title: Inactivation of the glycoside hydrolase NagZ attenuates antipseudomonal beta-lactam resistance in Pseudomonas aeruginosa.
abstract: The overproduction of chromosomal AmpC beta-lactamase poses a serious challenge to the successful treatment of Pseudomonas aeruginosa infections with beta-lactam antibiotics. The induction of ampC expression by beta-lactams is mediated by the disruption of peptidoglycan (PG) recycling and the accumulation of cytosolic 1,6-anhydro-N-acetylmuramyl peptides, catabolites of PG recycling that are generated by an N-acetyl-beta-D-glucosaminidase encoded by nagZ (PA3005). In the absence of beta-lactams, ampC expression is repressed by three AmpD amidases encoded by ampD, ampDh2, and ampDh3, which act to degrade these 1,6-anhydro-N-acetylmuramyl peptide inducer molecules. The inactivation of ampD genes results in the stepwise upregulation of ampC expression and clinical resistance to antipseudomonal beta-lactams due to the accumulation of the ampC inducer anhydromuropeptides. To examine the role of NagZ on AmpC-mediated beta-lactam resistance in P. aeruginosa, we inactivated nagZ in P. aeruginosa PAO1 and in an isogenic triple ampD null mutant. We show that the inactivation of nagZ represses both the intrinsic beta-lactam resistance (up to 4-fold) and the high antipseudomonal beta-lactam resistance (up to 16-fold) that is associated with the loss of AmpD activity. We also demonstrate that AmpC-mediated resistance to antipseudomonal beta-lactams can be attenuated in PAO1 and in a series of ampD null mutants using a selective small-molecule inhibitor of NagZ. Our results suggest that the blockage of NagZ activity could provide a strategy to enhance the efficacies of beta-lactams against P. aeruginosa and other gram-negative organisms that encode inducible chromosomal ampC and to counteract the hyperinduction of ampC that occurs from the selection of ampD null mutations during beta-lactam therapy.
authors.affiliation: Department of Microbiology, University of Manitoba, 418 Buller Building, Winnipeg, Manitoba, Canada R3T 2N2.
authors.forename: Azizah
authors.initials: A
authors.lastname: Asgarali
authors.forename: Keith A
authors.initials: KA
authors.lastname: Stubbs
authors.forename: Antonio
authors.initials: A
authors.lastname: Oliver
authors.forename: David J
authors.initials: DJ
authors.lastname: Vocadlo
authors.forename: Brian L
authors.initials: BL
authors.lastname: Mark
mesh_terms: D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D001426:Bacterial Proteins / Q000096:biosynthesis; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D008826:Microbial Sensitivity Tests; D010091:Oximes / Q000494:pharmacology; D048448:Phenylcarbamates / Q000494:pharmacology; D011550:Pseudomonas aeruginosa / Q000187:drug effects* / Q000201:enzymology; D018440:beta-Lactam Resistance; D001618:beta-Lactamases / Q000096:biosynthesis
chemical_list: D001426:Bacterial Proteins; D010091:Oximes; D048448:Phenylcarbamates; C068836:N-acetylglucosaminono-1,5-lactone O-(phenylcarbamoyl)oxime; D006026:Glycoside Hydrolases; C048795:AmpC beta-lactamases; D001618:beta-Lactamases; D000117:Acetylglucosamine