Invasion of human keratinocytes by Staphylococcus aureus and intracellular bacterial persistence represent haemolysin-independent virulence mechanisms that are followed by features of necrotic and apoptotic keratinocyte cell death

Background Colonization of human skin by Staphylococcus aureus is a characteristic feature of several inflammatory skin diseases, which is often followed by tissue invasion and severe cell damage. A crucial role has been attributed to staphylococcal haemolysins in the cytotoxicity to epidermal structures. Objectives To investigate haemolysin-independent virulence to human keratinocytes. Methods The stable alpha-haemolysin, beta-haemolysin double-negative S. aureus mutant DU 5720 was compared with the fully virulent parent strain 8325-4 and with its isogenic fibronectin-binding protein A/B-negative variant DU 5883 in an invasion model. Results This assay showed dose-dependent internalization of all the strains investigated by human HaCaT keratinocytes, with reduced internalization of DU 5883. Transmission electron microscopy revealed adhesion of staphylococci to cellular pilus-like extrusions, followed by the embedding of the bacteria in cellular grooves. Following attachment to the keratinocytes the staphylococci were engulfed into vesicles within the cytoplasm where some bacteria persisted for 24-48 h. Addition of cytochalasin D strongly reduced the bacterial uptake, suggesting an active keratinocyte process. Bacterial invasion was followed by severe keratinocyte cell damage showing the morphological changes of cytotoxic and, to a lesser extent, apoptotic cell death as determined by the trypan blue exclusion test and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling assay. The highest levels of lethal cytotoxicity were observed in haemolysin-producing strains, whereas the induction of apoptosis seemed to depend on internalization. Conclusions Staphylococcal invasion of human keratinocytes represents a potent staphylococcal virulence factor, which, independently of alpha- and beta-haemolysins, leads to necrotic and apoptotic cell damage.