?-Lactam resistance in Acinetobacter baumannii presents one of the greatest challenges to contemporary antimicrobial chemotherapy. The production of ?-lactamases is singularly responsible for this phenotype. Added to this "arsenal of resistance" is the emerging importance of efflux pumps. The goal of this R01 renewal is to embark upon a multidisciplinary and integrative approach to gain insight into the molecular details that result in ?-lactam resistance by studying the AmpC cephalosporinase, ADC-7 (Acinetobacter derived cephalosporinase), and the efflux pumps of A. baumannii. Our work addresses the most important barriers to successful ?-lactam therapy. To address this significant problem, we will analyze how specific boronic acid inhibitors (BAIs) that contain different R1 and R2 side chains inactivate ADC-7. Our approach will entail kinetic and biophysical (isothermal calorimetry, protein thermal stability, and circular dichroism) measurements that will complement structural characterization. Together, we will gain unique insights into the mechanism of inactivation, the energetic requirements that define a potent inhibitor, and understand the evolution of ?-lactamase specificity. Once the BAIs are characterized and the structures of each BAI/ADC-7 complex are determined, we will also test our conclusions by performing site-directed mutagenesis of amino acid residues that interact with the BAI and test the impact of these changes on susceptibility, kinetics, protein stability, and structure. This comprehensive approach will reveal which amino acid positions are: (a) important for binding of BAI and ?-lactam compounds;and (b) critical for enzyme structure, stability, and/or function. Our efforts will provide a rational basis for the development and optimization of novel BAIs. In concert with these investigations, we will define the role of heretofore uncharacterized efflux pumps in ?-lactam/BAI resistance. Using "knockouts" of AdeABC and AdeIJK (adeABC/adeIJK), we will explore the role of "secondary" efflux pumps and transporters in ?-lactam resistance. Achieving these aims in two areas affecting ?-lactam resistance will help us design more potent ?-lactams against A. baumannii. Additionally, our work is a "first step" to understanding why the efflux systems of A. baumannii present a formidable barrier to antibiotics. Integrating this knowledge will provide the necessary insights regarding ?-lactam resistance that will assist medicinal chemists and physicians faced with this serious infectious disease threat.
Resistance to ?-lactam antibiotics in Acinetobacter baumannii is one of the most serious problems facing physicians who treat patients in the hospital. This proposal seeks to understand the molecular details of how the ?-lactamase; Acinetobacter Derived Cephalosporinase; ADC -7 and efflux systems of Acinetobacter confer resistance to penicillins and cephalosporins.
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