DOCSLIB.ORG

Kinetic Behavior of the Major Multidrug Efflux Pump Acrb of Escherichia Coli

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Kinetic Behavior of the Major Multidrug Efflux Pump Acrb of Escherichia Coli Kinetic behavior of the major multidrug efflux pump AcrB of Escherichia coli Keiji Nagano1 and Hiroshi Nikaido2 Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720 Communicated by Alexander N. Glazer, University of California System, Oakland, CA, February 13, 2009 (received for review November 26, 2008) Multidrug efflux transporters, especially those that belong to the With intact cells, it is difficult to determine the substrate resistance-nodulation-division (RND) family, often show very concentration in the periplasm, a location where most of the broad substrate specificity and play a major role both in the substrates are thought to be captured (1, 8–10). When ␤-lactams intrinsic antibiotic resistance and, with increased levels of expres- are used as substrates, however, their periplasmic concentrations sion, in the elevated resistance of Gram-negative bacteria. How- can be calculated from their hydrolysis rate by intact cells and ever, it has not been possible to determine the kinetic behavior of from the kinetic constants of periplasmic ␤-lactamases (11). these important pumps so far. This is partly because these pumps Using this approach, we determined the kinetic behavior of form a tripartite complex traversing both the cytoplasmic and several cephalosporins in AcrB-catalyzed efflux and indeed outer membranes, with an outer membrane channel and a obtained indications of positive cooperativity in the ligand– periplasmic adaptor protein, and it is uncertain if the behavior of pump interaction. an isolated component protein reflects that of the protein in this multiprotein complex. Here we use intact cells of Escherichia coli Results containing the intact multiprotein complex AcrB-AcrA-TolC, and Principle of the Assay. When a ␤-lactam is added to the external measure the kinetic constants for various cephalosporins, by as- medium at a given concentration Co (␮M), it diffuses sponta- sessing the periplasmic concentration of the drug from their rate of neously across the outer membrane into the periplasm (Fig. 1). ␤ hydrolysis by periplasmic -lactamase and the rate of efflux as the The influx Vin is given by Fick’s first law of diffusion difference between the influx rate and the hydrolysis rate. Nitro- ϭ ͑ Ϫ ͒ MICROBIOLOGY cefin efflux showed a Km of about 5 ␮M with little sign of Vin P * A * Co Cp cooperativity. For other compounds (cephalothin, cefamandole, and cephaloridine) that showed lower affinity to the pump, how- where P, A, and Cp denote the permeability coefficient (cm/s) of 2 ever, kinetics showed strong positive cooperativity, which is con- the outer membrane, the surface area of bacterial cells (cm /mg ␮ sistent with the rotating catalysis model of this trimeric pump. For dry weight), and the periplasmic drug concentration ( Mor 3 2 the very hydrophilic cefazolin there was little sign of efflux. nmol/cm ). We used 132 cm /mg (12) for A. We measure the rate of hydrolysis by the periplasmic ␤-lactamase (Vh) spectropho- cephalosporin ͉ drug efflux pump ͉ multidrug resistance tometrically with suspensions of intact cells. We then solve the Michaelis-Menten equation for hydrolysis for Cp, using the values of Km (determined earlier) and Vmax (determined by using ultidrug efflux pumps of the resistance-nodulation- cell extracts). Mdivision (RND) family in Gram-negative bacteria can With nitrocefin, P was determined by coupling influx with the pump out a surprisingly wide range of antimicrobial compounds enzymatic hydrolysis in periplasm (11), with the active efflux (1, 2). For example, AcrB of Escherichia coli, which has been inactivated by deenergization of the cytoplasmic membrane with studied most extensively as a prototype of similar pumps, exports a proton conductor, 40 ␮M carbonyl cyanide m-chlorophenyl- a number of dyes, detergents, chloramphenicol, tetracyclines, hydrazone (CCCP). When this was done in the presence of, e.g., macrolides, novobiocin, fluoroquinolones, and organic solvents. ␮ ␤ 25 M of nitrocefin, the rate of hydrolysis increased severalfold Importantly, it also pumps out -lactams, some of which cannot upon the addition of CCCP, giving us qualitative confirmation easily cross the cytoplasmic membrane and thus stay in the that proton-motive-force-dependent pump(s) were pumping out periplasm (3). However, in the 13 years since their discovery (4, much of the nitrocefin from the periplasm in nonpoisoned cells. 