Studying the interactions between Staphylococcus aureus and atopic dermatitis skin cells using atomic force microscopy

With the constant increase in the resistance of Staphylococcus au- reus to antibiotics, it has become a priority to better understand how this pathogen interacts with host tissues, and to develop alternative strategies to fight staphylococcal infections. S. aureus plays an im- portant role in atopic dermatitis (AD), a chronic disorder that mostly affects children. Colonization of the skin of AD patients by S. aureus exacerbates the disease, and reduced levels of natural moisturizing factor (NMF) in the stratum corneum have been shown to be associa- ted with more severe AD symptoms. Today, two unsolved questions are : i) what are the bacterial adhesins involved in S. aureus - skin adhesion, and ii) is the skin protein corneodesmosin (CDSN) a major ligand for S. aureus adhesion ? The aim of this master thesis, perfor- med in collaboration with Dr. J. Geoghegan (Trinity College, Dublin), was to use atomic force microscopy (AFM) to study the interaction of S. aureus with AD skins, with a particular focus on the above open questions. We first used single-cell force spectroscopy (SCFS) to measure the adhesion forces between different strains of S. aureus and AD skin samples with a low NMF level. Using mutant strains of S. aureus with deleted adhesins, we have identified the involve- ment of three major adhesins in the interaction with AD skins : ClfB, FnBPA and FnBPB. In agreement with previous studies, we found that ClfB is responsible for high adhesion forces, ∼ 1600pN, whereas FnBPs are associated with lower forces ∼ 500pN. Next we used single molecule force spectroscopy (SMFS) with tips functionalized with an anti-CDSN antibody to detect and map the distribution of CDSN on the surface of AD skins, demonstrating that this protein is more abun- dant on low NMF skins compared to high NMF skins. Using SCFS, we were also able to partially block the interaction of S. aureus with low NMF skin by injecting Fab fragments of the anti-CDSN antibody at increasing concentrations. Both low and high forces were inhibited, suggesting that CDSN might be a ligand for both FnBPs and ClfB on low NMF skins. All together, these results shed new light onto the mo- lecular determinants of S. aureus-AD skin interactions : i) ClfB and FnBPs are the main adhesins involved; ii) the binding strengths of ClfB and FnBPs are high and moderate, respectively, suggesting that two different binding mechanisms are involved; iii) CDSN seems to be an important ligand for FnBPs, but also for ClfB. Our study opens up new possibilities for the development of anti-adhesive therapies to prevent S. aureus from attaching to human skins.