Molecular mechanisms of antibiotic resistance

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Bacterial infections pose serious threats to human health. Furthermore, viral infections such as the flu are frequently accompanied by bacterial infections which is often a deadly combination. Due to the development of resistance, the options for treating infections have dwindled substantially. This resistance is in large part due to the bacterial expression of beta-lactamases that degrade beta-lactam antibiotics. Our main goal is to structurally understand the molecular basis of resistance against the clinically available ceftazidime antibiotic and how certain mutations in beta-lactamases confer resistance to this antibiotic and other mutations confer resistance to the beta-lactamase inhibitors tazobactam, sulbactam, and clavulanic acid. Our structure-function studies involve a novel synergy between X-ray and time-resolved Raman crystallography. Our innovative inter-disciplinary approach allows us to identify and track reaction intermediates inside crystals prior to X-ray analysis and provides a unique advantage to accomplish our Specific Aims: Specific Aim 1: To test the hypothesis that SHV-1 resistance variants at position M69 cause active site changes that affect the level and stability of the intermediate formation involving the 3 inhibitors thus allowing them to overcome the inhibition. Specific Aim 2: To test the hypothesis that the extended-spectrum beta-lactamase SHV-2 variant (G238S) and SHV-5 variant (G238S & E240K) have evolved to hydrolyze the third-generation cephalosporin ceftazidime by widening the active site and forming novel interactions with this compound. Specific Aim 3: To test the hypothesis that the changes at position D179 which are present in SHV-6, -8, and -24 have evolved to hydrolyze ceftazidime by shifting the omega loop thereby extending the active site to accommodate ceftazidime. The structural knowledge gained from the proposed research could aid in the design of new antibiotic drugs.