5), no success was achieved in the determination of kinetic The influx rate, Vin, can then be calculated from the values of behavior of these pumps, although the affinity of some substrates P, A, and C , as described above. In our model, the rate of efflux, could be assessed on a relative scale by their activity as com- p V , is the difference between the (calculated) V and the petitive inhibitors in a proteoliposome reconstitution assay (6). e in (measured) V (Fig. 1). Recently an attempt to measure the binding of dyes to purified h One possible complication is that some of the periplasmic AcrB was made by using fluorescence polarization (7); however, cephalosporin may cross the cytoplasmic membrane [V (dashed it is unclear if the experiments measured binding to active sites c arrow) in Fig. 1]. Such diffusion is known to occur with relatively or to generally hydrophobic pockets in the protein. lipophilic cephalosporins such as cephalothin (3, 13). The first- The knowledge of kinetic constants is essential in our attempts order rate constant for this influx into cytosol can be calculated to understand the contribution of the pumps to drug resistance (14) from the half-equilibration time determined earlier (13). in a quantitative manner. Furthermore, recent crystallographic Ϫ For cephalothin, this is 2.9 ϫ 10 9 cm3/s/mg cells. When C ϭ 10 studies of AcrB (8–10) suggest that each protomer in this p trimeric transporter undergoes a succession of conformational changes that are dependent on the conformations of the neigh- Author contributions: H.N. designed research; K.N. performed research; K.N. and H.N. bors, that is, a functionally rotating mechanism. This mechanism analyzed data; and H.N. wrote the paper. predicts that positive cooperativity may exist in the export of The authors declare no conflict of interest. drugs by AcrB. In this study we measured the kinetic constants 1Present address: Department of Microbiology, Aichi-gakuin University School of Dentistry, of AcrB. We used intact cells, because AcrB, which exists as a Nagoya 464-8650, Japan. tripartite complex with the periplasmic protein AcrA and the 2To whom correspondence should be addressed. E-mail: [email protected]. outer membrane channel TolC (1, 2), might be altered in its This article contains supporting information online at www.pnas.org/cgi/content/full/ behavior when it is separated from these physiological partners. 0901695106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0901695106 PNAS Early Edition ͉ 1of5 Downloaded by guest on September 30, 2021 Co 0.04 V 0.03 0.05 in V (mM) Cp Cp e 0.04 β- Tol C- Cp V lactamase , Cp h AcrA- OM e 0.03 V , V h h 0.02 Ve Cp AcrB V 0.02 0.01 Ve 0 04080120Co (nmol/mg/s); e , V IM h 0.01 V V c Vh Fig. 1. Principle of the assay. A ␤-lactam compound is added to the external medium bathing the intact cells of E. coli at a concentration, Co. At steady state, the drug crosses the outer membrane at the net rate Vin, which, 0 Ϫ 0 50 100 150 200 according to Fick’s first law of diffusion, is equal to P * A *(Co Cp) (see text). Co This influx is balanced by the sum of 3 processes: (i) hydrolysis by the periplas- (µM) ␤ mic -lactamase at the rate Vh,(ii) periplasmic capture followed by efflux into Fig. 2. Changes of various processes at different external concentrations of the external medium through AcrAB-TolC complex at the rate Ve, and (iii) slow nitrocefin. Results from one typical experiment with strain HN1157 are shown. diffusion across the cytoplasmic membrane into cytosol at the rate Vc. The Inset shows similar results from an experiment with HN1159 (a ⌬acrAB derivative of HN1157), where efflux is undetectable at least within the range of C shown. Ϫ5 p ␮M, Vc will then be 2.9 ϫ 10 nmol/s/mg. This is about 1,000 times smaller than the rates we are concerned with here, Ve, Vin, and Vh (see below), and we neglected Vc in our analysis. rate Vh and the efflux rate Ve, it was not a simple function of Co. In practice, however, this approach demands certain condi- Examination of Vh and Ve (obtained by subtracting Vh from Vin) ␤ tions. For example, the Km of the -lactamase must be reason- showed that the values of Ve had a shape suggesting some ably high, so that Vh becomes a sensitive indicator of Cp, in the saturation, but those of Vh did not in this concentration range of range of the concentrations we are interested in. Ideally, Ve, Vh, Cp, as expected from the large value (340 ␮M) of Km. Efflux and Vin should all be of the same order of magnitude so that high represented by Ve was dominant in the low concentration range precision can be attained in our calculation. The Vh values must for Co, but the enzymatic hydrolysis represented by Vh became be reasonably high to allow for precise measurement of hydrolysis. more important at high values of Co. As an important control, a similar analysis was carried out by Optimization of Assay Conditions for Nitrocefin. In our initial stud- using the isogenic strain HN1159, where the main efflux genes ␤ ies, we used nitrocefin (15) as the -lactam substrate, because the acrAB were absent. As expected, at least up to 40 ␮M Cp, the changes in absorption occur at 486 nm, where the light scattering efflux rate Ve was insignificant (Fig.
