Activity-Related Conformational Changes in d,d-Carboxypeptidases Revealed by In Vivo Periplasmic Förster Resonance Energy Transfer Assay in Escherichia coli.

Nils Y Meiresonne,Mark A Hink,Tanneke Den Blaauwen,René Van Der Ploeg

doi:10.1128/mbio.01089-17

Abstract

ABSTRACTOne of the mechanisms of β-lactam antibiotic resistance requires the activity of d,d-carboxypeptidases (d,d-CPases) involved in peptidoglycan (PG) synthesis, making them putative targets for new antibiotic development. The activity of PG-synthesizing enzymes is often correlated with their association with other proteins. The PG layer is maintained in the periplasm between the two membranes of the Gram-negative cell envelope. Because no methods existed to detect in vivo interactions in this compartment, we have developed and validated a Förster resonance energy transfer assay. Using the fluorescent-protein donor-acceptor pair mNeonGreen-mCherry, periplasmic protein interactions were detected in fixed and in living bacteria, in single samples or in plate reader 96-well format. We show that the d,d-CPases PBP5, PBP6a, and PBP6b of Escherichia coli change dimer conformation between resting and active states. Complementation studies and changes in localization suggest that these d,d-CPases are not redundant but that their balanced activity is required for robust PG synthesis.

Highlights

One of the mechanisms of ␤-lactam antibiotic resistance requires the activity of D,D-carboxypeptidases (D,D-CPases) involved in peptidoglycan (PG) synthesis, making them putative targets for new antibiotic development
Class C PBPs have PG-modifying activities and are further classified by their ability to hydrolyze the peptide bond between the peptide side chains and the PG glycan strands through endopeptidase (D,D-EPase) activity (PBP4, PBP4b, and PBP7) or carboxypeptidase (D,DCPase) activity (PBP5, PBP6a, and PBP6b) that cleaves off the pentapeptide terminal residue D-Ala
Because to our knowledge such an assay did not exist for the periplasm, we developed a Förster resonance energy transfer (FRET) assay that works in this compartment

Summary

Introduction

One of the mechanisms of ␤-lactam antibiotic resistance requires the activity of D,D-carboxypeptidases (D,D-CPases) involved in peptidoglycan (PG) synthesis, making them putative targets for new antibiotic development. KEYWORDS FRET, PBP5, PBP6a, PBP6b, antibiotics, mCherry, mNeonGreen, periplasm, protein interactions The periplasmic FRET assay was used to show the in vivo interaction dynamics of the D,D-CPases PBP5, PBP6a, and PBP6b.

Results

Conclusion