Understanding the structure and function of proteins involved in the inducible expression of AmpC β-lactamase
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Date
2013, 2015, 2016, 2017
Authors
Vadlamani, Grishma
Journal Title
Journal ISSN
Volume Title
Publisher
Journal of Biological Chemistry
Chemical Communications
Protein Science
Chemical Communications
Protein Science
Abstract
The inducible expression of AmpC β-lactamase is a major cause of β-lactam antibiotic resistance in several clinically relevant Gram-negative bacteria, including the opportunistic pathogen Pseudomonas aeruginosa. AmpC induction is regulated by the transcriptional regulator AmpR, which binds to the divergent ampR-ampC operon and is activated by 1,6-anhydromuramoyl-peptide – an anabolite of peptidoglycan (PG) recycling that is generated by the N-acetyl--glucosaminidase NagZ. To investigate the molecular basis of ampC induction, the archetypal AmpR protein Citrobacter freundii (CfAmpR) was structurally and biophysically characterized. CfAmpR forms a homotetramer that is stabilized by binding the intergenic region of the ampR-ampC operon, and it interacts with up to four repressor ligands (UDP-MurNAc-pentapeptide) in an apparent stepwise manner. Moreover, protocols were developed to measure in vitro gene expression from the ampR-ampC operon by CfAmpR using either radiolabelled mRNA synthesis, or qPCR. Since NagZ generates the AmpR activator ligand, blocking its activity enhances β-lactam efficacy against bacteria with inducible AmpC systems. Crystal structures of NagZ from Burkholderia cenocepacia were determined in complex with the glycosidase inhibitor O-(2-acetamido-2- deoxy-D-glucopyranosylidene)-amino-N-phenylcarbamate (PUGNAc) and its NagZ-selective derivative ethylbutyryl-PUGNAc, 3-acetamido-4,5,6-trihydroxyazepane (MM-124) and its NagZ-selective derivative MM-156, showing that plasticity within the NagZ active site could be exploited to improve the design of inhibitors that selectively bind NagZ over functionally related human N-acetyl--glucosaminidases from glycoside hydrolase family 20 (GH20). An improved understanding of the catalytic mechanism of GH20 enzyme function was also explored by determining the crystal structure of a GH20 N-acetyl--hexosaminidase from Streptomyces plicatus in complex with inhibitors from the N-acetyl glycal class, namely N-acetyl-D-glucal and N-thioacetyl-D-glucal. Furthering our understanding of the role of NagZ inhibition on -lactam resistance in P. aeruginosa, it was found that the NagZ inhibitor PUGNAc could prevent the emergence of high-level AmpC-mediated β-lactam resistance, and significantly enhanced β-lactam susceptibility in synergy with the potent β-lactamase inhibitor avibactam in an ampC derepressed P. aeruginosa strain. Finally, the potency of avibactam was also structurally defined in a recently discovered class A serine carbapenemase from Vibrio cholerae. Collectively, this research explores a number of small molecule based strategies to potentiate β-lactam efficacy against Gram-negative bacterial pathogens.
Description
Keywords
AmpC, Beta-lactamase