Overexpression and functional characterization of an ABC (ATP-binding cassette) transporter encoded by the genes drrA and drrB of Mycobacterium tuberculosis.
The genes encoding ATP-binding cassette (ABC) transporters occupy 2.5% of the genome of Mycobacterium tuberculosis. However, none of these putative ABC transporters has been characterized so far. We describe the development of expression systems for simultaneous expression of the ATP-binding protein DrrA and the membrane integral protein DrrB which together behave as a functional doxorubicin efflux pump. Doxorubicin uptake in Escherichia coli or Mycobacterium smegmatis expressing DrrAB was inhibited by reserpine, an inhibitor of ABC transporters. The localization of DrrA to the membrane depended 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 expressed alone in the absence of DrrA. Simultaneous expression of the two polypeptides was essential to obtain a functional doxorubicin efflux pump. Expression of DrrAB in E. coli conferred 8-fold increased resistance to ethidium bromide, a cationic compound. 2',7'-bis-(2-Carboxyethyl)-5(6)-carboxyfluorescein (BCECF), a neutral compound, also behaved as a substrate of the reconstituted efflux pump. When expressed in M. smegmatis, DrrAB conferred resistance to a number of clinically relevant, structurally unrelated antibiotics. The resistant phenotype could be reversed by verapamil and reserpine, two potent inhibitors of ABC transporters.
Choudhuri
Baisakhee Saha
BS
Department of Chemistry, Bose Institute, 93/1 Acharya Prafulla Chandra Road, Calcutta 700 009, India.
Bhakta
Sanjib
S
Barik
Rajib
R
Basu
Joyoti
J
Kundu
Manikuntala
M
Chakrabarti
Parul
P
D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D018759:Adrenergic Uptake Inhibitors / Q000494:pharmacology; D000595:Amino Acid Sequence; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D001426:Bacterial Proteins; D001483:Base Sequence; D001692:Biological Transport; D002121:Calcium Channel Blockers / Q000494:pharmacology; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D003630:Daunorubicin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000494:pharmacology; D004351:Drug Resistance; D004926:Escherichia coli / Q000378:metabolism; D050356:Lipid Metabolism; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D012110:Reserpine / Q000494:pharmacology; D014700:Verapamil / Q000494:pharmacology
D018528:ATP-Binding Cassette Transporters; D018759:Adrenergic Uptake Inhibitors; D000903:Antibiotics, Antineoplastic; D001426:Bacterial Proteins; D002121:Calcium Channel Blockers; D004268:DNA-Binding Proteins; C101895:DrrA protein, Bacteria; C474312:DrrB protein, Mycobacterium tuberculosis; D004317:Doxorubicin; D012110:Reserpine; D000255:Adenosine Triphosphate; D014700:Verapamil; D003630:Daunorubicin