Complexities of a protonatable substrate in measurements of Hoechst 33342 transport by multidrug transporter LmrP

www.nature.com/scientificreports OPEN Complexities of a protonatable substrate in measurements of Hoechst 33342 transport by multidrug transporter LmrP Brendan M. Swain1, Dawei Guo1, Himansha Singh1, Philip B. Rawlins2, Mark McAlister2 & Hendrik W. van Veen1* 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. Hoechst 33342 (referred to as Hoechst) is a bis-benzamide dye that is relatively non-toxic and cell-permeable, making it suitable for a wide range of applications in cell biology. As its fluorescence increases when bound to adenine–thymine-rich sequences in the minor groove of double-stranded DNA, Hoechst is commonly used to stain nuclei, track chromatin condensation, and monitor the cell cycle phase in eukaryotic c ells1–3. Hoechst fluorescence also increases when the dye intercalates between lipid molecules in biological m embranes4. Hoechst is a substrate of multidrug transporters, which translocate a wide range of structurally unrelated compounds from cells in a metabolic energy-dependent f ashion4–6 and reduce Hoechst fluorescence in the transport process. In various mammalian cell lines and tissues, Hoechst efflux by the ATP-binding cassette (ABC) multidrug transporters ABCB1 and ABCG2 shows the presence of a ‘side population’ with decreased Hoechst fl uorescence5,7. The interactions of bacterial multidrug transporters with Hoechst is also documented in a wide range of publications. For example, Hoechst was used in studies of drug efflux-based antibiotic resistance in Salmonella enterica serovar Typhimurium8,9 and Acinetobacter baumannii10,11, and in transport measurements for the ABC multidrug transporters LmrA from Lactococcus. lactis12–14, Sav1866 from Staphylococcus aureus15, MsbA and YbhFSR from E. coli16,17, and PatAB from Streptococcus pneumoniae18. Hoechst transport assays were used in studies of secondary-active multidrug and toxic compound extrusion (MATE) transporters VcmA from Vibrio cholerae19 and AbeM from A. baumannii20, and in studies of a novel natural product inhibitor of the major facilitator superfamily (MFS) multidrug transporter NorA from Staphylococcus aureus21. Hoechst transport has also been reported for the MFS multidrug transporter LmrP from L. lactis, which can efflux a wide range of clinically relevant antibiotics and cytotoxic compounds and Ca2+ from cells22,23. LmrP exports monovalent cationic ethidium and divalent cationic propidium by electrogenic exchange with 2 protons and 3 protons, respectively24,25. These transport reactions are dependent on the transmembrane chemical proton gradient (ΔpH, interior alkaline) and membrane potential (Δψ, interior negative) components of the 1 Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK. 2Structure, Biophysics and Fragment‑Based Lead Generation, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Cambridge CB4 0WG, UK. *email: Scientific Reports | (2020) 10:20026 | https://doi.org/10.1038/s41598-020-76943-0 1 Vol.:(0123456789) www.nature.com/scientificreports/ Figure 1.  Protonation states of Hoechst. (A) Structure of Hoechst 33342 in which the three nitrogen atoms with predicted changes in protonation in the physiological pH range are labelled. (B) Calculated distribution of Hoechst species at varying pH. For simplicity in presentation, species with a maximum prevalence of less than 1% are not shown. proton-motive force (PMF = Δψ − ZΔpH in which Z = 59 mV at 24 °C). Therefore, the coupling stoichiometry in LmrP is variable and dependent on the charge and physico-chemical properties of the substrate. This phenomenon reflects the different mechanisms of drug binding in the interior chamber of LmrP, some of which alter the availability of catalytic carboxylates for proton interactions24,25. Hoechst transport and binding have also been used to characterise drug interactions in LmrP6,23,26, to define steps in the transport cycle in structural analyses27 and, most recently, to trap the protein in a conformation that could be crystallised, thus allowing the determination of the three-dimensional protein s tructure28. In spite of the frequent use of Hoechst as a reporter for drug resistance and efflux, the quantitative interpretation of the transport-associated fluorescence change of Hoechst is unclear and complicated by two significant factors. Firstly, complex equilibria will exist between pools of Hoechst in the cytoplasm, bound to DNA and intercalated in biological membranes, in which the dye exhibits different levels of fluorescence emission. Secondly, three basic nitrogen atoms are present in Hoechst’s chemical structure. The protonation state of Hoechst, therefore, varies within the physiological range of pH inside and outside of cells, leading to pH-dependent fluorescence and interaction with macromolecular structures29. 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. To facilitate the quantitative analysis of Hoechst transport by LmrP and deconvolute the physical and environmental effects on Hoechst fluorescence, we investigated the fluorescence properties of dissolved, and lipid or DNA-bound Hoechst as a function of pH, and developed an assay for the direct determination of the amount of Hoechst associated with cells that is independent of its fluorescence in situ. We compared this assay to the conventional cell-based fluorescence (...truncated)