Inactivation of the Glycoside Hydrolase NagZ Attenuates Antipseudomonal beta-Lactam Resistance in Pseudomonas aeruginosa
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Pseudomonas aeruginosa is a versatile Gram-negative opportunistic pathogen notorious for its ability to chronically colonize and deteriorate the pulmonary function of the cystic fibrosis lung. It exhibits high resistance to beta-lactam antibiotics, including cephalosporins and monobactams, via induction of their chromosomally encoded AmpC beta-lactamase. Regulation of ampC expression is coupled to the bacterial cell wall recycling pathway by the activity of NagZ, a glycosidase that produces 1,6-anhydroMurNAc-tri-(or penta-) peptides from internalized peptidoglycan metabolites. During beta-lactam therapy, this tripeptide rapidly concentrates in the bacterial cytosol to levels sufficient for it to bind and activate AmpR, the transcriptional activator of ampC. P. aeruginosa also encodes three ampD genes, each expressing an N-acetylmuramyl-L-amidase that cleaves the peptide stems from 1,6-anhydroMurNAc or GlcNAc 1,6-anhydroMurNAc. AmpD thus suppresses 1,6-anhydroMurNAc-peptide accumulation and moderates ampC induction. Selection of AmpD null mutants during therapy thus causes chronic hyperproduction of beta-lactamase, presumably from an increase in NagZ product, and have been identified in P. aeruginosa strains isolated from chronically infected CF patients. Mutants harboring an inactivated nagZ gene in a wild-type P. aeruginosa background were isolated and were found to have increased antibiotic susceptibility to antipseudomonal beta-lactams. Inactivating nagZ in a triple ampD mutant substantially decreased the expression of ampC and rendered these high-level resistant strains susceptible to antipseudomonal beta-lactams at wild-type strain levels. This brings the susceptibility of the P. aeruginosa strains down to the beta-lactam therapy range accepted by CLSI for use in cystic fibrosis patients suffering from chronic Pseudomonas aeruginosa infections. To assess whether P. aeruginosa expresses more than one N-acetyl-beta-glucosaminidase that could contribute to the production of the activating tripeptide, residual activity assays were conducted on nagZ deficient mutants. Mutants were devoid of activity so it was concluded that P. aeruginosa expresses only the one N-acetyl-beta-glucosaminidase in study, NagZ. Complementation studies using the wild type nagZ gene restored the wild type phenotypes, particularly evident in the triple ampD null mutants. These findings suggest that NagZ activity is required for ampC induction, and that an intricate balance exists between NagZ and AmpD activity to regulate the concentration of the inducer molecule 1,6-anhydroMurNAc-tripeptide.