In response to the critical and growing threat posed by microbial antibiotic resistance, the NIAID has made basic research leading to better understanding of resistance factors a top priority. The Gram-negative non-fermenting microbe Acinetobacter baumannii has developed resistance to multiple classes of antibiotics in the last 15-20 years, leading the Center for Disease Control to label it a serious domestic threat. While many of the basic resistance mechanisms have been extensively studied, newly emerging mechanisms threaten the dwindling therapeutic options. We plan to study how modifications in three A. baumannii enzyme families are enhancing this organism's ability to destroy (or become insensitive) to our most potent antimicrobial agents including carbapenems, advanced generation cephalosporins and monobactams. Preliminary data in our lab has shown that mutations in OXA-51-like class D ?-lactamases have led to enhanced hydrolytic activity against carbapenems. In our proposed studies we will use mutagenesis, kinetic assays and X-ray crystallography to understand how these variant enzymes lead to stronger enzymatic activity. We will explore similar clinical mutations in the OXA-23 and OXA-24/40 class D ? -lactamase subfamilies, investigating in particular how these mutations affect the breakdown of multiple classes of ? -lactam antibiotics. Lastly, we will study known mutations in one of the key target proteins for ? -lactam antibiotics in Acinetobacter spp: penicillin-binding protein 3 (PBP3). It i likely that these clinical mutations are making PBP3 less sensitive to these antibiotics, and thus making the organism less responsive to treatment. Ultimately, by discovering the structural and mechanistic details of how these enzymes are mutating to become more dangerous, we hope to provide direction for the design of more effective antibiotics and -lactamase inhibitors. 1
Bacterial resistance to -lactam antibiotics continues to grow at an alarming rate, rapidly threatening the efficacy of old and new treatments alike. Treating physicians are especially concerned about emerging threats in the problematic Gram-negative species Acinetobacter baumannii. The research proposed in this grant will illuminate how proteins in this species are evolving to become more dangerous, and will aid in the design of more effective antibiotics and -lactamase inhibitors. 1
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