Load more

Recommended publications
  • Natural Products As Alternative Choices for P-Glycoprotein (P-Gp) Inhibition
    Review Natural Products as Alternative Choices for P-Glycoprotein (P-gp) Inhibition Saikat Dewanjee 1,*, Tarun K. Dua 1, Niloy Bhattacharjee 1, Anup Das 2, Moumita Gangopadhyay 3, Ritu Khanra 1, Swarnalata Joardar 1, Muhammad Riaz 4, Vincenzo De Feo 5,* and Muhammad Zia-Ul-Haq 6,* 1 Advanced Pharmacognosy Research Laboratory, Department of Pharmaceutical Technology, Jadavpur University, Raja S C Mullick Road, Kolkata 700032, India; [email protected] (T.K.D.); [email protected] (N.B.); [email protected] (R.K.); [email protected] (S.J.) 2 Department of Pharmaceutical Technology, ADAMAS University, Barasat, Kolkata 700126, India; [email protected] 3 Department of Bioechnology, ADAMAS University, Barasat, Kolkata 700126, India; [email protected] 4 Department of Pharmacy, Shaheed Benazir Bhutto University, Sheringal 18050, Pakistan; [email protected] 5 Department of Pharmacy, Salerno University, Fisciano 84084, Salerno, Italy 6 Environment Science Department, Lahore College for Women University, Jail Road, Lahore 54600, Pakistan * Correspondence: [email protected] (S.D.); [email protected] (V.D.F.); [email protected] (M.Z.-U.-H.) Academic Editor: Maria Emília de Sousa Received: 11 April 2017; Accepted: 15 May 2017; Published: 25 May 2017 Abstract: Multidrug resistance (MDR) is regarded as one of the bottlenecks of successful clinical treatment for numerous chemotherapeutic agents. Multiple key regulators are alleged to be responsible for MDR and making the treatment regimens ineffective. In this review, we discuss MDR in relation to P-glycoprotein (P-gp) and its down-regulation by natural bioactive molecules. P-gp, a unique ATP-dependent membrane transport protein, is one of those key regulators which are present in the lining of the colon, endothelial cells of the blood brain barrier (BBB), bile duct, adrenal gland, kidney tubules, small intestine, pancreatic ducts and in many other tissues like heart, lungs, spleen, skeletal muscles, etc.
    [Show full text]
  • Cell Volume Change Through Water Efflux Impacts Cell Stiffness And
    Cell volume change through water efflux impacts cell PNAS PLUS stiffness and stem cell fate Ming Guoa,1, Adrian F. Pegoraroa, Angelo Maoa,b, Enhua H. Zhouc,2, Praveen R. Aranyd,e, Yulong Hana,f, Dylan T. Burnetteg, Mikkel H. Jensena,h, Karen E. Kaszaa,i, Jeffrey R. Moorej, Frederick C. Mackintoshk,l,m, Jeffrey J. Fredbergc, David J. Mooneya,b, Jennifer Lippincott-Schwartzn,3, and David A. Weitza,o,3 aJohn A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138; bWyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, MA 02138; cHarvard T. H. Chan School of Public Health, Boston, MA 02115; dDepartment of Oral Biology, University at Buffalo, Buffalo, NY 14214; eDepartment of Biomedical Engineering, University at Buffalo, Buffalo, NY 14214; fBiomedical Engineering and Biomechanics Center, Xi’an Jiaotong University, Xi’an 710049, China; gDepartment of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232; hDepartment of Physics and Astronomy, California State University, Sacramento, CA 95819; iDepartment of Mechanical Engineering, Columbia University, New York, NY 10027; jDepartment of Biological Sciences, University of Massachusetts at Lowell, Lowell, MA 01854; kDepartment of Physics and Astronomy, VU University, 1081 HV Amsterdam, The Netherlands; lDepartment of Chemical and Biomolecular Engineering, Rice University, Houston, TX 77030; mCenter for Theoretical Biophysics, Rice University, Houston, TX 77030; nHoward Hughes Medical Institute, Janelia Research Campus, Ashburn, VA 20147; and oDepartment of Physics, Harvard University, Cambridge, MA 02138 Contributed by Jennifer Lippincott-Schwartz, August 26, 2017 (sent for review March 29, 2017; reviewed by Daniel A. Fletcher and Valerie M.
