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

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.