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Research GBB Molecular Microbiology

Multidrug resistance

Mechanism and function of multidrug transporters in pro- and eukaryotic cells

The development of resistance to multiple drugs is a major problem in the treatment of cancer cells and infections by pathogenic microorganisms. One of the mechanisms by which human cells and bacteria can acquire multidrug resistance involves the expression of membrane proteins which mediate the active extrusion of drugs from the cell. Current research focuses on the molecular properties of four distinct multidrug transport proteins.

Multidrug transporter LmrP
In Lactococcus lactis LmrP mediates drug resistance by extruding amphiphilic, organic compounds from the inner leaflet of the cytoplasmic membrane. LmrP is a secondary multidrug transporter which is typical for prokaryotes. The antibiotic specificity of LmrP is exceptionally broad and includes tetracyclins, quinolones, aminoglucosides, lincosamides, macrolides, streptogramins and others. To facilitate functional and structural studies, histidine-tagged LmrP protein was overexpressed in L. lactis, solubilized with dodecylmaltoside, purified to homogeneity by nickel chelate affinity chromatography, and functionally reconstituted in proteoliposomes. Interestingly, LmrP is able to transport detergents such as Triton X-100. Therefore, the choice of the detergent used for solubilization of the protein was critical.

Multidrug transporter LmrA
A second protein in L. lactis, LmrA, mediates antibiotic resistance via a similar mechanism, and with a similar specificity as the secondary multidrug transporter LmrP. Unlike other known bacterial multidrug resistance proteins, LmrA is an ATP-binding cassette (ABC) transporter. LmrA is homologous to the human multidrug resistance P-glycoprotein, encoded by the MDR1 gene, overexpression of which is one of the major causes of resistance of human cancers to chemotherapy. In collaboration with Prof. C.F. Higgins in Oxford, LmrA was expressed in human lung fibroblast cells to compare the pharmacological properties of LmrA with those of P-glycoprotein. Surprisingly, LmrA was targeted to the plasma membrane and conferred typical multidrug resistance on these human cells. The pharmacological characteristics of LmrA and P-glycoprotein- expressing lung fibroblasts were very similar, and the affinities of both proteins for vinblastine and magnesium-ATP indistinguishable. Blockers of P-glycoprotein-mediated multidrug resistance also inhibited LmrA-dependent drug resistance. Kinetic analysis of drug dissociation from LmrA, expressed in plasma membranes of insect cells, revealed the presence of two allosterically-linked drug binding sites indistinguishable from those of P-glycoprotein. These findings have implications for the reversal of antibiotic resistance in pathogenic microorganisms. Taken together, these observations demonstrate that bacterial LmrA and human P-glycoprotein are functionally interchangeable and that this type of multidrug resistance efflux pump is conserved from bacteria to man. LmrA has been overexpressed in L. lactis up to a level of 35% of total membrane protein. This protein is functional, and has been purified and reconstituted into proteoliposomes to facilitate detailed structure-function analyses. Currently, mg quantities of reconstituted LmrA can be obtained rather easily.

Multidrug transporter HorA
Lactococcal LmrA is homologous to other prokaryotic ABC transporters such as the hop-resistance protein HorA in the beer-spoilage bacterium Lactobacillus brevis. To study HorA in more detail, the protein was functionally expressed in L. lactis. Studies in whole cells and membrane vesicles derived thereof demonstrate that HorA is a multidrug transporter able to transport hop compounds (iso-a-acids), with a substrate specificity similar to that of LmrA. Recently, the protein has been purified and reconstituted into proteoliposomes.

Cholate transporter
Micro-organisms able to colonize the gastrointestinal tract in mammals must tolerate high levels of bile salts, powerful detergents that disrupt biological membranes. Physiological evidence has been obtained for the presence of an ATP-dependent cholate transporter in L. lactis. Interestingly, this transport activity may be related to the transport activities for fluorescent anionic dyes BCECF and FTUG in this organism. Current work aims at the isolation of the gene(s) encoding the cholate transporter.

Multidrug resistance-associated protein
Multidrug resistance in humans is caused by the overexpression of two multidrug transporters: P-glycoprotein and the Multidrug Resistance-Associated Protein (MRP1). Although MRP1 contains two nucleotide binding domains (NBDs), it is not known whether both NBDs can function as an active ATPase, and whether drug-protein interactions in these domains play a role in the drug-stimulated ATPase activity of MRP1. The NBDs of MRP1 were expressed in fusion with glutathione S-transferase, purified by affinity chromatography, cleaved from the fusion partner by thrombine, and purified to homogeneity by gel filtration. Both NBDs hydrolyze ATP, with a Km of 340 mM Mg-ATP and Vmax of 6.0 nmol Pi/mg/min for NBD1, and a Km of 910 mM Mg-ATP and Vmax of 7.5 nmol Pi/mg/min for NBD2. The properties of NBD1 were further studied. Surprisingly, the Vmax of the NBD1 ATPase reaction was stimulated more than 3-fold by the MRP substrate decyl glutathione but not by decyl maltoside or decanol, whereas the Km of the reaction was not affected. The interaction between S-alkyl glutathione conjugates and NBD1 was analyzed by using the intrinsic tryptophan fluorescence of NBD1 as a reporter. The tryptophan fluorescence was quenched in a concentration-dependent manner by S-alkyl glutathione conjugates, but not by alkyl maltosides or alkanols. The apparent KD of NBD1 for S-alkyl glutathione conjugates decreased with an increasing length of the alkyl chain. Thus, NBD1 is suggested to have a high-affinity binding site for S-alkyl glutathione conjugates.

Last modified:15 December 2021 4.20 p.m.