nottingham.
Abstract
Colonisation and chronic lung infection by the opportunistic pathogen Pseudomonas aeruginosa (PA) is the leading cause of morbidity and mortality in cystic fibrosis patients. A critical key determinant of PA pathogenicity is the switch from planktonic to the biofilm mode of growth, which facilitates chronic infections and makes PA eradication extremely difficult. Biofilms are aggregates of microorganisms in which the cells are enclosed in an extracellular polymeric substance (EPS) that contains proteins, extracellular DNA (eDNA) and exopolysaccharides. Psl and Pel are two important exopolysaccharides utilised by PA to construct biofilms. While Psl is composed of D-mannose, D-glucose, and L-rhamnose, Pel contains N-acetylgalactosamine (GalNAc) and N-acetylglucosamine (GlcNAc).
C-type lectins receptors (CLRs) are pattern recognition receptors (PRRs) that recognise carbohydrate motifs and are expressed by innate immune cells, including dendritic cells (DCs), macrophages and neutrophils. The overarching aim of this thesis is to investigate the interplay between PA and innate immune cells and how it is affected by biofilm formation. We hypothesise that engagement of CLRs through recognition of carbohydrate structures within PA biofilms could modulate the immune response and interfere with innate immune cells function, hence facilitating the persistence of infection.
Previously, our lab demonstrated that PA biofilms and biofilm-associated carbohydrates purified from the Pel-deficient mutant ΔwspF Δpel, engage two CLRs, Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN, CD209) and Mannose receptor (MR, CD206). In this study, we show that Dectin-2 (CLEC6A) is another CLRs that binds PA biofilms and purified biofilm carbohydrates; Dectin-2 binds to biofilms formed by PA strains with different EPS composition as well as biofilms formed by PA wound isolates. Similarly to MR and DC-SIGN, Dectin-2 also recognised purified biofilm-associated carbohydrates. High and low molecular fractions of purified biofilm carbohydrates were tested in this study and Dectin-2, together with DC-SIGN and MR preferentially bound to the HMW preparations. Biofilm-associated HMW carbohydrates did not induce Dectin-2 signalling on huDectin-2 reporter cells or HEK-Blue™ mDectin-2 cells but showed antagonistic activity blocking the response of muDectin-2 reporter cells to the Dectin-2 ligand zymosan.
To microscopically examine the distribution of CLRs ligands within the biofilms and to study the interaction of immune cells with biofilm, we optimised biofilm generation in substrates suitable for microscopic examination. Confocal analysis shows DC-SIGN, Dectin-2 and, weakly, MR ligands within PAO1 biofilms concentrated into discrete clusters with additional DC-SIGN ligands dispersed among bacteria aggregates. Monocyte-derived DCs (moDCs) (DC-SIGN+ and MR+) cultured with biofilms formed by strains that overexpress Psl (ΔwspF and ΔwspF Δpel) tend to display round morphology and similar findings were noted when moDCs were incubated with purified biofilm carbohydrates. In a different set of experiments cytokine production by human moDCs in response to biofilms was tested and no differences were seen among biofilms with different carbohydrate compositions or after addition of purified biofilm carbohydrates. Taken together, these data indicate that DC-SIGN, MR and Dectin-2 are receptors for PA biofilms and biofilm-associated carbohydrates. The results of this research support the idea that CLRs engagements upon biofilm recognition by innate immune cells might modulate immune cells function.
As the first responder cells of the innate immune system, neutrophils possess a unique set of migration features. Several studies found that neutrophils were not able to eradicate infection caused by biofilm-forming PA, which might indicate that biofilm formation was hindering neutrophil’s activity. Here, we describe an in vitro assay that allows us to examine neutrophil behaviour, using live and confocal microscopy, following their interaction with PA, as a planktonic or biofilm cultures. Using live microscopy analysis, we observed that (1) biofilms reduced neutrophil velocity, net distance covered and the mean square displacement. Using confocal microscopy analysis, we observed that (2) neutrophils appeared unable to release granules when incubated with biofilms. Finally, (3) preliminary observations indicated that cells exhibit higher circularity when incubated with biofilm formed by strains expressing Psl or Psl-deficient biofilms in the presence of purified biofilm carbohydrates.
The findings from this study have gone some way towards enhancing our understanding of the potential impact of biofilm formation on innate immune cells which might open new avenues for therapeutic approaches.
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