Tryptophan Fluorescence Quenching in β-Lactam-Interacting Proteins Is Modulated by the Structure of Intermediates and Final Products of the Acylation Reaction

In most bacteria, β-lactam antibiotics inhibit the last cross-linking step of peptidoglycan synthesis by acylation of the active-site Ser of d,d-transpeptidases belonging to the penicillin-binding protein (PBP) family. In mycobacteria, cross-linking is mainly ensured by l,d-transpeptidases (LDTs), which are promising targets for the development of β-lactam-based therapies for multidrug-resistant tuberculosis. For this purpose, fluorescence spectroscopy is used to investigate the efficacy of LDT inactivation by β-lactams but the basis for fluorescence quenching during enzyme acylation remains unknown. In contrast to what has been reported for PBPs, we show here using a model l,d-transpeptidase (Ldt) that fluorescence quenching of Trp residues does not depend upon direct hydrophobic interaction between Trp residues and β-lactams. Rather, Trp fluorescence was quenched by the drug covalently bound to the active-site Cys residue of Ldt. Fluorescence quenching was not quantitatively determined by the size of the drug and was not specific of the thioester link connecting the β-lactam carbonyl to the catalytic Cys as quenching was also observed for acylation of the active-site Ser of β-lactamase BlaC from M. tuberculosis. Fluorescence quenching was extensive for reaction intermediates containing an amine anion and for acylenzymes containing an imine stabilized by mesomeric effect, but not for acylenzymes containing a protonated β-lactam nitrogen. Together, these results indicate that the extent of fluorescence quenching is determined by the status of the β-lactam nitrogen. Thus, fluorescence kinetics can provide information not only on the efficacy of enzyme inactivation but also on the structure of the covalent adducts responsible for enzyme inactivation

Copper inhibits peptidoglycan LD-transpeptidases suppressing β-lactam resistance due to bypass of penicillin-binding proteins

The peptidoglycan (PG) layer stabilizes the bacterial cell envelope to maintain the integrity and shape of the cell. Penicillin-binding proteins (PBPs) synthesize essential 4-3 cross-links in PG and are inhibited by \u3b2-lactam antibiotics. Some clinical isolates and laboratory strains of Enterococcus faecium and Escherichia coli achieve high-level \u3b2-lactam resistance by utilizing \u3b2-lactam-insensitive LD-transpeptidases (LDTs) to produce exclusively 3-3 cross-links in PG, bypassing the PBPs. In E. coli, other LDTs covalently attach the lipoprotein Lpp to PG to stabilize the envelope and maintain the permeability barrier function of the outermembrane. Here we show that subminimal inhibitory concentration of copper chloride sensitizes E. coli cells to sodium dodecyl sulfate and impair survival upon LPS transport stress, indicating reduced cell envelope robustness. Cells grown in the presence of copper chloride lacked 3-3 cross-links in PG and displayed reduced covalent attachment of Braun's lipoprotein and reduced incorporation of a fluorescent d-amino acid, suggesting inhibition of LDTs. Copper dramatically decreased the minimal inhibitory concentration of ampicillin in E. coli and E. faecium strains with a resistance mechanism relying on LDTs and inhibited purified LDTs at submillimolar concentrations. Hence, our work reveals how copper affects bacterial cell envelope stability and counteracts LDT-mediated \u3b2-lactam resistance