Abstract
The increasing spread of plasmid-borne ampC-ampR operons is of considerable medical importance, since the AmpC beta-lactamases they encode confer high level resistance to many third generation cephalosporins. Induction of AmpC beta-lactamase from endogenous or plasmid-borne ampC-ampR operons is mediated by a catabolic inducer molecule, 1,6-anhydro-N-acetylmuramic acid (MurNAc) tripeptide, an intermediate of the cell wall recycling pathway derived from the peptidoglycan. Here we describe a strategy for attenuating the antibiotic resistance associated with the ampC-ampR operon by blocking the formation of the inducer molecule using small molecule inhibitors of NagZ, the glycoside hydrolase catalyzing the formation of this inducer molecule. The structure of the NagZ-inhibitor complex provides insight into the molecular basis for inhibition and enables the development of inhibitors with 100-fold selectivity for NagZ over functionally related human enzymes. These PUGNAc-derived inhibitors reduce the minimal inhibitory concentration (MIC) values for several clinically relevant cephalosporins in both wild-type and AmpC-hyperproducing strains lacking functional AmpD.
Highlights
Onto plasmids (4 – 8) as well as other transposable elements has greatly increased the prevalence of this type of resistance mechanism in Gram-negative organisms [9]
A major achievement in the field of antibiotic research has been the discovery of -lactamase inhibitors
There are currently three main -lactamase inhibitors available on the market, each having the same -lactam core found in -lactam antibiotics
Summary
Enzymes, Chemical Techniques, and Bacterial Strains—All media components were obtained from Bioshop. Crystals of VcNagZ in complex with PUGNAc were obtained by first mixing PUGNAc with concentrated VcNagZ at a 10:1 molar ratio of inhibitor to protein for 30 min at room temperature and subjecting the mixture to hanging drop vapor diffusion using 13% polyethylene glycol 20,000, and 100 mM BisTris, pH 6.1, as mother liquor. An aliquot of cells (4 ml) was used to inoculate the appropriate MuellerHinton broth medium mixture (4 ml) containing EtBuPUG (2 mM) (type a) or no inhibitor (type b). These mixtures were cultured for 60 min at 37 °C. Protein concentrations of the supernatant were quantified by Bradford assay using bovine serum albumin as a standard
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