PROJECT SUMMARY The continued and inevitable emergence of antibiotic resistance demands a vigorous and sustained effort to identify fundamentally new targets and strategies for innovative antimicrobial therapeutics. Antibiotic-resistant enterococci are major causes of hospital-acquired infections. Enterococci are successful hospital-acquired pathogens in part because of their intrinsic resistance to commonly used antibiotics that target the bacterial cell envelope, such as cephalosporins. However, many questions remain regarding the genetic and biochemical basis for cephalosporin resistance in enterococci. Previous work revealed key roles for two signal transduction systems - the IreK transmembrane kinase and the CroS/R two-component system - in regulation of cephalosporin resistance, but the downstream effectors in the signaling pathways that drive cephalosporin resistance remain unknown. In preliminary studies we showed that two penicillin-binding proteins – enzymes that synthesize peptidoglycan – are each essential for cephalosporin resistance, yet are functionally distinct from each other. The mechanisms by which the activity of these penicillin-binding proteins are regulated in enterococci are unknown, although current models point to the possibility that these penicillin-binding proteins exist as components of multiprotein peptidoglycan synthase complexes. Our data suggest that the IreK and CroS/R signaling systems are responsible for regulation of penicillin-binding protein activity to promote cephalosporin resistance. The major knowledge gaps to be addressed are that (i) the composition and activity of the peptidoglycan synthases in response to cephalosporin stress are unknown; (ii) a definitive link between IreK or CroS/R and the peptidoglycan synthases has not been established; and (iii) the mechanisms by which cephalosporins induce lethality when one peptidoglycan synthase is impaired is unknown. The research proposed here is designed to elucidate new insights into the roles and regulation of peptidoglycan synthases in the biological processes that drive enterococcal cephalosporin resistance. By doing so, we will provide new insights into the fundamental biological processes that drive key antibiotic resistance in enterococci and define new targets for innovative therapeutics designed to impair enterococcal cephalosporin resistance.