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

Abstract

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. Pseudomonas aeruginosa is a frequent cause of nosocomial infections, especially affecting patients in intensive care units (ICUs) with mechanical ventilation-associated pneumonia or burn wound infections, both of which are associated with a high mortality rate (1). This pathogen is also the major cause of chronic respiratory infections in patients with cystic fibrosis and other underlying chronic respiratory diseases (2). One of the most striking features of P. aeruginosa is its extraordinary capacity for developing resistance to almost any available antibiotic by the selection of mutations in chromosomal genes (3). Among the mutationmediated ␤-lactam resistance mechanisms, particularly noteworthy are those leading to the constitutive overexpression of the inducible chromosomal cephalosporinase AmpC, which confers resistance to penicillins, cephalosporins, and monobactams (4). Additionally, mutations that lead to the repression or inactivation of the porin OprD, acting synergistically with inducible or cons...

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

“…However, its role as a regulator of carboxypeptidases in P. aeruginosa is yet to be investigated. An additional increase of pentapeptides is also seen upon deletion of dacB and pbpG, confirming the presence of multiple carboxypeptidases [125]. PBP4/DacB and PBP7/PbpG also act as endopeptidases [122,124,125].…”

“…aeruginosa has three LMM PBPs (PBP4, PBP5 and PBP7) whose role in b-lactam resistance has been investigated [125,216]. Clinical mutants of PBP4 (PA3047) are associated with increased b-lactam resistance [216].…”

“…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].…”

Deciphering the Resistome of the Widespread Pseudomonas aeruginosa Sequence Type 175 International High-Risk Clone through Whole-Genome Sequencing

“…The effect of this mutation still needs to be experimentally addressed, but it is located in a highly conserved residue and is documented in the isolate showing the highest resistance overall to cephalosporins, penicillins, and monobactams ( Table 2). The effect, if any, of a unique mutation in PBP5 documented in one of the isolates is uncertain (Table 2), although PBP5 has been shown to contribute to beta-lactam resistance to some extent through its role in AmpC induction, ␤-lactam trapping, and/or ␤-lactamase activity (44,45). Involvement of efflux pumps.…”