M Putman

Molecular Properties of Bacterial Multidrug Transporters

SUMMARY One of the mechanisms that bacteria utilize to evade the toxic effects of antibiotics is the active extrusion of structurally unrelated drugs from the cell. Both intrinsic and acquired multidrug transporters play an important role in antibiotic resistance of several pathogens, including Neisseria gonorrhoeae, Mycobacterium tuberculosis, Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Vibrio cholerae. Detailed knowledge of the molecular basis of drug recognition and transport by multidrug transport systems is required for the development of new antibiotics that are not extruded or of inhibitors which block the multidrug transporter and allow traditional antibiotics to be effective. This review gives an extensive overview of the currently known multidrug transporters in bacteria. Based on energetics and structural characteristics, the bacterial multidrug transporters can be classified into five distinct families. Functional reconstitution in liposomes of purified multidrug transport proteins from four families revealed that these proteins are capable of mediating the export of structurally unrelated drugs independent of accessory proteins or cytoplasmic components. On the basis of (i) mutations that affect the activity or the substrate specificity of multidrug transporters and (ii) the three-dimensional structure of the drug-binding domain of the regulatory protein BmrR, the substrate-binding site for cationic drugs is predicted to consist of a hydrophobic pocket with a buried negatively charged residue that interacts electrostatically with the positively charged substrate. The aromatic and hydrophobic amino acid residues which form the drug-binding pocket impose restrictions on the shape and size of the substrates. Kinetic analysis of drug transport by multidrug transporters provided evidence that these proteins may contain multiple substrate-binding sites.

RESTRICTIVE USE OF DETERGENTS IN THE FUNCTIONAL RECONSTITUTION OF THE SECONDARY MULTIDRUG TRANSPORTER LMRP

The histidine-tagged secondary multidrug transporter LmrP was overexpressed in Lactococcus lactis, using a novel protein expression system for cytotoxic proteins based on the tightly regulated, nisin-inducible nisA promoter. LmrP-mediated H+/drug antiport activity in inside-out membrane vesicles was inhibited by detergents, such as Triton X-100, Triton X-114, and Tween 80, at low concentrations that did not affect the magnitude or composition of the proton motive force. The inhibition of the activity of LmrP by detergents restricted the range of compounds that could be used for the solubilization and reconstitution of the protein because low concentrations of detergent are retained in proteoliposomes. Surprisingly, dodecyl maltoside did not modulate the activity of LmrP. Therefore, LmrP was solubilized with dodecyl maltoside, purified by nickel-chelate affinity chromatography, and reconstituted in dodecyl maltoside-destabilized, preformed liposomes prepared from Escherichia coli phospholipids and egg phosphatidylcholine. Reconstituted LmrP mediated the transport of multiple drugs in response to an artificially imposed pH gradient, demonstrating that the protein functions as a proton motive force-dependent multidrug transporter, independent of accessory proteins. These observations are relevant for the effective solubilization and reconstitution of multidrug transporters belonging to the major facilitator superfamily, which, in view of their broad drug specificity, may strongly interact with detergents.

Antibiotic resistance : era of the multidrug pump

Over the past years, concerns have heightened over the escalating numbers of pathogenic microorganisms isolated that are resistant to multiple antibiotics (Berkelman et al. 1994, Science 264: 368±370). This phenomenon poses major problems in the treatment of patients with hospitalor community-acquired infections caused by bacteria, yeasts, fungi, or parasitic organisms. Drug expulsion, mediated by membrane-associated drug efflux pumps, is one of the ingenious mechanisms that microorganisms use to evade the toxic effects of antibiotics. Particularly intriguing are the so-called multidrug transporters, which have specificity for compounds with very different chemical structures and cellular targets. Here, we report that a single ATP-dependent multidrug transporter of Lactococcus lactis, a bacterium used in dairy fermentations, confers resistance to a record of eight classes of clinically relevant broad-spectrum antibiotics. Although most microbial multidrug efflux systems known to date mediate drug±proton exchange (Paulsen et al. 1996, Microbiol Rev 60: 575±608), the lactococcal multidrug transporter LmrA uses the free energy of ATPhydrolysis to drive the export of toxic compounds from the inner leaflet of the cytoplasmic membrane (Bolhuis et al. 1996, EMBO J 15: 4239±4245). LmrA, a 590-amino-acid integral membrane protein, is a member of the ATPbinding Cassette (ABC) Superfamily (Higgins, 1992, Annu Rev Cell Biol 8: 67±113; van Veen et al. 1996, Proc Natl Acad Sci USA 93: 10668±10672). LmrA shares significant sequence identity with the hop resistance protein HorA of the beer-spoilage bacterium Lactobacillus brevis, and with ABC transporters in pathogenic microorganisms (van Veen and Konings, 1998, Biochim Biophys Acta 1365: 31±36). Furthermore, LmrA is a structural and functional homologue of the human multidrug transporter P-glycoprotein (vanVeenet al. 1998, Nature391: 291±295). To address the role of LmrA in antibiotic resistance, LmrA was expressed in Escherichia coli strain CS1562, which is hypersensitive to drugs due to a deficiency of the TolC porin. The difference in in vivo antibiotic susceptibility between cells harbouring the lmrA containing plasmid pGKLmrA (van Veen et al. 1996, Proc Natl Acad Sci USA 93: 10668±10672) or control plasmid pGK13 was studied in liquid cultures. LmrA expression resulted in an increased resistance to aminoglycosides, lincosamides, macrolides, quinolones, streptogramins, tetracyclines and chloramphenicol (Table 1). The resistance to trimethoprim and the glycopeptide vancomycin was unaltered. Surprisingly, LmrA expression also increased the resistance to certain b-lactam antibiotics. Because the targets of b-lactam antibiotics are extracellular transpeptidase enzymes involved in the biosynthesis of the cell wall, the role of multidrug transporters in b-lactam resistance is not immediately evident. However,

