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Changes to its peptidoglycan-remodeling enzyme repertoire modulate ?-lactam resistance in Pseudomonas aeruginosa.

ABSTRACT: Pseudomonas aeruginosa is a leading cause of hospital-acquired infections and is resistant to many antibiotics. Among its primary mechanisms of resistance is expression of a chromosomally encoded AmpC ?-lactamase that inactivates ?-lactams. The mechanisms leading to AmpC expression in P. aeruginosa remain incompletely understood but are intricately linked to cell wall metabolism. To better understand the roles of peptidoglycan-active enzymes in AmpC expression-and consequent ?-lactam resistance-a phenotypic screen of P. aeruginosa mutants lacking such enzymes was performed. Mutants lacking one of four lytic transglycosylases (LTs) or the nonessential penicillin-binding protein PBP4 (dacB) had altered ?-lactam resistance. mltF and slt mutants with reduced ?-lactam resistance were designated WIMPs (wall-impaired mutant phenotypes), while highly resistant dacB, sltB1, and mltB mutants were designated HARMs (high-level AmpC resistant mutants). Double mutants lacking dacB and sltB1 had extreme piperacillin resistance (>256 ?g/ml) compared to either of the single knockouts (64 ?g/ml for a dacB mutant and 12 ?g/ml for an sltB1 mutant). Inactivation of ampC reverted these mutants to wild-type susceptibility, confirming that AmpC expression underlies resistance. dacB mutants had constitutively elevated AmpC expression, but the LT mutants had wild-type levels of AmpC in the absence of antibiotic exposure. These data suggest that there are at least two different pathways leading to AmpC expression in P. aeruginosa and that their simultaneous activation leads to extreme ?-lactam resistance.

SUBMITTER: Cavallari JF

PROVIDER: S-EPMC3697359 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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Changes to its peptidoglycan-remodeling enzyme repertoire modulate β-lactam resistance in Pseudomonas aeruginosa.

Cavallari Joseph F JF Lamers Ryan P RP Scheurwater Edie M EM Matos Andrea L AL Burrows Lori L LL

Antimicrobial agents and chemotherapy 20130422 7

Pseudomonas aeruginosa is a leading cause of hospital-acquired infections and is resistant to many antibiotics. Among its primary mechanisms of resistance is expression of a chromosomally encoded AmpC β-lactamase that inactivates β-lactams. The mechanisms leading to AmpC expression in P. aeruginosa remain incompletely understood but are intricately linked to cell wall metabolism. To better understand the roles of peptidoglycan-active enzymes in AmpC expression-and consequent β-lactam resistance- ...[more]

PMID: 23612194

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Project description:This study aimed to characterize the role of Pseudomonas aeruginosa low-molecular-mass penicillin-binding proteins (LMM PBPs), namely, PBP4 (DacB), PBP5 (DacC), and PBP7 (PbpG), in peptidoglycan composition, ?-lactam resistance, and ampC regulation. For this purpose, we constructed all single and multiple mutants of dacB, dacC, pbpG, and ampC from the wild-type P. aeruginosa PAO1 strain. Peptidoglycan composition was determined by high-performance liquid chromatography (HPLC), ampC expression by reverse transcription-PCR (RT-PCR), PBP patterns by a Bocillin FL-binding test, and antimicrobial susceptibility by MIC testing for a panel of ?-lactams. Microscopy and growth rate analyses revealed no apparent major morphological changes for any of the mutants compared to the wild-type PAO1 strain. Of the single mutants, only dacC mutation led to significantly increased pentapeptide levels, showing that PBP5 is the major dd-carboxypeptidase in P. aeruginosa. Moreover, our results indicate that PBP4 and PBP7 play a significant role as dd-carboxypeptidase only if PBP5 is absent, and their dd-endopeptidase activity is also inferred. As expected, the inactivation of PBP4 led to a significant increase in ampC expression (around 50-fold), but, remarkably, the sequential inactivation of the three LMM PBPs produced a much greater increase (1,000-fold), which correlated with peptidoglycan pentapeptide levels. Finally, the ?-lactam susceptibility profiles of the LMM PBP mutants correlated well with the ampC expression data. However, the inactivation of ampC in these mutants also evidenced a role of LMM PBPs, especially PBP5, in intrinsic ?-lactam resistance. In summary, in addition to assessing the effect of P. aeruginosa LMM PBPs on peptidoglycan structure for the first time, we obtained results that represent a step forward in understanding the impact of these PBPs on ?-lactam resistance, apparently driven by the interplay between their roles in AmpC induction, ?-lactam trapping, and dd-carboxypeptidase/?-lactamase activity.

Dataset Information

Changes to its peptidoglycan-remodeling enzyme repertoire modulate ?-lactam resistance in Pseudomonas aeruginosa.

Publications

Changes to its peptidoglycan-remodeling enzyme repertoire modulate β-lactam resistance in Pseudomonas aeruginosa.

Similar Datasets

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