    [Show full text]
  • Involvement of Multiple Influx and Efflux Transporters in the Accumulation of Cationic Fluorescent Dyes by Escherichia Coli Srijan Jindal1,2,3, Lei Yang4, Philip J
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by University of Liverpool Repository Jindal et al. BMC Microbiology (2019) 19:195 https://doi.org/10.1186/s12866-019-1561-0 RESEARCH ARTICLE Open Access Involvement of multiple influx and efflux transporters in the accumulation of cationic fluorescent dyes by Escherichia coli Srijan Jindal1,2,3, Lei Yang4, Philip J. Day2,3 and Douglas B. Kell1,2,4,5* 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.
    [Show full text]
  • Blood-Brain Barrier Active Efflux Transporters
    NeuroRx௡: The Journal of the American Society for Experimental NeuroTherapeutics Blood-Brain Barrier Active Efflux Transporters: ATP-Binding Cassette Gene Family Wolfgang Lo¨scher and Heidrun Potschka Department of Pharmacology, Toxicology, and Pharmacy, University of Veterinary Medicine Hannover, Hannover D-30559, Germany Summary: The blood-brain barrier (BBB) contributes to brain efflux transporters in the BBB include minimizing or avoiding homeostasis by protecting the brain from potentially harmful neurotoxic adverse effects of drugs that otherwise would pen- endogenous and exogenous substances. BBB active drug efflux etrate into the brain. However, ABC efflux transporters may transporters of the ATP-binding cassette (ABC) gene family also limit the central distribution of drugs that are beneficial to are increasingly recognized as important determinants of drug treat CNS diseases. Furthermore, neurological disorders such distribution to, and elimination from, the CNS. The ABC efflux as epilepsy may be associated with overexpression of ABC transporter P-glycoprotein (Pgp) has been demonstrated as a efflux transporters at the BBB, resulting in pharmacoresistance key element of the BBB that can actively transport a huge to therapeutic medication. Therefore, modulation of ABC ef- variety of lipophilic drugs out of the brain capillary endothelial flux transporters at the BBB forms a novel strategy to enhance cells that form the BBB. In addition to Pgp, other ABC efflux the penetration of drugs into the brain and may yield new transporters such as members of the multidrug resistance pro- therapeutic options for drug-resistant CNS diseases. Key tein (MRP) family and breast cancer resistance protein (BCRP) Words: P-glycoprotein, multidrug resistance proteins, epi- seem to contribute to BBB function.
    [Show full text]
  • Targeting the Zinc Transporter ZIP7 in the Treatment of Insulin Resistance and Type 2 Diabetes
    nutrients Review Targeting the Zinc Transporter ZIP7 in the Treatment of Insulin Resistance and Type 2 Diabetes John Adulcikas, Sabrina Sonda, Shaghayegh Norouzi, Sukhwinder Singh Sohal and Stephen Myers * College of Health and Medicine, School of Health Sciences, University of Tasmania, TAS 7005, Australia; [email protected] (J.A.); [email protected] (S.S.); [email protected] (S.N.); [email protected] (S.S.S.) * Correspondence: [email protected]; Tel.: +61-3-6324-5459 Received: 20 December 2018; Accepted: 12 February 2019; Published: 15 February 2019 Abstract: Type 2 diabetes mellitus (T2DM) is a disease associated with dysfunctional metabolic processes that lead to abnormally high levels of blood glucose. Preceding the development of T2DM is insulin resistance (IR), a disorder associated with suppressed or delayed responses to insulin. The effects of this response are predominately mediated through aberrant cell signalling processes and compromised glucose uptake into peripheral tissue including adipose, liver and skeletal muscle. Moreover, a major factor considered to be the cause of IR is endoplasmic reticulum (ER) stress. This subcellular organelle plays a pivotal role in protein folding and processes that increase ER stress, leads to maladaptive responses that result in cell death. Recently, zinc and the proteins that transport this metal ion have been implicated in the ER stress response. Specifically, the ER-specific zinc transporter ZIP7, coined the “gate-keeper” of zinc release from the ER into the cytosol, was shown to be essential for maintaining ER homeostasis in intestinal epithelium and myeloid leukaemia cells. Moreover, ZIP7 controls essential cell signalling pathways similar to insulin and activates glucose uptake in skeletal muscle.
