Unravelling the triad of penicillin-binding proteins, β-lactamase activity, and mRNA dynamics in Pseudomonas aeruginosa AmpC induction

Abstract

Objectives: The main objective of the present work was to dissect the interplay between penicillin-binding protein (PBP) occupancy, ampC transcriptional dynamics, and β-lactamase activity in Pseudomonas aeruginosa. Methods: Using wild-type PAO1 and isogenic LMW-PBP knockout mutants (PAOΔdacB, PAOΔdacBdacC, and PAOΔdacBdacCpbpG), we assessed ampC induction following exposure to 18 β-lactams and β-lactamase inhibitors. PBP binding (IC50) was quantified via Bocillin-FL labelling. AmpC mRNA levels were measured by qRT-PCR, and β-lactamase activity was determined in crude extracts, periplasmic and extracellular fractions using nitrocefin and cefalotin substrates. Results: Carbapenems and cefoxitin, the drugs that maximally inhibited PBP4, induced strong ampC transcription and β-lactamase activity, while ceftazidime, aztreonam, and penicillins (PBP3-binding) triggered mRNA upregulation without enzymatic output. Furthermore, LMW-PBP-deficient mutants showed stepwise ampC overexpression (up to ∼7000-fold), but activity increases plateaued after ∼1100-fold, even after a significant mRNA induction following incubation with ceftazidime. Conclusions: These findings highlight that ampC transcription does not predict functional enzyme levels, especially for PBP3-binding drugs. Full induction appears to require PBP4 targeting and CreBC activation. These findings underscore the need for integrated assessment of PBP IC50, transcription, and enzymatic activity to guide rational β-lactam and β-lactamase inhibitor therapy, and lay the groundwork for future studies on alternative ampC regulatory pathways.