Smuggling small molecules probes into bacteria cell walls: a strategy for the selective fluorescent labelling of Vancomycin resistant strains (360G-Wellcome-206930_Z_17_Z)

Vancomycin inhibits bacterial cell wall synthesis by binding to the D-Ala-D-Ala motif integral to peptidoglycan synthesis. However, in a unique resistance mechanism the VanA operon reduces Vancomycin binding affinity for peptidoglycan by exchanging an amide bond for a depsipeptide ester bond; D-Ala-D-Ala into D-Ala-D-Lac. D-Ala is used by bacteria only in peptidoglycan synthesis, which has enabled fluorescent labelling of peptidoglycan cell walls in a range of bacteria, by feeding cells unnatural azides, or alkynes. The azide/or alkynes are incorporated into peptidoglycan and then tagged with fluorescent markers and subsequently imaged. Our strategy is to feed bacteria unnatural D-Lac mimics, as opposed to D-Ala, which should only be metabolically incorporated into peptidoglycan if the VanA operon is present. We will synthesise a range of simple small molecule probes, containing azide, alkynes or even whole fluorophores, in a D-Lac or depsipeptide scaffold and then screen enzymes from the VanA operon in vitro to determine their affinity for unnatural substrates. We will then use these substrates for metabolic feeding to Vancomycin resistant bacteria, using Van resistant Enterococci strains, before finally labelling the peptidoglycan on the cell surface and performing fluorescence miscroscopy imaging studies.