Abstract
Multidrug transporters can confer drug resistance on cells by extruding structurally unrelated compounds from the cellular interior. In transport assays, Hoechst 33342 (referred to as Hoechst) is a commonly used substrate, the fluorescence of which changes in the transport process. With three basic nitrogen atoms that can be protonated, Hoechst can exist as cationic and neutral species that have different fluorescence emissions and different abilities to diffuse across cell envelopes and interact with lipids and intracellular nucleic acids. Due to this complexity, the mechanism of Hoechst transport by multidrug transporters is poorly characterised. We investigated Hoechst transport by the bacterial major facilitator superfamily multidrug-proton antiporter LmrP in Lactococcus lactis and developed a novel assay for the direct quantitation of cell-associated Hoechst. We observe that changes in Hoechst fluorescence in cells do not always correlate with changes in the amount of Hoechst. Our data indicate that chemical proton gradient-dependent efflux by LmrP in cells converts populations of highly fluorescent, membrane-intercalated Hoechst in the alkaline interior into populations of less fluorescent, cell surface-bound Hoechst in the acidic exterior. Our methods and findings are directly relevant for the transport of many amphiphilic antibiotics, antineoplastic agents and cytotoxic compounds that are differentially protonated within the physiological pH range.
Highlights
Multidrug transporters can confer drug resistance on cells by extruding structurally unrelated compounds from the cellular interior
Hoechst is a substrate of multidrug transporters, which translocate a wide range of structurally unrelated compounds from cells in a metabolic energy-dependent fashion[4,5,6] and reduce Hoechst fluorescence in the transport process
Hoechst was used in studies of drug efflux-based antibiotic resistance in Salmonella enterica serovar Typhimurium[8,9] and Acinetobacter baumannii[10,11], and in transport measurements for the ATP-binding cassette (ABC) multidrug transporters LmrA from Lactococcus. lactis[12,13,14], Sav1866 from Staphylococcus aureus[15], MsbA and YbhFSR from E. coli[16,17], and PatAB from Streptococcus pneumoniae[18]
Summary
Multidrug transporters can confer drug resistance on cells by extruding structurally unrelated compounds from the cellular interior. With three basic nitrogen atoms that can be protonated, Hoechst can exist as cationic and neutral species that have different fluorescence emissions and different abilities to diffuse across cell envelopes and interact with lipids and intracellular nucleic acids. Due to this complexity, the mechanism of Hoechst transport by multidrug transporters is poorly characterised. Hoechst is a substrate of multidrug transporters, which translocate a wide range of structurally unrelated compounds from cells in a metabolic energy-dependent fashion[4,5,6] and reduce Hoechst fluorescence in the transport process. Measurements of Hoechst transport are further complicated for LmrP and other proton-coupled multidrug transporters, where the pH difference across the plasma membrane provides a driving force for Hoechst extrusion by drug/proton antiport
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