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Antimicrobial Agents and Chemotherapy, June 2009, p. 2274-2282, Vol. 53, No. 6
0066-4804/09/$08.00+0 doi:10.1128/AAC.01617-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Inactivation of the Glycoside Hydrolase NagZ Attenuates Antipseudomonal β-Lactam Resistance in Pseudomonas aeruginosa
Azizah Asgarali,1
Keith A. Stubbs,2,
Antonio Oliver,3
David J. Vocadlo,2 and
Brian L. Mark1*
Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2,1 Department of Chemistry, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia, Canada V5A 1S6,2 Servicio de Microbiología, Hospital Son Dureta, Palma de Mallorca, Spain3
Received 5 December 2008/ Returned for modification 25 January 2009/ Accepted 1 March 2009
The overproduction of chromosomal AmpC β-lactamase poses a serious challenge to the successful treatment of Pseudomonas aeruginosa infections with β-lactam antibiotics. The induction of ampC expression by β-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-β-D-glucosaminidase encoded by nagZ (PA3005). In the absence of β-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 β-lactams due to the accumulation of the ampC inducer anhydromuropeptides. To examine the role of NagZ on AmpC-mediated β-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 β-lactam resistance (up to 4-fold) and the high antipseudomonal β-lactam resistance (up to 16-fold) that is associated with the loss of AmpD activity. We also demonstrate that AmpC-mediated resistance to antipseudomonal β-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 β-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 β-lactam therapy.
* Corresponding author. Mailing address: Department of Microbiology, University of Manitoba, 418 Buller Building, Winnipeg, Manitoba, Canada R3T 2N2. Phone: (204) 480-1430. Fax: (204) 474-7603. E-mail: brian_mark{at}umanitoba.ca
Published ahead of print on 9 March 2009.
Present address: Chemistry M313, School of Biomedical, Biomolecular and Chemical Sciences, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia.
Antimicrobial Agents and Chemotherapy, June 2009, p. 2274-2282, Vol. 53, No. 6
0066-4804/09/$08.00+0 doi:10.1128/AAC.01617-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
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