Drug-Lipid A Interactions on the Escherichia coli ABC Transporter MsbA

• DOI: 10.1128/jb.187.18.6363-6369.2005
• Publication Date: 2005-09-02T21:57:53Z
• Authors: Barbara Woebking; Galya Reuter; Richard A. Shilling; Saroj Velamakanni; Sanjay Shahi; Henrietta Venter; Lekshmy Balakrishnan; Hendrik W. van Veen
• Keywords: Lactococcus lactis; 0301 basic medicine; 03 medical and health sciences; Lipid A; Bacterial Proteins; Pharmaceutical Preparations; Escherichia coli; ATP-Binding Cassette Transporters; Biological Transport; Multidrug Resistance-Associated Proteins; 3. Good health

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 μM, respectively. We also detected a simple noncompetitive inhibition of Hoechst 33342 transport by free lipid A with a

K i

of 57 μM, 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).