Project 2 - SUMMARY Background: Staphylococcus aureus remains clinically problematic due to continually evolving antimicrobial resistance. Pathogenesis is attributed to production of potent extracellular toxins, and recent evidence from our laboratory has demonstrated that beta-lactam antibiotics, such as nafcillin, both increase and prolong toxin production in methicillin-sensitive and methicillin-resistant S. aureus (MRSA). The current study investigates detailed molecular mechanisms by which cell-wall active antibiotics alter the S. aureus divisional apparatus (divisome) and stimulate toxin production. Hypothesis: We hypothesize that a) cell-wall active antibiotics induce toxin expression via a unique and uncharacterized pathway, orchestrated directly or indirectly by effects on the bacterial cell wall divisome; b) antibiotic-induced toxin expression involves divisome elements PBP1 and/or PknB; and c) the conserved extracellular PASTA domain of PBP1 and PknB will serve as a therapeutic target for S. aureus disease. Specific Aim 1: To determine the transcriptional profile of a clinically relevant S. aureus strain during different phases of growth and in the presence of nafcillin. Genome-wide expression analysis will be used to elucidate the transcriptional response to nafcillin throughout the normal bacterial growth cycle. Specific Aim 2: To determine the role of three divisome proteins, PBP1, FtsZ and PknB Ser/Thr kinase in nafcillin-induced toxin expression. S. aureus mutants and recombinant proteins will be used to evaluate the role of divisome elements in the direct, or indirect, recognition and response to nafcillin. Specific Aim 3: To test the efficacy of immunization against PASTA domain-containing proteins in prevention of experimental S. aureus disease. Survival and pathogenesis studies will evaluate protective effects of immunization with S. aureus PASTA domain-containing antigens. This aim will heavily utilize the proposed HPIC facility. Impact on Human Health: Understanding molecular mechanisms that drive S. aureus toxin production in response to cell-wall active antibiotics will identify novel targets for disease intervention. Contribution to Multi-disciplinary Infectious Diseases Research Program: Dr. Bolz brings significant expertise in intracellular signaling pathways of the host response, and in diverse experimental models and technical approaches for studying host/pathogen interactions. Under Dr. Bolz's direction, the proposed studies will establish the use of high-throughput genomic technologies in the Infectious Diseases laboratory. In addition, targeting divisome proteins for potential new treatment modalities will expand our approach to antimicrobial development and vaccine design.