    [Show full text]
  • Role of the Efflux Pumps in Antimicrobial Resistance in E
    Role of the Efflux Pumps in Antimicrobial Resistance in E. coli Patrick Plésiat Bacteriology Department Teaching Hospital Besançon, France ANTIBIOTIC TARGET Bacterial targets for antibiotics Chromosome Cell wall Ribosomes Cytoplasmic membrane Main resistance mechanisms to drugs Inactivation Modification ANTIBIOTIC Efflux Impermeability Protection TARGET Reduced affinity Substitution - mutations Amplification - recombinaisons - enzymatic modification Gram-negative species with known efflux systems Escherichia coli Pseudomonas aeruginosa Salmonella Typhimurium Pseudomonas putida Burkholderia cepacia Shigella dysenteriae Burkholderia pseudomallei Klebsiella pneumoniae Stenotrophomonas maltophilia Enterobacter aerogenes Alcaligenes eutrophus... Serratia marcescens Proteus vulgaris Haemophilus influenzae Citrobacter freundii... Campylobacter jejuni Helicobacter pylori Vibrio parahaemolyticus Vibrio cholerae Bacteroides fragilis... Neisseria gonorrhoeae... Efflux mechanisms: practical implications Do efflux systems produce clinically relevant levels of resistance ? Does the expression of drug transporters somewhat impair the virulence of bacterial pathogens ? What is the prevalence of efflux systems relative to other resistance mechanisms among the clinical isolates ? How to recognize efflux mutants in laboratory practice ? What recommendations can be made to the physician for the treatment of patients infected with mdr strains ? Drug accumulation experiments S ATP glucose R Intracellular accumulation CCCP Time Structure of bacterial efflux
    [Show full text]
  • Overview of P-Glycoprotein Inhibitors: a Rational Outlook
    Brazilian Journal of Pharmaceutical Sciences vol. 48, n. 3, jul./sep., 2012 Article Overview of P-glycoprotein inhibitors: a rational outlook Kale Mohana Raghava Srivalli, P. K. Lakshmi* Department of Pharmaceutics, G. Pulla Reddy College of Pharmacy, Osmania University, India P-glycoprotein (P-gp), a transmembrane permeability glycoprotein, is a member of ATP binding cassette (ABC) super family that functions specifically as a carrier mediated primary active efflux transporter. It is widely distributed throughout the body and has a diverse range of substrates. Several vital therapeutic agents are substrates to P-gp and their bioavailability is lowered or a resistance is induced because of the protein efflux. Hence P-gp inhibitors were explored for overcoming multidrug resistance and poor bioavailability problems of the therapeutic P-gp substrates. The sensitivity of drug moieties to P-gp and vice versa can be established by various experimental models in silico, in vitro and in vivo. Ever since the discovery of P-gp, the research plethora identified several chemical structures as P-gp inhibitors. The aim of this review was to emphasize on the discovery and development of newer, inert, non-toxic, and more efficient, specifically targeting P-gp inhibitors, like those among the natural herb extracts, pharmaceutical excipients and formulations, and other rational drug moieties. The applications of cellular and molecular biology knowledge, in silico designed structural databases, molecular modeling studies and quantitative structure-activity relationship (QSAR) analyses in the development of novel rational P-gp inhibitors have also been mentioned. Uniterms: P-glycoprotein/inhibitors. Multidrug resistence. Cluster of differentiation 243. Sphingolipids. Competitive inhibitors.
