Drug-lipid A interactions on the Escherichia coli ABC transporter MsbA.
MsbA is an essential ATP-binding cassette half-transporter in the cytoplasmic membrane of the gram-negative Escherichia coli and is required for the export of lipopolysaccharides (LPS) to the outer membrane, most likely by transporting the lipid A core moiety. Consistent with the homology of MsbA to the multidrug transporter LmrA in the gram-positive Lactococcus lactis, our recent work in E. coli suggested that MsbA might interact with multiple drugs. To enable a more detailed analysis of multidrug transport by MsbA in an environment deficient in LPS, we functionally expressed MsbA in L. lactis. MsbA expression conferred an 86-fold increase in resistance to the macrolide erythromycin. A kinetic characterization of MsbA-mediated ethidium and Hoechst 33342 transport revealed apparent single-site kinetics and competitive inhibition of these transport reactions by vinblastine with K(i) values of 16 and 11 microM, respectively. We also detected a simple noncompetitive inhibition of Hoechst 33342 transport by free lipid A with a K(i) of 57 microM, in a similar range as the K(i) for vinblastine, underscoring the relevance of our LPS-less lactococcal model for studies on MsbA-mediated drug transport. These observations demonstrate the ability of heterologously expressed MsbA to interact with free lipid A and multiple drugs in the absence of auxiliary E. coli proteins. Our transport data provide further functional support for direct LPS-MsbA interactions as observed in a recent crystal structure for MsbA from Salmonella enterica serovar Typhimurium (C. L. Reyes and G. Chang, Science 308:1028-1031, 2005).
Woebking
Barbara
B
Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, United Kingdom.
Reuter
Galya
G
Shilling
Richard A
RA
Velamakanni
Saroj
S
Shahi
Sanjay
S
Venter
Henrietta
H
Balakrishnan
Lekshmy
L
van Veen
Hendrik W
HW
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D001692:Biological Transport; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism*; D013294:Lactococcus lactis / Q000378:metabolism; D008050:Lipid A / Q000378:metabolism*; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics / Q000378:metabolism*; D004364:Pharmaceutical Preparations / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D008050:Lipid A; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D004364:Pharmaceutical Preparations; C101824:LmrA protein, Lactococcus lactis; C079726:MsbA protein, Bacteria