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. Our preliminary data reveal previously unknown roles for an intracellular nucleotide signal and nucleotide metabolite in control of enterococcal cephalosporin resistance. Little is known about how the activity of the enzymes responsible for controlling levels of these intracellular nucleotides is regulated in response to cell wall stress. Moreover, the targets for the nucleotide signal in enterococci are largely unknown. Our preliminary data suggest a novel model for regulation in which sensory input from physical association with other membrane-bound proteins regulates synthesis of the nucleotide signal in response to cell wall stress. The major knowledge gaps to be addressed are that (i) the mechanisms by which nucleotide signal synthesis is controlled in enterococci are unknown; (ii) the molecular target(s) for nucleotide signal in the context of antimicrobial resistance are unknown; and (iii) the mechanisms by which nucleotide metabolites impact cephalosporin resistance are unknown. The research proposed here is designed to elucidate new insights into the roles and regulation of intracellular nucleotides 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.