    [Show full text]
  • Drug Delivery That Overcomes P-Glycoprotein Mediated Drug Efflux
    Technology Available For License Drug Delivery that Overcomes P-glycoprotein Mediated Drug Efflux A drug delivery system that overcomes the problems associated with P-glycoprotein mediated drug efflux. Disadvantages of Current Treatments: P-Glycoprotein (P-gp) is a transport protein that effluxes a wide variety of structurally unrelated drugs out of cells. The bioavailability of various anticancer drugs, anti-HIV drugs, calcium channel drugs, and other drugs which are substrates is limited by this efflux transporter. Over-expression of P-gp by tumor cells confers multi-drug resistance. Efflux of many anticancer drugs including taxol, vincristine, vinblastine, actinomycin D, colchicines, and daunorubicin, from tumor cells makes P-gp a major barrier to chemothera- py. High expression of this transporter on the blood-brain-barrier (BBB) restricts the entry into the brain of P-gp substrates such as anti -HIV drugs such as ritonavir, saquinavir, nelfinavir, and various anticancer drugs, and thus imposes a major challenge in the treat- ment of various diseases of the brain. Expression of the efflux transporter on various body tissues and cells not only influences the in vivo disposition of various therapeuti- cally active drugs, but also greatly influences the drugs' pharmacokinetics. It has been known that inhibition of P-gp by various modulators can lead to improved bioavailability of drugs across the intestines, the kidneys, and the BBB. Various modulators that inhibit P-gp are often co-administered with other bioactive agents to increase bioavailability. However, use of these compounds is limited by their toxicity. To achieve P-gp inhibition, doses that result in high serum concentrations of the toxic inhibitor are required.
    [Show full text]
  • In Situ Structure and Assembly of the Bacterial Multidrug Efflux Pump
    1304 Microsc. Microanal. 25 (Suppl 2), 2019 doi:10.1017/S1431927619007256 © Microscopy Society of America 2019 In Situ Structure and Assembly of the Bacterial Multidrug Efflux Pump Xiaodong Shi1, Muyuan Chen1, James Michael Bell2, Zhili Yu1, Steven J. Ludtke1 and Zhao Wang1,3* 1. Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, USA. 2. Graduate Program in Quantitative and Computational Biosciences, Baylor College of Medicine, Houston, Texas, USA 3. Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA. * Corresponding author: [email protected] With the increasing use of antibiotics, multidrug resistance in pathogenic bacteria has grown to become a medical crisis to public health. Multidrug efflux pumps play an important role in conferring drug resistance, as they are capable of expelling a variety of toxic substrates from the cell. Targeting assembly of efflux pumps is a promising strategy to overcoming antibiotic resistance [1]. In Gram- negative bacteria, these pumps form tripartite assemblies that span both the inner and outer membranes making them difficult for traditional structural studies [2]. AcrAB-TolC is one of the tripartite pumps that are constitutively expressed in Escherichia coli (E. coli) [3]. As the main multidrug efflux machinery, AcrAB-TolC is comprised of the outer membrane protein TolC, the periplasmic adaptor protein AcrA, and the inner membrane transporter AcrB from the Resistance-Nodulation-cell Division (RND) superfamily [2]. The AcrAB-TolC efflux pump transports diverse compounds, conferring resistance to a broad spectrum of antibiotics [4]. Structural studies of this pump have been limited to individual components by X-ray crystallography [5-8] or fully assembled pumps by cryo-electron microscopy (cryo-EM) single particle analysis [9, 10].
