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

Abstract

ABSTRACTThis study aimed to characterize the role ofPseudomonas aeruginosalow-molecular-mass penicillin-binding proteins (LMM PBPs), namely, PBP4 (DacB), PBP5 (DacC), and PBP7 (PbpG), in peptidoglycan composition, β-lactam resistance, andampCregulation. For this purpose, we constructed all single and multiple mutants ofdacB,dacC,pbpG, andampCfrom the wild-typeP. aeruginosaPAO1 strain. Peptidoglycan composition was determined by high-performance liquid chromatography (HPLC),ampCexpression 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, onlydacCmutation led to significantly increased pentapeptide levels, showing that PBP5 is the majordd-carboxypeptidase inP. aeruginosa. Moreover, our results indicate that PBP4 and PBP7 play a significant role asdd-carboxypeptidase only if PBP5 is absent, and theirdd-endopeptidase activity is also inferred. As expected, the inactivation of PBP4 led to a significant increase inampCexpression (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 theampCexpression data. However, the inactivation ofampCin these mutants also evidenced a role of LMM PBPs, especially PBP5, in intrinsic β-lactam resistance. In summary, in addition to assessing the effect ofP. aeruginosaLMM 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, anddd-carboxypeptidase/β-lactamase activity.