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, β-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 β-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 β-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 β-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 β-lactam resistance, apparently driven by the interplay between their roles in AmpC induction, β-lactam trapping, and dd-carboxypeptidase/β-lactamase activity.

FIG 1

Bocillin FL binding test of PAO1 wild-type and derived mutants. (A) Conventional cell membrane preparation protocol. (B) Modified protocol to avoid AmpC contamination of cell membrane preparations leading to Bocillin FL hydrolysis. The PBP pattern (at left) of all the constructed P. aeruginosa mutants and the wild-type PAO1 (lanes 1 to 18) were visualized by fluorescence scanning using the Typhoon 9410 variable-mode imager at 588 nm, with a 520 BP 40 emission filter, after an SDS-PAGE run of the reaction samples in 8% acrylamide gels, in which each reaction involved an incubation of 100 μg of cell membrane protein with 10 μM Bocillin FL at 37°C for 30 min. Lanes 1 and 10, wild-type PAO1; lane 2, PAO ΔdacB; lane 3, PAO ΔdacC; lane 4, PAO ΔpbpG; lane 5, PAO ΔdacB ΔdacC; lane 6, PAO ΔdacB ΔpbpG; lane 7, PAO ΔdacC ΔpbpG; lane 8, PAO ΔdacB ΔdacC ΔpbpG; lane 9, PAO ΔampC; lane 11, PAO ΔdacB ΔampC; lane 12, PAO ΔdacC ΔampC; lane 13, PAO ΔpbpG ΔampC; lane 14, PAO ΔdacB ΔdacC ΔampC; lane 15, PAO ΔdacB ΔpbpG ΔampC; lane 16, PAO ΔdacC ΔpbpG ΔampC; lane 17, PAO ΔdacB ΔpbpG ΔampC ΔdacC; and lane 18, PAO ΔdacB ΔdacC ΔpbpG ΔampC.

FIG 2

High-performance liquid chromatograms of peptidoglycan muropeptides of the wild-type and constructed PAO1 mutants. Each series displays peaks corresponding to the common muropeptides in peptidoglycan of the given PAO1 strain (indicated at right). Each peak corresponds to a muropeptide whose name (in bold) and retention time (RT) (in minutes) are indicated at the top. M3, disaccharide tripeptide; M4G, disaccharide tetrapeptide with Gly at position 4; M4, disaccharide tetrapeptide; M5, disaccharide pentapeptide in which l-Ala, d-Glu, Dap (meso-diaminopimelic acid), d-Ala, and d-Ala occupy positions 1, 2, 3, 4, and 5, respectively, and l-Ala is linked to N-acetylmuramic acid; M5G, disaccharide pentapeptide with Gly at position 5; D44, cross-linked dimer of disaccharide tetrapeptide-disaccharide tetrapeptide; D43, cross-linked dimer of disaccharide tetrapeptide-disaccharide tripeptide; D45, cross-linked dimer of disaccharide tetrapeptide-disaccharide pentapeptide; T444, cross-linked trimer of disaccharide tetrapeptide-disaccharide tetrapeptide-disaccharide tetrapeptide; T445, cross-linked trimer of disaccharide tetrapeptide-disaccharide tetrapeptide-disaccharide pentapeptide; anhydro-muropeptides D44N, D45N, T444N, and T445N have the same structures as muropeptides D44, D45, T444, and T445, respectively, but with anhydro-N-acetylmuramic acid instead of N-acetylmuramic acid. Each disaccharide is composed of N-acetylglucosamine and N-acetylmuramic acid.