339 Staphylococcus aureus from atopic dermatitis binds to keratin 6 in the stratum corneum of human skin

Staphylococcus aureus is a predominant bacterium on lesions in severe cases of atopic dermatitis and preferentially binds via the adhesins Clumping factor B (ClfB) and fibronectin binding protein B (FnBPB) to glycine loop containing proteins corneodesmosin, loricrin and keratin 10 on the surface of corneocytes. The objective of this study was to discover new epidermal proteins as adhesion sites for S. aureus. A yeast double hybrid study using a reconstructed differentiated epidermis library as prey and the ligand binding domain (N1-N3) of ClfB as bait showed that ClfB bound to a glycine rich internal domain of keratin 6A and 6B. In bacterial adhesion assays the atopic dermatitis S. aureus strain CC1 bound recombinant keratin 6B in a dose dependent manner. Analyses using S. aureus CC1 mutant strains for ClfB, FnBPA and FnBPB showed that these adhesins mediate the binding of CC1 strain to keratin 6. Furthermore, the CC1 strain does not bind keratin 6 in stationary phase. In addition, the SH1000 4X S. aureus strain which does not express ClfB, FnBPA and FnBPB did not bind keratin 6. However, when the gene for each of the adhesins were cloned and expressed in this strain, binding was restored to keratin 6. The binding of ClfB recombinant protein to keratin 6 was also confirmed by ELISA. Western blot analysis showed that keratin 6 was present in the stratum corneum. Pull-down experiments using the S. aureus strain ATCC 25904 which expresses ClfB and protein extracts from stratum corneum plantar were performed and subsequent proteomic analyses identified keratin 6 as one of the proteins that bound to S. aureus. Taken together the data show that the atopic dermatitis strain CC1 binds to keratin 6 present in the stratum corneum of human skin and this interaction could be a new therapeutic target to improve the symptoms associated with AD.