Staphylococci are the leading cause of hospital-acquired infections, especially nosocomial bacteremia. The two most effective and widely used anti-staphylococcal therapeutic agents are glycopeptides and beta-lactams, both of which target cell wall biosynthesis. However, therapy with these agents is becoming less effective as resistance has developed, first to beta-lactams and, more recently, to glycopeptides. The most important mechanism of resistance to beta-lactams is the acquisition of a new target, a cell wall transpeptidase or penicillin binding protein (PBP2a) that is not inactivated by the antibiotic. This type is called methicillin or oxacillin resistance (OR) and the gene that mediates this resistance, mecA, is encoded within a pathogenicity island called SCCmec. The following proposal seeks to continue studies that explore the origin, dissemination and regulation of genes that mediate OR and genomic adaptations required for staphylococci to become resistant to agents that damage their cell walls. The First Specific Aim will be to investigate the transfer of SCCmec between strains of Staphylococcus aureus (SA) and from a different staphylococcal species, S. epidermidis (SE), to SA. There is evidence that a new SCCmec type, Type IV, has recently moved into SA isolates prevalent in the community and it is present in the majority of SE isolates. The excision, transfer (by plasmid and phage) and reinsertion of this element will be investigated. The Second Specific Aim will be to continue studies on the induction of mecA transcription through the sensor/transducer, MecR1, resulting in the release of the transcriptional repressor, Mecl, from its DNA binding site. The basis of signal transduction and role of proteolytic cleavage of inducer and repressor will be assessed by constructing chimeric molecules, determining the crystal structure of repressors and identifying additional chromosomal genes required for induction. The Third Specific Aim will be to confirm and expand observations made by microarray transcriptional profiling that purine biosynthesis is altered in strains that develop high level resistance to vancomycin and oxacillin, but in opposite directions (increased and decreased respectively). These two phenotypes appear to be mutually exclusive. The purine biosynthetic operons will be genetically manipulated and correlated with development of VR and OR. In addition, microarray and proteomic studies will be pursued on other agents that perturb the cell wall.
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