Expanded Substrate Activity of OXA-24/40 in Carbapenem-Resistant Acinetobacter baumannii Involves Enhanced Binding Loop Flexibility.

Gram-negative bacteria resist ß-lactam antibiotics primarily by deploying ß-lactamase proteins that hydrolytically destroy the antibiotics. In clinical settings, these bacteria are producing variant ß-lactamases with "gain-of-activity" mutations that inactivate a broader range of ß-lactams. Learning how these mutations broaden substrate activity is important for coping with ß-lactam resistance. Here, we investigate a gain of activity mutation in OXA-24/40, a carbapenem-hydrolyzing class D ß-lactamase (CHDL) in Acinetobacter baumannii. OXA-24/40 was originally active against penicillin and carbapenem classes of ß-lactams, but a clinical variant of OXA-24/40, the single-site substitution mutant P227S, has emerged with expanded activity that now includes advanced cephalosporins and the monobactam aztreonam. Using solution-state nuclear magnetic resonance (NMR) spectroscopy, we have compared the site-specific backbone dynamics of wild-type OXA-24/40 and the P227S variant. P227S changes local backbone flexibility in segments that are important for both binding and hydrolysis of carbapenem and cephalosporin substrates. Our results suggest that mutation-induced changes in sequence-specific dynamics can expand substrate activity and thus highlight the role of protein conformational dynamics in antibiotic resistance. To the best of our knowledge, this is the first NMR study of CHDL conformational dynamics and its impact on the expansion of ß-lactam antibiotic resistance.

Investigation of signal transduction routes within the sensor/transducer protein BlaR1 of Staphylococcus aureus.

The transmembrane antibiotic sensor/signal transducer protein BlaR1 is part of a cohort of proteins that confer ß-lactam antibiotic resistance in methicillin-resistant Staphylococcus aureus (MRSA) [Fisher, J. F., Meroueh, S. O., and Mobashery, S. (2005) Chem. Rev. 105, 395-424; Llarrull, L. I., Fisher, J. F., and Mobashery, S. (2009) Antimicrob. Agents Chemother. 53, 4051-4063; Llarrull, L. I., Toth, M., Champion, M. M., and Mobashery, S. (2011) J. Biol. Chem. 286, 38148-38158]. Specifically, BlaR1 regulates the inducible expression of ß-lactamases that hydrolytically destroy ß-lactam antibiotics. The resistance phenotype starts with ß-lactam antibiotic acylation of the BlaR1 extracellular domain (BlaRS). The acylation activates the cytoplasmic protease domain through an obscure signal transduction mechanism. Here, we compare protein dynamics of apo versus antibiotic-acylated BlaRS using nuclear magnetic resonance. Our analyses reveal inter-residue interactions that relay acylation-induced perturbations within the antibiotic-binding site to the transmembrane helix regions near the membrane surface. These are the first insights into the process of signal transduction by BlaR1.

Arginine Modulates Carbapenem Deactivation by OXA-24/40 in Acinetobacter baumannii.

The resistance of Gram-negative bacteria to ß-lactam antibiotics stems mainly from ß-lactamase proteins that hydrolytically deactivate the ß-lactams. Of particular concern are the ß-lactamases that can deactivate a class of ß-lactams known as carbapenems. Carbapenems are among the few anti-infectives that can treat multi-drug resistant bacterial infections. Revealing the mechanisms of their deactivation by ß-lactamases is a necessary step for preserving their therapeutic value. Here, we present NMR investigations of OXA-24/40, a carbapenem-hydrolyzing Class D ß-lactamase (CHDL) expressed in the gram-negative pathogen, Acinetobacter baumannii. Using rapid data acquisition methods, we were able to study the "real-time" deactivation of the carbapenem known as doripenem by OXA-24/40. Our results indicate that OXA-24/40 has two deactivation mechanisms: canonical hydrolytic cleavage, and a distinct mechanism that produces a ß-lactone product that has weak affinity for the OXA-24/40 active site. The mechanisms issue from distinct active site environments poised either for hydrolysis or ß-lactone formation. Mutagenesis reveals that R261, a conserved active site arginine, stabilizes the active site environment enabling ß-lactone formation. Our results have implications not only for OXA-24/40, but the larger family of CHDLs now challenging clinical settings on a global scale.