title: Optimized Nile Red efflux assay of AcrAB-TolC multidrug efflux system shows competition between substrates.
abstract: AcrAB-TolC is the major constitutively expressed efflux pump system that provides resistance to a variety of antimicrobial agents and dyes in Escherichia coli. However, no systematically optimized real-time dye efflux assay has been published for the measurement of its activity and for detection of possible competition between substrates. Here, we report on the development of such an assay using a lipophilic dye, Nile Red. Energy-depleted cells were loaded with the dye in the presence of low (10 microM or less) concentrations of the proton conductor carbonyl cyanide m-chlorophenylhydrazone (CCCP). The CCCP was then removed, and efflux was triggered by energization with glucose. Various known efflux pump inhibitors and antimicrobials were checked for the ability to slow down Nile Red efflux, presumably through competition. Besides the known inhibitors Phe-Arg-beta-naphthylamide and 1-naphthyl-methylpiperazine, several tetracyclic compounds (doxorubicin, minocycline, chlortetracycline, doxycycline, and tetracycline) and tetraphenylphosphonium chloride were found to interfere with dye efflux. This inhibition could not be explained by the depletion of proton motive force. None of the other tested antimicrobials, including macrolides, fluoroquinolones, and beta-lactams, had any impact on Nile Red efflux, even at concentrations of up to 1 mM.
authors.affiliation: University of California, Berkeley, Department of Molecular and Cell Biology, Berkeley, CA 94720-3202, USA.
authors.forename: Jürgen A
authors.initials: JA
authors.lastname: Bohnert
authors.forename: Brian
authors.initials: B
authors.lastname: Karamian
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nikaido
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002751:Chlortetracycline / Q000494:pharmacology; D004151:Dipeptides / Q000494:pharmacology; D004317:Doxorubicin / Q000494:pharmacology; D004318:Doxycycline / Q000494:pharmacology; D024901:Drug Resistance, Multiple, Bacterial; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D008911:Minocycline / Q000494:pharmacology; D009861:Onium Compounds / Q000494:pharmacology; D009943:Organophosphorus Compounds / Q000494:pharmacology; D013752:Tetracycline / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D004151:Dipeptides; D029968:Escherichia coli Proteins; D009861:Onium Compounds; D009943:Organophosphorus Compounds; C474925:phenylalanylarginine-naphthylamide; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D004317:Doxorubicin; D013752:Tetracycline; D008911:Minocycline; D004318:Doxycycline; C013289:tetraphenylphosphonium; D002751:Chlortetracycline