Understanding the structure and function of proteins involved in the inducible expression of AmpC β-lactamase
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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.