Research Project
7643649
DESCRIPTION (provided by applicant): Class D (-lactamases confer resistance to penicillin, cephalosporin and carbapenem (-lactam antibiotics. The threat that these enzymes pose is particularly troublesome in problematic Gram negative species such as Pseudomonas aeruginosa, Acinetobacter baumannii and Escherichia coli. While members of the class D family share some critical amino acid residues, their efficacy toward various antibiotics varies markedly. Moreover, a general mechanism by which they hydrolyze the lactam ring in these drugs has not been established in all members. Work in our laboratory has contributed to a better understanding of the mechanisms of substrate selection and turnover in the class D (-lactamase OXA-1. In the studies outlined in this grant, we propose to further define the determinants of substrate selectivity in OXA-1 and the related carbapenemase OXA-24. We will use mutagenesis of key active site residues in each enzyme followed by Minimum Inhibitory Concentration (MIC) analysis to accomplish this goal. We will also compare and contrast the acylation/deacylation turnover mechanism of OXA-1 and OXA-24 to establish if a carboxy-lysine is present and active as a general base in OXA-24 as it is the OXA-1. We will use a gel-based fluorescence assay to characterize OXA-1/OXA-24 mutants that are deacylation-deficient (ie. those that can become covalently attached to substrate, but cannot release it as product). A proper understanding of the modes by which class D (-lactamases select and break down various classes of antibiotics will help us anticipate future resistance trends as the genes for these enzymes spread and mutate. Also, information about active site/substrate interactions will hopefully contribute to the design of more effective class D inhibitors, as well as (-lactam antibiotics that are more resistant to these dangerous enzymes. PUBLIC HEALTH RELEVANCE: There has been a striking rise in reports of multi-drug resistant Acinetobacter baumannii infections world-wide, with many associated fatalities. One of the most worrying trends is the rise of strains resistant to several frontline (-lactam antibiotics including extended spectrum cephalosporins (ie. cefepime;ceftazidime) and carbapenems (ie. imipenem;meropenem). Of particular concern is the extent of carbapenem-resistant infections in soldiers returning from Iraq, Kuwait and Afghanistan [1, 2]. It has been reported that over 80% of the A.baumannii infections at some military hospitals are resistant to imipenem, and in some cases, the only remaining treatment option is the toxic antibiotic, colistin [3]. One of the key mechanisms of resistance in these Acinetorbacter infections, and in infections caused by a variety of other Gram negative pathogens, is expression of one or more class D (-lactamases [4, 5]. Despite this emerging threat, class D (-lactamases are the least understood of the four subfamilies. The studies proposed in this grant will explore the mechanism by which these dangerous enzymes select various penicillins, cephalosporins and carbapenems as substrates. We also aim to illuminate the catalytic mechanism, with particular emphasis on how the active site stabilizes the unusual carboxy-lysine modification that is critical for activity. The knowledge gained from these studies will inform future experiments in three critical ways: a. Details of substrate selection will be potentially useful for the design of new (-lactam antibiotics that are resistant to hydrolysis by (-lactamases, as well the design of new class D (-lactamase inhibitors. b. Understanding the effects of active site substitutions will help predict future evolution of class D (-lactamases and the potential threats they pose with regard to broadened specificity. c. A thorough understanding of the formation of stable acyl-enzyme intermediates will lead directly to x-ray crystallography studies of catalytic intermediates. These studies have been designed to explore two different subtype class D (-lactamases, OXA-1 and OXA-24. The former is a typical oxacillinase/penicillinase, while the latter is a carbapenemase from Acinetobacter baumannii.
