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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. Simultaneous expression mycobacteria, virulence. INTRODUCTION binding proteins, bind nucleotides [7,8]. The doxorubicin- resistance operon, first identified in Streptomyces peucetius [9], Mycobacterium tuberculosis accounts for the largest number of shares similarity with the eukaryotic P-glycoprotein. Two trans- deaths caused by a single human pathogen with predictions of an lationally coupled open reading frames, drrA and drrB, encode excess of 80 million new cases and 20 million deaths in the an ABC-type transporter, with drrA encoding the nucleotide- coming decade. The appearance of drug-resistant strains of M. binding domain and drrB encoding the membrane integral tuberculosis and the HIV pandemic have exacerbated this situ- component [10]. A monomer of DrrA and DrrB forms a molecule ation. Effective treatment of tuberculosis infections requires the about half the size of P-glycoprotein. The likely stoichiometry identification of new drugs, drug targets and drug-resistance of the functional transporter is DrrA#B# [10]. By analogy to factors. The ATP-binding cassette (ABC) transporters [1] con- S. peucetius, the completed M. tuberculosis genome [11] also stitute a large superfamily of multi-subunit permeases that contains a doxorubicin-resistance operon, drr. In addition, the transport diverse molecules (ions, amino acids, peptides, drugs, drr operon has been identified in the genomes of Mycobacterium antibiotics, lipids, polysaccharides, proteins etc.) at the expense leprae and Mycobacterium aium. As powerful techniques of of ATP [2,3]. The genes encoding the ABC transporters occupy molecular biology have become available, the drr operon has about 2.5% of the genome of M. tuberculosis [4]. At least 37 assumed particular significance in relation to its implications in complete and incomplete ABC transporters have been identified the virulence of M. tuberculosis. The biosynthesis of phthiocerol in M. tuberculosis based on the structural similarities of the dimycocerosates (DIMs) involves several genes [12]. The genes typical subunits of ABC transporters present in all living organ- pps A-E encode a type I modular polyketide synthase responsible isms. Knowledge of these M. tuberculosis ABC transporters is for the synthesis of phthiocerol and phenolphthiocerol. Another necessary both for understanding their involvement in the gene, mas, encodes an iterative type I polyketide synthase that development of multidrug resistance in M. tuberculosis as well as produces mycocerosic acid. The gene fadD28 is probably involved in the export of the unique antigenic cell-surface components in the release and transfer of mycocerosic acid from Mas on to the of this organism, such as the phthiocerol mycocerosates and diols. These seven genes are clustered on a 50 kb fragment of lipoarabinomannan. the chromosome containing, among other genes, the open reading The prototype eukaryotic ABC transporter is P-glycoprotein frames of the drr operon and mmpl7, encoding polypeptides [5]. The 170 kDa P-glycoprotein is characterized by two mem- similar to ABC transporters. Signature-tagged transposon muta- brane integral domains with six membrane-spanning helices each genesis has shown that transposon insertions in the drr operon or connected with two ATP-hydrolysing domains and the presence mmpl7 lead to a strong growth defect of M. tuberculosis in the of ‘Walker sites A and B’ [6] in the primary structure of the ATP- lungs of intravenously infected mice [13,14] and in export of hydrolysing domains, which, by analogy with ATP-and GTP- DIM to the cell surface [15]. Abbreviations used: ABC, ATP-binding cassette; BCECF-AM, 2h,7h-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetomethoxyl ester; DIM, phthiocerol dimycocerosate; IPTG, isopropyl β-D-thiogalactoside; MIC, minimum inhibitory concentration. 1 To whom correspondence should be addressed (e-mail parul!bosemain.boseinst.ac.in). # 2002 Biochemical Society 280 B. S. Choudhuri and others The biochemical characterization of the Drr transporter the KpnI and EcoRI sites (underlined) to generate the plasmid assumes obvious importance in the light of these recent observa- pCKB104. pCKB104 was sequenced and the drrA gene was tions. Its association with virulence suggests that it presents an excised and cloned between the NdeI (shown above in bold) and attractive drug target, which if inactivated will probably disable EcoRI sites of pET28a+ to give pCKB105. the pathogen in terms of its ability to export complex molecules The drrB gene was amplified from plasmid pCKB101 using the such as DIM, a surface-exposed antigenic lipid present in seven following primer pair: 5h-GAAGATCTCATATGAGCGGCC- pathogenic species of mycobacteria [16], to the cell surface. In CG-3h (sense) and 5h-CCCGAATTCGTCGTGATCATGGGC- addition, the likely importance of the drr operon in multidrug CGCCTAG-3h (antisense) with asymmetric BglII and EcoRI resistance deserves evaluation. The biochemical characterization sites, cloned in the vector pK18 to give pCKB107 and sequenced. of the drr operon was undertaken before most of these recent The drrB gene was excised and cloned between the NdeI and observations came to light, envisaging its likely importance in EcoRI sites of pET28a+ to give pCKB108, as well as between multidrug resistance of M. tuberculosis. The goal was to develop the BglII and EcoRI sites of pBAD-HisA to give pCKB109. expression systems expressing a functional DrrAB transporter in order to evaluate the role of the transporter in drug resistance as Construction of an artificial operon well as to exploit the expression systems in further characteriz- ation of the transporter in relation to the roles of the conserved pCKB108 was digested with NcoI and NdeI to eliminate a 58 bp domains in transport function. fragment. This was replaced by oligonucleotide 5h-CATGGCT- GGTACCGGGGTCAAGGAGATAACA-3h and its reverse h EXPERIMENTAL complement strand, with overhangs of CATG at the 5 -end and AT at the 3h end to complement NcoI and NdeI respectively. The Materials resulting plasmid, termed pCKB110, contained a KpnI site ["%C]Doxorubicin and the Thermosequenase cycle sequencing kit (underlined) and a Shine–Dalgarno sequence (AGGA). were purchased from Amersham Bioscience (Little Chalfont, The drrA gene was amplified from pCKB105 using the primers 5h-GAAGATCTCATATGCGCAACGACGACATGGC-3h Bucks., U.K.). Restriction enzymes and antibiotics were pur- h chased from Life Technologies (Gaithersberg, MD, U.S.A.), [α- (sense) and 5 -GGAATTCGGTACCAGATGGGTCAGAGA- $# CTCGGT-3h (antisense) with asymmetric NdeI and EcoRI sites P]ATP was purchased from NEN Life Sciences (Boston, MA, + U.S.A.) and 2h,7h-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein and cloned between the NdeI and EcoRI sites of pET28a to give acetomethoxyl ester (BCECF-AM) was a product of Molecular pCKB111. Plasmid pCKB110 was digested with KpnI and EcoRI Probes (Eugene, OR, U.S.A.). Doxorubicin, daunorubicin, and the fragment containing the drrB gene with the upstream ethidium bromide, reserpine, verapamil, chloramphenicol, tetra- Shine–Dalgarno sequence was cloned between the KpnI (shown cycline, erythromycin, ethambutol, rifampicin, norfloxacin, above in bold) and EcoRI sites of pCKB111 to give pCKB112. streptomycin and puromycin were products of Sigma Chemicals pCKB112 therefore contains the translationally coupled drrAB (St. Louis, MO, U.S.A.). All other reagents were of analytical genes, with the artificial ribosome-binding site AGGA upstream grade. of the drrB gene, under the control of the T7 promoter. The following cloning steps were performed to introduce the Myc epitope at the C-terminal end of DrrB. Using pCKB112 as Strains a
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