Optimized Nile Red Efflux Assay of AcrAB-TolC Multidrug Efflux System Shows Competition between Substrates
- 1 September 2010
- journal article
- Published by American Society for Microbiology in Antimicrobial Agents and Chemotherapy
- Vol. 54 (9), 3770-3775
- https://doi.org/10.1128/aac.00620-10
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 μM 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-β-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 β-lactams, had any impact on Nile Red efflux, even at concentrations of up to 1 mM.This publication has 35 references indexed in Scilit:
- Kinetic Parameters of Efflux of Penicillins by the Multidrug Efflux Transporter AcrAB-TolC of Escherichia coliAntimicrobial Agents and Chemotherapy, 2010
- Mechanism of recognition of compounds of diverse structures by the multidrug efflux pump AcrB of Escherichia coliProceedings of the National Academy of Sciences of the United States of America, 2010
- Identification of Nile red as a fluorescent substrate of the Candida albicans ATP-binding cassette transporters Cdr1p and Cdr2p and the major facilitator superfamily transporter Mdr1pAnalytical Biochemistry, 2009
- A coordinated network of transporters with overlapping specificities provides a robust survival strategyProceedings of the National Academy of Sciences of the United States of America, 2009
- The assembled structure of a complete tripartite bacterial multidrug efflux pumpProceedings of the National Academy of Sciences of the United States of America, 2009
- Kinetic behavior of the major multidrug efflux pump AcrB of Escherichia coliProceedings of the National Academy of Sciences of the United States of America, 2009
- Site-Directed Mutagenesis Reveals Putative Substrate Binding Residues in the Escherichia coli RND Efflux Pump AcrBJournal of Bacteriology, 2008
- Substrate Competition Studies Using Whole-Cell Accumulation Assays with the Major Tripartite Multidrug Efflux Pumps of Escherichia coliAntimicrobial Agents and Chemotherapy, 2007
- Drug Export Pathway of Multidrug Exporter AcrB Revealed by DARPin InhibitorsPLoS Biology, 2006
- Structural Asymmetry of AcrB Trimer Suggests a Peristaltic Pump MechanismScience, 2006