Asgarali Azizah, Stubbs Keith A, Oliver Antonio, Vocadlo David J, Mark Brian L
Department of Microbiology, University of Manitoba, 418 Buller Building, Winnipeg, Manitoba, Canada R3T 2N2.
Antimicrob Agents Chemother. 2009 Jun;53(6):2274-82. doi: 10.1128/AAC.01617-08. Epub 2009 Mar 9.
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.
染色体AmpCβ-内酰胺酶的过量产生对使用β-内酰胺类抗生素成功治疗铜绿假单胞菌感染构成了严峻挑战。β-内酰胺类药物对ampC表达的诱导是由肽聚糖（PG）循环的破坏以及胞质1,6-脱水-N-乙酰胞壁酰肽的积累介导的，PG循环的这些分解代谢产物是由nagZ（PA3005）编码的N-乙酰-β-D-氨基葡萄糖苷酶产生的。在没有β-内酰胺类药物的情况下，ampC的表达受到ampD、ampDh2和ampDh3编码的三种AmpD酰胺酶的抑制，这些酶的作用是降解这些1,6-脱水-N-乙酰胞壁酰肽诱导分子。ampD基因的失活导致ampC表达的逐步上调以及由于ampC诱导剂无水肽聚糖的积累而产生的对抗假单胞菌β-内酰胺类药物的临床耐药性。为了研究NagZ在铜绿假单胞菌中AmpC介导的β-内酰胺耐药性中的作用，我们在铜绿假单胞菌PAO1和同基因三重ampD缺失突变体中使nagZ失活。我们发现，nagZ的失活既抑制了内在的β-内酰胺耐药性（高达4倍），也抑制了与AmpD活性丧失相关的高抗假单胞菌β-内酰胺耐药性（高达16倍）。我们还证明，使用NagZ的选择性小分子抑制剂，可以在PAO1和一系列ampD缺失突变体中减弱AmpC介导对抗假单胞菌β-内酰胺类药物的耐药性。我们的结果表明，阻断NagZ的活性可能提供一种策略，以提高β-内酰胺类药物对铜绿假单胞菌和其他编码可诱导染色体ampC的革兰氏阴性菌的疗效，并抵消在β-内酰胺治疗期间因ampD缺失突变的选择而发生的ampC过度诱导。
