Staphylococcal Corneocyte Adhesion: Assay Optimization and Roles of Aap and SasG Adhesins in the Establishment of Healthy Skin Colonization

Staphylococcus aureus is an opportunistic pathogen that causes the majority of wound and soft tissue infections. The accumulation-associated protein (Aap) from S. epidermidis and surface protein G (SasG) from S. aureus are cell wall-anchored (CWA) proteins known to be important in adhesion to healthy corneocytes from human skin. We investigated the mechanisms by which S. aureus colonizes healthy human skin by developing an optimized corneocyte adhesion assay. Trypan blue was used for enhanced red autofluorescent visualization of corneocytes with an overlay of green-fluorescent bacteria. The percent area of bacterial adhesion for images acquired by a fluorescence microscope was quantified using Fiji ImageJ. Using this optimized imaging procedure, differences in adhesion between various species and strains of staphylococci were measured. The ability of purified SasG to reduce Staphylococcus epidermidis adhesion was investigated in order to determine if these CWA proteins can compete for binding sites. To further test CWA-mediated adhesion, we engineered a nonadhering S. carnosus strain to express full-length SasG from two methicillin-resistant S. aureus (MRSA) strains. Finally, we demonstrated that the SasG A domain was a critical region of this surface protein for adherence to healthy human corneocytes. The developed imaging and expression methods are useful for studying staphylococcal adhesion to healthy human skin and have the potential to be used with a wide variety of fluorescently labeled organisms on both healthy and disease-state (such as atopic dermatitis) corneocytes.