Microbial Multidrug Efflux: Introduction

J. Mol. Microbiol. Biotechnol. (2001) 3(2): 143-144. MicrobialJMMB Multidrug Symposium Efflux 143

Ian T. Paulsen1*, and Kim Lewis2 to structural determination. The article by Alex Neyfakh presents this approach with the crystallographic analysis 1The Institute for Genomic Research, 9712 Medical of the purified ligand-binding domain of BmrR, the regulator Center Drive, Rockville, MD, 20850, USA of expression of the multidrug efflux transporter Bmr. BmrR 2Biotechnology Center, Department of Chemical and was shown to bind multiple hydrophobic substrates with Biological Engineering, Tufts University, 4 Colby Street, micromolar affinities through multiple electrostatic and Medford, MA 02155 hydrophobic interactions. The work on BmrR is the first important contribution to our understanding of the general principles of multidrug recognition. The phenomenon of multidrug efflux, whereby a single Another approach for examining the basis of drug transporter is capable of recognizing and transporting binding is to generate transporter mutants with alterations multiple drugs, with no apparent common structural in substrate specificity. Two examples of this are presented similarity, was first described in higher eukaryotes where in the articles by Bibi et al. and Brown and Skurray which P-glycoprotein was found to provide resistance to describe the mutagenesis and characterization of the anticancer chemotherapeutic agents via an ATP-driven multidrug efflux proteins MdfA of Escherichia coli and QacA efflux process. In the late 1980s and early 1990s, it became of Staphylococcus aureus, respectively. In each case, apparent that multidrug efflux systems were also present mutations have been isolated that affect the binding of only in microorganisms, with the identification of bacterial particular substrates and hence implicate particular multidrug transporters such as Bmr from Bacillus subtilis, residues in determining substrate specificity. Shimon QacA from Staphylococcus aureus and EmrB from Schuldiner’s group presents an extensive mutagenic Escherichia coli. Since that time, the number of analysis of the small multidrug efflux protein EmrE, using characterized multidrug efflux transporters has expanded cysteine-scanning mutagenesis. Approximately half of dramatically, and it appears from genomic analyses that amino acid residues in this transporter have been targeted multidrug efflux systems are probably essentially using this approach and biochemical characterization of ubiquitous. This symposium includes sixteen reviews that the mutants has identified residues involved in substrate together provide a comprehensive overview of the current binding. From these and mutagenic studies of other types status of research in the field of microbial multidrug efflux. of transporters it is becoming apparent that only a few Since the first studies on microbial multidrug efflux, residues in each transporter appear to directly interact with one focus of research and passionate debate has been on the transported substrate. how multidrug pumps function, i.e., what is the molecular Van Veen and colleagues have used Lactococcus mechanism of recognizing and transporting multiple lactis LmrA as a model for understanding the function of different drugs with dissimilar structures? This puzzle is ABC multidrug efflux pumps and have obtained evidence further compounded by the necessity for a transporter to that it may function by an alternating two-site mechanism discriminate between its substrate drugs and all cellular of transport. In this model, hydrolysis of ATP is coupled to compounds. drug efflux through the movement of an inward-facing high- One of the main barriers towards an understanding of affinity binding site with bound drug to an outward-facing the molecular mechanism of drug efflux systems in low-affinity binding site, from which the drug can dissociate. particular and membrane transporters in general is the lack Another interesting feature of LmrA, which it appears to of a high resolution structure for these proteins. Membrane share with a variety of other multidrug efflux systems, is proteins are notoriously difficult to purify and crystallize. that substrates can be extruded directly from the The article from Peter Henderson and collaborators phospholipid bilayer. presents an overview of the art of membrane transport Another related topic is how do drug efflux systems protein purification with details on the purification of composed of multiple protein constituents function? Nikaido multidrug, sugar and other transporters. In recent years and Zgurskaya have examined the molecular mechanism there have been high resolution structures determined for of the E. coli multicomponent AcrB-AcrA-TolC multidrug representatives of porins, electron transporters, channels efflux system, which is composed of an inner membrane and ion pumps, thus there has been a gradual transition drug efflux protein, AcrB; an accessory membrane fusion from structural membrane proteins to transporters for small protein, AcrA; and the TolC outer membrane channel. ligands and then towards larger ligands. While there are Crosslinking studies have suggested that AcrA and AcrB still no structures available for multidrug transporters or interact with each other, but that TolC may only be for transporters of other large solutes, at least this apparent transiently associated with the AcrAB complex. Evidence progression provides hope for the future. is accumulating that the role of AcrA is to bring the inner One approach around this problem is to examine the and outer membranes closer together. multidrug binding capabilities of proteins more amenable Another contentious question in the study of multidrug efflux pumps is: what is the native physiological role of drug efflux systems? Is it in detoxification? Or do multidrug *For correspondence. Email [email protected]; Tel. 1-301-8383531; efflux pumps have other physiologically relevant substrates Fax. 1-301-8380208.

