Staphylococcus aureus is responsible for many diseases ranging from superficial skin infections to arthritis, osteomyelitis, intravascular infection, endocarditis, and sepsis. Of particular interest are diseases complicated by persistent infection. Osteomyelitis and arthritis, as well as infective endocarditis, are frequently long-lasting and difficult to treat, particularly in patients with prosthetic devices, and these patients often relapse after treatment. S. aureus infections are also a significant cause of upper airway infection particularly in patients with cystic fibrosis Initial colonization followed by recurrent clonal infections and ineffective antibiotic therapy strongly suggests that S. aureus can establish an intracellular niche in these patients. The focus of our research with S. aureus was the development of laboratory models of intracellular bacterial survival with the aim of uncovering clues to its survival strategies. Virtually nothing is known how S. aureus reprogrammes its gene expression to survive intracellularly and to thwart innate immune responses. The long term goal of the grant was to provide these details, or new research leads, so that ultimately, intracellular S. aureus infections can be eradicated by effective therapeutic regimens. Initially, our work established an understanding of bacterial uptake in two human airway cell lines, subcellular fate after internalization, and the discovery that not all cells that take up S. aureus undergo apoptosis or show overt cytotoxic effects. A second research phase outlined a screen of a large insertional mutant library to define bacterial genes necessary for intracellular survival. The protocol was revised following concerns over the use of flow cytometry with live S. aureus. A microarray study was ultimately accomplished in its place. Our studies used a variety of cellular biological methods (confocal microscopy, immunofluoresescence, electron microscopy, cytotoxicity assays, and standard lysostaphin-gentamicin protection assays to measure intracellular bacterial survival) and molecular genetic and genomic methods.An outgrowth of this grant was the initiation of new studies asking how S. aureus senses its environment. Alterations in signaling systems of the two-component variety, which are highly conserved in bacteria, were observed in our microarray study with intracellular S. aureus, in S. aureus biofilm formation, and in collaborative projects of drug resistance. Several collaborative studies were realized that permitted publication of studies including the unambiguous detection of intracellular S. aureus in nasal tissue of patients with recurrent rhinosinusitis, the identification of over twenty new genes implicated in S. aureus biofilm formation, and new insights into Listeria monocytogenes actin-based intracellular motility. In total, eight manuscripts published in peer-reviewed journals were funded wholly, or in part, by this grant and a ninth publication is anticipated.