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.

FIG. 1.

Schematic of the PG recycling pathway and its role in AmpC β-lactamase induction. During growth, GlcNAc-1,6-anhydro-MurNAc tri-, tetra-, and pentapeptides (only the tripeptide species is shown) are excised from the PG and transported into the cytoplasm via the AmpG permease. The removal of GlcNAc by NagZ produces 1,6-anhydro-MurNAc peptide (boxed at the right), and either the tri- or pentapeptide species is believed to be responsible for the activation of AmpR to express ampC from the ampC-ampR operon. AmpD clears the muropeptide from the cytoplasm by removing the stem peptides from both GlcNAc-1,6-anhydro-MurNAc and 1,6-anhydro-MurNAc. These PG degradation products are eventually recycled into UDP-MurNAc pentapeptide (boxed at the left), a precursor molecule of PG synthesis and a repressor of AmpR. Exposure to β-lactams causes an increased cytosolic concentration of the 1,6-anhydro-MurNAc peptide that is sufficient to convert AmpR into an activator of ampC transcription.

FIG. 2.

NagZ activity assay of wild-type P. aeruginosa and deletion mutants. NagZ activity was determined from sonicated cultures by monitoring 4-MU liberation, as described in Materials and Methods. ○, PAO1; ▪, PAΔnagZ; ▵, PAΔDDh2Dh3; ⧫, PAΔDDh2Dh3nagZ. The level of 4-MU liberation from PAΔnagZ (▪) and PAΔDDh2Dh3nagZ (⧫) is the same as that from thermally denatured PAO1, PAΔnagZ, PAΔDDh2Dh3, and PAΔDDh2Dh3nagZ, confirming the absence of NagZ activity in these strains.

FIG. 3.

Structure of the NagZ selective inhibitor EtBuPUG. Designed to resemble the putative oxocarbenium ion-like transition state used by NagZ, EtBuPUG is a potent inhibitor of P. aeruginosa NagZ (K_i = 3.5 μM) and is highly selective for this enzyme and other CAZy family 3 β-glucosaminidases (42).