Development of a Coupled VanA/VanX Assay: Screening for Inhibitors of Glycopeptide Resistance
Resistance in Enterococcus faecium to the glycopeptide antibiotics vancomycin and teicoplanin is encoded by five genes: vanR, vanS, vanH, vanA, and vanX.1 The mechanism of resistance involves replacement of the dipeptide D-Ala-D-Ala, destined for the peptidoglycan layer with the depsipeptide D-Ala-D-lactate. This alteration lowers the binding affinity of vancomycin for the bacterial cell wall by a factor of 1000. The functions of VanA and VanX are the ligation of D-Ala and D-lactate, and the hydrolysis of D-Ala-D-Ala, respectively. We report here the overexpression of both genes as well as the D-Ala-D-Ala ligase (Ddl) from Enterococcus faecium, development of a coupled assay and several inhibitors obtained by high-throughput screening (HTS). All genes were expressed in E. coli by translational coupling to kdsB, the CMP-KDO synthetase gene, under control of a modified lac promoter. The coupled VanA/VanX assay employs colorimetric detection of inorganic phosphate (Pi) released in the VanA ligation reaction, with the VanX dipeptidase activity providing the D-Ala substrate for VanA. A secondary VanX assay uses cadmium-ninhydrin calorimetric detection of free amino acid released by the dipeptidase activity of the enzyme on D-Ala-D-Ala. We have also developed an assay using Ddl ligase. Over 250,000 compounds have been screened to date using the coupled assay.