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Structural analysis of the role of Pseudomonas aeruginosa penicillin-binding protein 5 in ?-lactam resistance.

ABSTRACT: Penicillin-binding protein 5 (PBP5) is one of the most abundant PBPs in Pseudomonas aeruginosa. Although its main function is that of a cell wall dd-carboxypeptidase, it possesses sufficient ?-lactamase activity to contribute to the ability of P. aeruginosa to resist the antibiotic activity of the ?-lactams. The study of these dual activities is important for understanding the mechanisms of antibiotic resistance by P. aeruginosa, an important human pathogen, and to the understanding of the evolution of ?-lactamase activity from the PBP enzymes. We purified a soluble version of P. aeruginosa PBP5 (designated Pa sPBP5) by deletion of its C-terminal membrane anchor. Under in vitro conditions, Pa sPBP5 demonstrates both dd-carboxypeptidase and expanded-spectrum ?-lactamase activities. Its crystal structure at a 2.05-Å resolution shows features closely resembling those of the class A ?-lactamases, including a shortened loop spanning residues 74 to 78 near the active site and with respect to the conformations adopted by two active-site residues, Ser101 and Lys203. These features are absent in the related PBP5 of Escherichia coli. A comparison of the two Pa sPBP5 monomers in the asymmetric unit, together with molecular dynamics simulations, revealed an active-site flexibility that may explain its carbapenemase activity, a function that is absent in the E. coli PBP5 enzyme. Our functional and structural characterizations underscore the versatility of this PBP5 in contributing to the ?-lactam resistance of P. aeruginosa while highlighting how broader ?-lactamase activity may be encoded in the structural folds shared by the PBP and serine ?-lactamase classes.

SUBMITTER: Smith JD

PROVIDER: S-EPMC3697341 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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Structural analysis of the role of Pseudomonas aeruginosa penicillin-binding protein 5 in β-lactam resistance.

Smith Jeffrey D JD Kumarasiri Malika M Zhang Weilie W Hesek Dusan D Lee Mijoon M Toth Marta M Vakulenko Sergei S Fisher Jed F JF Mobashery Shahriar S Chen Yu Y

Antimicrobial agents and chemotherapy 20130429 7

Penicillin-binding protein 5 (PBP5) is one of the most abundant PBPs in Pseudomonas aeruginosa. Although its main function is that of a cell wall dd-carboxypeptidase, it possesses sufficient β-lactamase activity to contribute to the ability of P. aeruginosa to resist the antibiotic activity of the β-lactams. The study of these dual activities is important for understanding the mechanisms of antibiotic resistance by P. aeruginosa, an important human pathogen, and to the understanding of the evolu ...[more]

PMID: 23629710

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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

Structural analysis of the role of Pseudomonas aeruginosa penicillin-binding protein 5 in ?-lactam resistance.

Publications

Structural analysis of the role of Pseudomonas aeruginosa penicillin-binding protein 5 in β-lactam resistance.

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