title: A liposomal method for evaluation of inhibitors of H(+)-coupled multidrug transporters.
abstract: BACKGROUND
  This paper describes a novel technique, Fluorosomes, applied to investigating the interaction of antimicrobials with proton driven microbial efflux transporters. These transporters remove toxic compounds from the cytoplasm, including antibiotics and are involved in antibiotic resistance.
  
  
  METHODS
  To assess transporter activity we developed a methodology to generate a proton gradient across Fluorosome membranes into which selected purified fully active efflux transporters were reconstituted. The interior of the Fluorosome particle (a unilamellar liposome) contains a fluorescent drug sensing probe whose fluorescence is quantitatively quenched by transporter substrates. Using an injecting fluorescence plate reader to initiate a proton gradient and to monitor subsequent fluorescence change, real time transport kinetics can be followed and transport inhibition characterized.
  
  
  RESULTS
  Fluorosomes containing the Escherichia coli EmrE efflux pump demonstrated transport of two known EmrE substrates, ethidium and methyl viologen upon creation of a proton gradient. For Fluorosomes containing the inactive EmrE mutant, E14Q, no transport was observed. When the gradient was fully collapsed by the addition of nigericin, full inhibition of substrate transport was observed. The IC50 for nigericin inhibition of ethidium was shown to be 0.71 μM.
  
  
  CONCLUSIONS
  We have for the first time prepared and validated a single bacterial efflux pump assay, Fluorosome-trans-EmrE, that faithfully mimics properties of the transporter in vivo. It is faster than whole cell screens, simple to use, amenable to robotics, and reports on very specific targets. We have demonstrated proof of principle with EmrE and have created the first of an intended series of proton driven Fluorosomes.
authors.affiliation: GLSynthesis Inc., One Innovation Drive, Worcester, MA 01605, USA. Electronic address: donald.melchior@glsynthesis.com.
authors.forename: Donald L
authors.initials: DL
authors.lastname: Melchior
authors.affiliation: Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Edmond J. Safra Campus, Hebrew University, Jerusalem 91904, Israel.
authors.forename: Shlomo
authors.initials: S
authors.lastname: Brill
authors.affiliation: GLSynthesis Inc., One Innovation Drive, Worcester, MA 01605, USA.
authors.forename: George E
authors.initials: GE
authors.lastname: Wright
authors.affiliation: Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Edmond J. Safra Campus, Hebrew University, Jerusalem 91904, Israel.
authors.forename: Shimon
authors.initials: S
authors.lastname: Schuldiner
mesh_terms: D000890:Anti-Infective Agents / Q000494:pharmacology*; D001692:Biological Transport / Q000187:drug effects*; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism; D004996:Ethidium / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D008081:Liposomes / Q000494:pharmacology*; D026901:Membrane Transport Proteins / Q000819:agonists / Q000378:metabolism*; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011522:Protons
chemical_list: D000890:Anti-Infective Agents; D004996:Ethidium; D005456:Fluorescent Dyes; D008081:Liposomes; D026901:Membrane Transport Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011522:Protons
keywords: Bacterial efflux pumps; EmrE; Fluorescence; Fluorosomes; Liposomes; Membrane reconstitution; Methods; Model mimetic systems; Proton driven drug transport; Transport inhibitors