Calprotectin is the most abundant protein in neutrophils. Our surprising discovery that calprotectin can bind and hydrolyze multiple β-lactam antibiotics forms the basis of this proposal. In particular, calprotectin hydrolyzes and inactivates the penicillin sub-class that includes ampicillin, amoxicillin and oxacillin, which are among our most important and most widely used antibiotics. The identification of calprotectin as the first known host protein with β-lactamase activity has major implications for the treatment of infection. Indeed, our preliminary data demonstrate that human neutrophils hydrolyze β-lactam antibiotics in tissue culture and that oxacillin treatment is more effective in calprotectin-deficient mice than in wild-type mice. In Aim 1, we will determine the impact of calprotectin’s β-lactamase activity on antibiotic efficacy in vivo through three sub-Aims. We will first assess the ability of calprotectin to hydrolyze β-lactams during E. coli and S. aureus infection in mice. We will determine how the β-lactamase activity of calprotectin alters antibiotic- mediated growth inhibition and killing of pathogens during bacteremia and wound infection. We have shown that the serine β-lactamase inhibitor, sulbactam, blocks the β-lactamase activity of calprotectin. Therefore, we will next examine the capacity of various approved β-lactamase inhibitors to inhibit the β-lactamase activity of calprotectin and determine the capacity of inhibitors to improve β-lactam efficacy in mice, against penicillin sensitive bacteria, lacking any native β-lactamase production. Finally, we will assess whether β-lactam binding or inhibition of β-lactamase activity of calprotectin affects the innate immune functions of calprotectin, including metal sequestration and TLR4 activation. In Aim 2, we will elucidate the molecular mechanism(s) of calprotectin’s β-lactamase activity through two sub-Aims. Our preliminary evidence suggest calprotectin has multiple β-lactam binding sites and binds β- lactams with a high affinity. We will first fully characterize the β-lactamase activity of calprotectin, including dependence on parameters such as metal ions, salts, pH, temperature, and multimeric state. We will test hypotheses concerning reaction mechanism through enzymatic characterization of site-directed mutants. We will complement kinetic methods by determining the co-crystal structure of calprotectin bound to various inhibitors of β-lactamase activity. The completion of our Specific Aims will comprehensively characterize the newly discovered β- lactamase activity of calprotectin, determine its contribution to antibiotic efficacy in vivo and identify new avenues for drug development. Our proposal is significant due to potential impact on multiple aspects of treatments for bacterial infections. Our proposal is conceptually innovative in establishing a new paradigm for how calprotectin affects the outcome of bacterial infection.