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 a key role for a transmembrane protein kinase (IreK) and its intracellular substrate (IreB) in regulation of cephalosporin resistance, but the downstream effectors in the signaling pathway remain unknown. In preliminary studies we showed that one (MurAA) of two UDP-GlcNAc 1-carboxyvinyltransferases encoded in E. faecalis (catalyzing the first committed step in peptidoglycan biosynthesis) is specifically required for cephalosporin resistance. The paralog of MurAA (MurAB) cannot drive cephalosporin resistance. Hence, MurAA possesses a specialized, specific ability to promote cephalosporin resistance. The major knowledge gaps to be addressed are that (i) a biochemical link between the IreK pathway and MurAA has not been established, and (ii) the mechanism by which MurAA drives cephalosporin resistance is unknown. The research proposed here is designed to elucidate new insights into the role of MurAA 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 may define new targets for innovative therapeutics designed to impair enterococcal cephalosporin resistance.