    [Show full text]
  • ATP-Binding Cassette
    Biochem. J. (2002) 367, 279–285 (Printed in Great Britain) 279 Overexpression and functional characterization of an ABC (ATP-binding cassette) transporter encoded by the genes drrA and drrB of Mycobacterium tuberculosis Baisakhee Saha CHOUDHURI, Sanjib BHAKTA, Rajib BARIK, Joyoti BASU, Manikuntala KUNDU and Parul CHAKRABARTI1 Department of Chemistry, Bose Institute, 93/1 Acharya Prafulla Chandra Road, Calcutta 700 009, India The genes encoding ATP-binding cassette (ABC) transporters of the two polypeptides was essential to obtain a functional occupy 2.5% of the genome of Mycobacterium tuberculosis. doxorubicin efflux pump. Expression of DrrAB in E. coli However, none of these putative ABC transporters has been conferred 8-fold increased resistance to ethidium bromide, a characterized so far. We describe the development of expression cationic compound. 2h,7h-bis-(2-Carboxyethyl)-5(6)-carboxyfluo- systems for simultaneous expression of the ATP-binding protein rescein (BCECF), a neutral compound, also behaved as a sub- DrrA and the membrane integral protein DrrB which together strate of the reconstituted efflux pump. When expressed in behave as a functional doxorubicin efflux pump. Doxorubicin M. smegmatis, DrrAB conferred resistance to a number of clini- uptake in Escherichia coli or Mycobacterium smegmatis ex- cally relevant, structurally unrelated antibiotics. The resistant pressing DrrAB was inhibited by reserpine, an inhibitor of ABC phenotype could be reversed by verapamil and reserpine, two transporters. The localization of DrrA to the membrane de- potent inhibitors of ABC transporters. pended on the simultaneous expression of DrrB. ATP binding was positively regulated by doxorubicin and daunorubicin. At the same time, DrrB appeared to be sensitive to proteolysis when Key words: antibiotic resistance, efflux pump, lipid transporter, expressed alone in the absence of DrrA.
    [Show full text]
  • Differential Expression of Zinc Transporters Accompanies the Differentiation of C2C12 Myoblasts
    University of Massachusetts Medical School eScholarship@UMMS Open Access Articles Open Access Publications by UMMS Authors 2018-9 Differential expression of zinc transporters accompanies the differentiation of C2C12 myoblasts Amanda L. Paskavitz University of Massachusetts Medical School Et al. Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/oapubs Part of the Cell Biology Commons, Cells Commons, Developmental Biology Commons, and the Molecular Biology Commons Repository Citation Paskavitz AL, Quintana J, Cangussu D, Tavera-Montanez C, Xiao Y, Ortiz-Mirnada S, Navea JG, Padilla- Benavides T. (2018). Differential expression of zinc transporters accompanies the differentiation of C2C12 myoblasts. Open Access Articles. https://doi.org/10.1016/j.jtemb.2018.04.024. Retrieved from https://escholarship.umassmed.edu/oapubs/3605 Creative Commons License This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License. This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in Open Access Articles by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. Journal of Trace Elements in Medicine and Biology 49 (2018) 27–34 Contents lists available at ScienceDirect Journal of Trace Elements in Medicine and Biology journal homepage: www.elsevier.com/locate/jtemb Molecular biology Differential expression of zinc transporters accompanies the differentiation
    [Show full text]
  • ATP Binding Cassette Transporters and Drug Resistance in Breast Cancer
    Endocrine-Related Cancer (2003) 10 43–73 REVIEW ATP binding cassette transporters and drug resistance in breast cancer F Leonessa and R Clarke1 Laboratory of Clinical Investigation,National Institute on Aging,National Institutes of Health,5600 Nathan Shock Drive,Baltimore,Maryland 21224,USA 1Departments of Oncology,Physiology and Biophysics,and Lombardi Cancer Center,Georgetown University School of Medicine,3970 Reservoir Road NW,Washington DC 20007,USA (Requests for offprints should be addressed to R Clarke, Department of Oncology, room W405A, Research Building, 3970 Reservoir Road NW, Washington DC 20007, USA; Email: [email protected]) Abstract Resistance to chemotherapy is a critical issue in the management of breast cancer patients. The nature of clinical drug resistance is likely to be multifactorial. However,in the last decade considerable attention has been dedicated to the role played by membrane transporter proteins belonging to the ATP binding cassette protein superfamily,and in particular by the MDR1 product P-glycoprotein (Pgp) and the multidrug resistance protein (MRP1). Heterogeneity of results is a common feature of studies evaluating the expression and prognostic role of these proteins,due to both methodological and biological factors. Nonetheless,Pgp and MRP1 are detected in a significant proportion of untreated breast cancers (on average 40 and 50% respectively,by immunohistochemistry),without a clear and consistent association with cancer stage. Exposure to chemotherapy increases the expression of both proteins. In vitro studies on primary cultures of breast cancer cells obtained at surgery consistently show an association between Pgp (protein) or MDR1 (mRNA) expression and resistance to chemotherapy. However,the correlation with clinical drug resistance is not as well defined.
    [Show full text]