title: 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.
authors.affiliation: Centro de Biología Molecular Severo Ochoa, Madrid, Spain.
authors.forename: Alaa
authors.initials: A
authors.lastname: Ropy
authors.affiliation: Servicio de Microbiología and Unidad de Investigación, Hospital Universitario Son Espases, Instituto de Investigación Sanitaria de Palma (IdISPa), Palma de Mallorca, Spain.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Cabot
authors.affiliation: Servicio de Microbiología and Unidad de Investigación, Hospital Universitario Son Espases, Instituto de Investigación Sanitaria de Palma (IdISPa), Palma de Mallorca, Spain.
authors.forename: Irina
authors.initials: I
authors.lastname: Sánchez-Diener
authors.affiliation: Centro de Biología Molecular Severo Ochoa, Madrid, Spain.
authors.forename: Cristian
authors.initials: C
authors.lastname: Aguilera
authors.affiliation: Servicio de Microbiología and Unidad de Investigación, Hospital Universitario Son Espases, Instituto de Investigación Sanitaria de Palma (IdISPa), Palma de Mallorca, Spain.
authors.forename: Bartolome
authors.initials: B
authors.lastname: Moya
authors.affiliation: Centro de Biología Molecular Severo Ochoa, Madrid, Spain jayala@cbm.csic.es antonio.oliver@ssib.es.
authors.forename: Juan A
authors.initials: JA
authors.lastname: Ayala
authors.affiliation: Servicio de Microbiología and Unidad de Investigación, Hospital Universitario Son Espases, Instituto de Investigación Sanitaria de Palma (IdISPa), Palma de Mallorca, Spain jayala@cbm.csic.es antonio.oliver@ssib.es.
authors.forename: Antonio
authors.initials: A
authors.lastname: Oliver
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics*; D001896:Boron Compounds / Q000378:metabolism; D002268:Carboxypeptidases / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D055786:Gene Knockout Techniques; D008826:Microbial Sensitivity Tests; D009154:Mutation / Q000235:genetics; D046915:Penicillin-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D010406:Penicillins / Q000378:metabolism; D010457:Peptidoglycan / Q000737:chemistry / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism*; D018440:beta-Lactam Resistance / Q000235:genetics*; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism; D047090:beta-Lactams / Q000494:pharmacology*
chemical_list: D000900:Anti-Bacterial Agents; C118961:BOCILLIN FL; D001426:Bacterial Proteins; D001896:Boron Compounds; D046915:Penicillin-Binding Proteins; D010406:Penicillins; D010457:Peptidoglycan; D047090:beta-Lactams; D002268:Carboxypeptidases; C048795:AmpC beta-lactamases; D001618:beta-Lactamases