PMID:16159769
| Citation |
Woebking, B, Reuter, G, Shilling, RA, Velamakanni, S, Shahi, S, Venter, H, Balakrishnan, L and van Veen, HW (2005) Drug-lipid A interactions on the Escherichia coli ABC transporter MsbA. J. Bacteriol. 187:6363-9 |
|---|---|
| Abstract |
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). |
| Links |
PubMed PMC1236644 Online version:10.1128/JB.187.18.6363-6369.2005 |
| Keywords |
ATP-Binding Cassette Transporters/metabolism; Bacterial Proteins/metabolism; Biological Transport; Escherichia coli/enzymology; Escherichia coli/metabolism; Lactococcus lactis/metabolism; Lipid A/metabolism; Multidrug Resistance-Associated Proteins/genetics; Multidrug Resistance-Associated Proteins/metabolism; Pharmaceutical Preparations/metabolism |
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