Structural Analysis of the Role of Pseudomonas aeruginosa Penicillin-Binding Protein 5 in β-Lactam Resistance

Abstract

b 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.

Role of Pseudomonas aeruginosa Low-Molecular-Mass Penicillin-Binding Proteins in AmpC Expression, β-Lactam Resistance, and Peptidoglycan Structure

“…The most highly expressed PBP in P. aeruginosa membranes has been documented to be PBP5 (40), consistent with the results of our work. It was recently shown that PBP5 is a DD-carboxypeptidase that preferentially degrades low-molecularweight substrates (28). In this work, we confirm that PBP5 is the major DD-carboxypeptidase in P. aeruginosa, as evidenced by the fact that of the three LMM PBP single mutants, only dacC mutation led to significantly increased pentapeptide levels.…”

“…Purified E. coli PBP5 failed to show significant ␤-lactamase activity, and therefore it was concluded that the role of this PBP in intrinsic ␤-resistance might be a consequence of ␤-lactam trapping. However, interestingly, the recently crystalized P. aeruginosa PBP5 does show certain broad-spectrum (including to penicillins, cephalosporins, and carbapenems) ␤-lactamase activity (28). Therefore, the observed effect of PBP5 in P. aeruginosa intrinsic resistance is expected to result from both trapping and hydrolysis of ␤-lactams.…”

“…Recent studies suggest that E. coli LMM PBPs, particularly PBP5, play a role in intrinsic ␤-lactam resistance (26,27). Moreover, the recently crystalized P. aeruginosa PBP5 shows certain ␤-lactamase activities, adding further interest to the role of LMM PBPs in antibiotic resistance (28).…”

Cell-wall recycling and synthesis in Escherichia coli and Pseudomonas aeruginosa – their role in the development of resistance

“…Loss of dacC results in increased pentapeptides establishing its role as the primary carboxypeptidase in P. aeruginosa [123,125,126]. P. aeruginosa encodes a homologue of BolA (PA0857), which shows a 46 % identity to their E. coli counterpart.…”

“…LMM PBPs in P. aeruginosa include PBP4/DacB (PA3047), PBP5/DacC (PA3999) and PBP7/PbpG (PA0869) [66,[122][123][124][125]. Loss of dacC results in increased pentapeptides establishing its role as the primary carboxypeptidase in P. aeruginosa [123,125,126].…”