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      Involvement of multiple influx and efflux transporters in the accumulation of cationic fluorescent dyes by Escherichia coli

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          Abstract

          Background

          It is widely believed that most xenobiotics cross biomembranes by diffusing through the phospholipid bilayer, and that the use of protein transporters is an occasional adjunct. According to an alternative view, phospholipid bilayer transport is negligible, and several different transporters may be involved in the uptake of an individual molecular type. We recognise here that the availability of gene knockout collections allows one to assess the contributions of all potential transporters, and flow cytometry based on fluorescence provides a convenient high-throughput assay for xenobiotic uptake in individual cells.

          Results

          We used high-throughput flow cytometry to assess the ability of individual gene knockout strains of E coli to take up two membrane-permeable, cationic fluorescent dyes, namely the carbocyanine diS-C3(5) and the DNA dye SYBR Green. Individual strains showed a large range of distributions of uptake. The range of modal steady-state uptakes for the carbocyanine between the different strains was 36-fold. Knockouts of the ATP synthase α- and β-subunits greatly inhibited uptake, implying that most uptake was ATP-driven rather than being driven by a membrane potential. Dozens of transporters changed the steady-state uptake of the dye by more than 50% with respect to that of the wild type, in either direction (increased or decreased); knockouts of known influx and efflux transporters behaved as expected, giving credence to the general strategy. Many of the knockouts with the most reduced uptake were transporter genes of unknown function (‘y-genes’). Similarly, several overexpression variants in the ‘ASKA’ collection had the anticipated, opposite effects. Similar results were obtained with SYBR Green (the range being approximately 69-fold). Although it too contains a benzothiazole motif there was negligible correlation between its uptake and that of the carbocyanine when compared across the various strains (although the membrane potential is presumably the same in each case).

          Conclusions

          Overall, we conclude that the uptake of these dyes may be catalysed by a great many transporters of putatively broad and presently unknown specificity, and that the very large range between the ‘lowest’ and the ‘highest’ levels of uptake, even in knockouts of just single genes, implies strongly that phospholipid bilayer transport is indeed negligible. This work also casts serious doubt upon the use of such dyes as quantitative stains for representing either bioenergetic parameters or the amount of cellular DNA in unfixed cells (in vivo) . By contrast, it opens up their potential use as transporter assay substrates in high-throughput screening.

          Electronic supplementary material

          The online version of this article (10.1186/s12866-019-1561-0) contains supplementary material, which is available to authorized users.

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          Most cited references 157

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          Multidrug-resistance efflux pumps - not just for resistance.

           Laura Piddock (2006)
          It is well established that multidrug-resistance efflux pumps encoded by bacteria can confer clinically relevant resistance to antibiotics. It is now understood that these efflux pumps also have a physiological role(s). They can confer resistance to natural substances produced by the host, including bile, hormones and host-defence molecules. In addition, some efflux pumps of the resistance nodulation division (RND) family have been shown to have a role in the colonization and the persistence of bacteria in the host. Here, I present the accumulating evidence that multidrug-resistance efflux pumps have roles in bacterial pathogenicity and propose that these pumps therefore have greater clinical relevance than is usually attributed to them.
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            Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacteria.

             Laura Piddock (2006)
            Efflux pump genes and proteins are present in both antibiotic-susceptible and antibiotic-resistant bacteria. Pumps may be specific for one substrate or may transport a range of structurally dissimilar compounds (including antibiotics of multiple classes); such pumps can be associated with multiple drug (antibiotic) resistance (MDR). However, the clinical relevance of efflux-mediated resistance is species, drug, and infection dependent. This review focuses on chromosomally encoded pumps in bacteria that cause infections in humans. Recent structural data provide valuable insights into the mechanisms of drug transport. MDR efflux pumps contribute to antibiotic resistance in bacteria in several ways: (i) inherent resistance to an entire class of agents, (ii) inherent resistance to specific agents, and (iii) resistance conferred by overexpression of an efflux pump. Enhanced efflux can be mediated by mutations in (i) the local repressor gene, (ii) a global regulatory gene, (iii) the promoter region of the transporter gene, or (iv) insertion elements upstream of the transporter gene. Some data suggest that resistance nodulation division systems are important in pathogenicity and/or survival in a particular ecological niche. Inhibitors of various efflux pump systems have been described; typically these are plant alkaloids, but as yet no product has been marketed.
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              Broad-specificity efflux pumps and their role in multidrug resistance of Gram-negative bacteria.

              Antibiotic resistance mechanisms reported in Gram-negative bacteria are causing a worldwide health problem. The continuous dissemination of 'multidrug-resistant' (MDR) bacteria drastically reduces the efficacy of our antibiotic 'arsenal' and consequently increases the frequency of therapeutic failure. In MDR bacteria, the overexpression of efflux pumps that expel structurally unrelated drugs contributes to the reduced susceptibility by decreasing the intracellular concentration of antibiotics. During the last decade, several clinical data have indicated an increasing involvement of efflux pumps in the emergence and dissemination of resistant Gram-negative bacteria. It is necessary to clearly define the molecular, functional and genetic bases of the efflux pump in order to understand the translocation of antibiotic molecules through the efflux transporter. The recent investigation on the efflux pump AcrB at its structural and physiological levels, including the identification of drug affinity sites and kinetic parameters for various antibiotics, may pave the way towards the rational development of an improved new generation of antibacterial agents as well as efflux inhibitors in order to efficiently combat efflux-based resistance mechanisms. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.
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                Author and article information

                Contributors
                srijan.jindal@postgrad.manchester.ac.uk
                leiya@biosustain.dtu.dk
                Philip.j.day@manchester.ac.uk
                dbk@liv.ac.uk , doukel@biosustain.dtu.dk
                Journal
                BMC Microbiol
                BMC Microbiol
                BMC Microbiology
                BioMed Central (London )
                1471-2180
                22 August 2019
                22 August 2019
                2019
                : 19
                Affiliations
                [1 ]ISNI 0000000121662407, GRID grid.5379.8, Department of Chemistry, , The University of Manchester, ; 131 Princess St, Manchester, M1 7DN UK
                [2 ]ISNI 0000000121662407, GRID grid.5379.8, Manchester Institute of Biotechnology, The University of Manchester, ; 131 Princess St, Manchester, M1 7DN UK
                [3 ]ISNI 0000000121662407, GRID grid.5379.8, Faculty of Biology, Medicine and Health, , The University of Manchester, ; Manchester, M13 9PT UK
                [4 ]ISNI 0000 0001 2181 8870, GRID grid.5170.3, Novo Nordisk Foundation Centre for Biosustainability, , Technical University of Denmark, ; Building 220, Kemitorvet, 2800 Kgs, Lyngby, Denmark
                [5 ]ISNI 0000 0004 1936 8470, GRID grid.10025.36, Department of Biochemistry, Institute of Integrative Biology, Faculty of Health and Life Sciences, , University of Liverpool, ; Crown St, Liverpool, L69 7ZB UK
                Article
                1561
                10.1186/s12866-019-1561-0
                6704527
                © The Author(s). 2019

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100000268, Biotechnology and Biological Sciences Research Council;
                Award ID: BB/R000093/1
                Award ID: BB/P009042/1
                Funded by: FundRef http://dx.doi.org/10.13039/501100009708, Novo Nordisk Fonden;
                Award ID: NNF10CC1016517
                Categories
                Research Article
                Custom metadata
                © The Author(s) 2019

                Microbiology & Virology

                membrane energisation, sybr green, carbocyanine, keio, drug transporters

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