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Order from caister.com/order 144 Paulsen and Lewis and only transport drugs fortuitously or opportunistically? which are missed by classical pharmaceutical screens. Shafer et al. describe efflux pumps in the human pathogen Finally, the article by Lomovskaya and Watkins reviews Neisseria gonorrhoea which not only are involved in the contribution of efflux pumps to clinically significant resistance to clinically useful antibiotics, but which may resistance for a range of chemotherapeutic agents, and also contribute to pathogenicity by providing resistance to then explores the possibilities of developing efflux pump antibacterial compounds that bathe mucosal surfaces in inhibitors with activity against a variety of multidrug efflux the host. The paper by Kim Lewis analyses substrate pumps. Clinical use of efflux pump inhibitors may serve to preferences of MDRs in an attempt to glean out their decrease intrinsic resistance in pathogens and lower the possible natural function; one of the conclusion from this frequency of emergence of resistant strains for various analysis is that amphipathic cations might have been the antibiotics. substances that originally fueled the evolution of MDR pumps. Pseudomonas aeruginosa is notoriously intrinsically resistant to a wide selection of antimicrobial agents, the article by Keith Poole reviews the major contribution to this resistance provided by the Mex multidrug efflux pumps. Ouellette et al. review the occurrence of drug efflux systems in parasitic protozoa, such as Leishmania and Plasmodium, where ABC efflux systems have been implicated in resistance to antimonial compounds in Leishmania, and chloroquine resistance in Plasmodium. Terry Krulwich and colleagues examine the eccentricities of the tetracycline efflux proteins Tet(L) and Tet(K), which in addition to transporting a tetracycline– divalent metal ion complex in exchange for protons, can also catalyze sodium and potassium ion exchange for protons, as well as sodium or potassium ion or tetracycline- metal cation for potassium ion. The multiple catalytic modes and different physiological roles (e.g., Tet(L) appears to be involved in potassium ion acquisition, as well as tetracycline, sodium ion and alkali resistance) of these transporters raises the possibility that selective pressures other than drugs may influence the dissemination and retention of drug efflux systems amongst microbial populations. The development of complete genome sequencing and the burgeoning of post-genomic approaches, such as microarray expression analysis and large scale gene knockout studies, holds much promise for providing insights into the phenomenon of multidrug efflux. Paulsen et al. presents an analysis of the overall transporter and the predicted drug transporter content of microorganisms based on their complete genome sequences. The ability to use bioinformatics to predict all likely multidrug efflux systems then allows experimental approaches to investigate or validate these transporters. Two examples of systematic knockout analysis of multidrug efflux genes are described in Rogers et al. and Davis et al. In the case of Rogers et al., a series of single and multiple deletions of 7 multidrug ABC transporters and 2 transcriptional activation factors in Saccharomyces cerevisiae were analyzed. Davis et al. presents the first published example of the systematic interruption of each of the multidrug efflux genes predicted in a single organism. They generated knockouts in each of the 31 predicted multidrug transporters in Enterococcus faecalis. As well as providing insight into the overall drug transport capabilities of these organisms, both of these studies have led to the generation of strains that are hypersensitive to a broad range of antibacterial compounds and hence may be useful in screens for novel antimicrobial compounds to detect low abundance or weakly-active lead structures,