The lactococcal secondary multidrug transporter LmrP confers resistance to lincosamides, macrolides, streptogramins and tetracyclines

The active efflux of toxic compounds by (multi)drug transporters is one of the mechanisms that bacteria have developed to resist cytotoxic drugs. The authors describe the role of the lactococcal secondary multidrug transporter LmrP in the resistance to a broad range of clinically important antibiotics. Cells expressing LmrP display an increased resistance to the lincosamide, streptogramin, tetracycline and 14- and 15-membered macrolide antibiotics. The streptogramin antibiotic quinupristin, present in the fourth-generation antibiotic RP 59500, can inhibit LmrP-mediated Hoechst 33342 transport, but is not transported by LmrP, indicating that quinupristin acts as a modulator of LmrP activity. LmrP-expressing Lactococcus lactis cells in which a proton-motive force is generated accumulate significantly less tetracycline than control cells without LmrP expression. In contrast, LmrP-expressing and control cells accumulate equal amounts of tetracycline in the absence of metabolic energy. These findings demonstrate that the increased antibiotic resistance in LmrP-expressing cells is a result of the active extrusion of antibiotics from the cell.

Structure-function analysis of multidrug transporters in Lactococcus lactis

The active extrusion of cytotoxic compounds from the cell by multidrug transporters is one of the major causes of failure of chemotherapeutic treatment of tumor cells and of infections by pathogenic microorganisms. A multidrug transporter in Lactococcus lactis, LmrA, is a member of the ATP-binding cassette (ABC) superfamily and a bacterial homolog of the human multidrug resistance P-glycoprotein. Another multidrug transporter in L. lactis, LmrP, belongs to the major facilitator superfamily, and is one example of a rapidly expanding group of secondary multidrug transporters in microorganisms. Thus, LmrA and LmrP are transport proteins with very different protein structures, which use different mechanisms of energy coupling to transport drugs out of the cell. Surprisingly, both proteins have overlapping specificities for drugs, are inhibited by the same set of modulators, and transport drugs via a similar transport mechanism. The structure-function relationships that dictate drug recognition and transport by LmrP and LmrA represent an intriguing area of research.

Multidrug resistance in lactic acid bacteria : molecular mechanisms and clinical relevance

The active extrusion of cytotoxic compounds from the cell by multidrug transporters is one of the major causes of failure of chemotherapeutic treatment of tumor cells and of infections by pathogenic microorganisms. The secondary multidrug transporter LmrP and the ATP-binding cassette (ABC) type multidrug transporter LmrA in Lactococcus lactis are representatives of the two major classes of multidrug transporters found in pro- and eukaryotic organisms. Therefore, knowledge of the molecular properties of LmrP and LmrA will have a wide significance for multidrug transporters in all living cells, and may enable the development of specific inhibitors and of new drugs which circumvent the action of multidrug transporters. Interestingly, LmrP and LmrA are transport proteins with very different protein structures, which use different mechanisms of energy coupling to transport drugs out of the cell. Surprisingly, both proteins have overlapping specificities for drugs, are inhibited by t he same set of modulators, and transport drugs via a similar transport mechanism. The structure-function relationships that dictate drug recognition and transport by LmrP and LmrA will represent an intriguing new area of research.

Molecular pharmacological characterization of two multidrug transporters in Lactococcus lactis

The active extrusion of cytotoxic compounds from the cell by multidrug transporters is one of the major causes of failure of chemotherapeutic treatment of tumor cells and of infections by pathogenic microorganisms. A multidrug transporter in Lactococcus lactis, LmrA, is a member of the ATP-binding cassette superfamily and a bacterial homolog of the human multidrug resistance P-glycoprotein. Another multidrug transporter in Lactococcus lactis, LmrP, belongs to the major facilitator superfamily, and is one example of a rapidly expanding group of secondary multidrug transporters in microorganisms. Thus, LmrA and LmrP are transport proteins with very different protein structures, which use different mechanisms of energy coupling to transport drugs out of the cell. Surprisingly, both proteins have overlapping specificities for drugs, are inhibited by the same set of modulators, and transport drugs via a similar transport mechanism. The structure-function relationships that dictate drug recognition and transport by LmrP and LmrA represent an intriguing area of research.

Chapter 8 Multidrug resistance in prokaryotes: Molecular mechanisms of drug efflux

Publisher Summary This chapter discusses the cell biological mechanisms through which prokaryotes develop multidrug resistance. The majority of bacterial multidrug transporters characterized thus far, operates via a secondary transport mechanism. A number of genes encoding TEXANs and Mini TEXANs are cloned and sequenced. Analysis of these primary sequences reveals a striking similarity in the overall structure, suggesting that the proteins may function via a similar mechanism. Transport studies in membrane vesicles of Lactococcus lactis ( L. lactis )and Escherichia coli ( E. coli )are demonstrated the drug/proton antiport mechanism of the TEXAN LmrP. Smr and EmrE are purified and characterized upon reconstitution into proteoliposomes. These studies exhibit that a single multidrug resistance protein is able to function as a drug pump, and show the Δp-dependence of drug transport via the Mini TEXANs. The discovery of the ABC-type multidrug transporter LmrA in L. lactis shows overlap with that of the human multidrug resistance P-glycoprotein, the TEXANs LmrP and Bmr, and Mini TEXANs Smr, EmrE and QacC. On the other hand, the substrate specificity of these multidrug transporters is very different from that of the TetA(B) and other transporters dedicated to the efflux of a specific drug or class of drugs.