DEVELOPMENT OF A COUPLED VANA VANX ASSAY - SCREENING FOR INHIBITORS OF GLYCOPEPTIDE RESISTANCE/

Resistance in Enterococcus faecium to the glycopeptide antibiotics van comycin and teicoplanin is encoded by five genes: vanR, vanS, vanH, va nA, and vanX.(1) The mechanism of resistance involves replacement of t he dipeptide D-Ala-D-Ala, destined for the peptidoglycan layer with th e depsipeptide D-Ala-D-lactate. This alteration lowers the binding aff inity of vancomycin for the bacterial cell wall by a factor of 1000. T he 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 o verexpression of both genes as well as the D-Ala-D-Ala ligase (Ddl) fr om Enterococcus faecium, development of a coupled assay and several in hibitors obtained by high-throughput screening (HTS). All genes were e xpressed in E. coli by translational coupling to kdsB, the CMP-KDO syn thetase gene, under control of a modified lac promoter. The coupled Va nA/VanX assay employs colorimetric detection of inorganic phosphate (P -i) released in the VanA ligation reaction, with the VanX dipeptidase activity providing the D-Ala substrate for VanA. A secondary VanX assa y uses cadmium-ninhydrin colorimetric detection of free amino acid rel eased by the dipeptidase activity of the enzyme on D-Ala-D-Ala. We hav e also developed an assay using Ddl ligase. Over 250,000 compounds hav e been screened to date using the coupled assay.