Microbial Multidrug Efflux Chapter Abstracts
Chapter 1: Microbial Multidrug Efflux: Introduction
Ian T. Paulsen, and Kim Lewis
Abstract
NO ABSTRACT is available. The first paragraph for this chapter is as follows:
The phenomenon of multidrug efflux, whereby a single transporter is capable of recognizing and transporting multiple drugs, with no apparent common structural similarity, was first described in higher eukaryotes where P-glycoprotein was found to provide resistance to anticancer chemotherapeutic agents via an ATP-driven efflux process. In the late 1980s and early 1990s, it became apparent that multidrug efflux systems were also present in microorganisms, with the identification of bacterial multidrug transporters such as Bmr from Bacillus subtilis, QacA from Staphylococcus aureus and EmrB from Escherichia coli. Since that time, the number of characterized multidrug efflux transporters has expanded dramatically, and it appears from genomic analyses that multidrug efflux systems are probably essentially ubiquitous. This volume includes sixteen reviews that together provide a comprehensive overview of the current status of research in the field of microbial multidrug efflux.
Chapter 2: Comparative Genomics of Microbial Drug Efflux Systems
Ian T. Paulsen, Joan Chen, Karen E. Nelson, and Milton H. Saier, Jr
Abstract
The complete genome sequences of 36 microorganisms have now been published and this wealth of genome data has enabled the development of comparative genomic and functional genomic approaches to investigate the biology of these organisms. Comparative genomic analyses of membrane transport systems have revealed that transporter substrate specificities correlate with an organism's lifestyle. The types and numbers of predicted drug efflux systems vary dramatically amongst sequenced organisms. Microarray and gene knockout studies to date have suggested that predicted drug efflux genes often appear to be a) non-essential and b) expressed at detectable levels under standard laboratory growth conditions.
Chapter 3: The Ostensible Paradox of Multidrug Recognition
Alex A. Neyfakh
Abstract
The ability of multidrug-efflux transporters to recognize scores of dissimilar organic compounds has always been considered paradoxical because of its apparent contradiction to some of the basic dogmas of biochemistry. In order to understand, at least in principle, how a protein can recognize multiple compounds, we analysed the transcriptional regulator of the Bacillus subtilis multidrug transporter Bmr. This regulator, BmrR, binds multiple dissimilar hydrophobic cations and, by activating expression of the Bmr transporter, causes their expulsion from the cell. Crystallographic analysis of the complexes of the inducer-binding domain of BmrR with some of its inducers revealed that ligands penetrate the hydrophobic core of the protein, where they form multiple van der Waals and stacking interactions with hydrophobic amino acids and an electrostatic bond with the buried glutamate. Mutational analysis of the binding site suggests that each ligand forms a unique set of atomic contacts with the protein: each tested mutation exerted disparate effects on the binding of different ligands. The example of BmrR demonstrates that a protein can bind multiple hydrophobic compounds with micromolar affinities by using only electrostatic and hydrophobic interactions. Its ligand specificity can be further broadened by the flexibility of the binding site. It appears, therefore, that the commonly expressed fascination with the relaxed substrate specificity of multidrug transporters is misdirected and originates from an almost exclusive familiarity with the more sophisticated processes of specific molecular recognition that predominate among proteins analyzed to date.
Chapter 4: Precious Things Come in Little Packages
Shimon Schuldiner, Dorit Granot, Sonia Steiner Mordoch, Shira Ninio, Dvir Rotem, Michael Soskin and Hagit Yerushalmi
Abstract
The 110-amino acid multidrug transporter from E. coli, EmrE, is a member of the family of MiniTexan or Smr drug transporters. EmrE can transport acriflavine, ethidium bromide, tetraphenylphosphonium (TPP+), benzalkonium and several other drugs with relatively high affinities. EmrE is an H+/drug antiporter, utilizing the proton electrochemical gradient generated across the bacterial cytoplasmic membrane by exchanging two protons with one substrate molecule.
The EmrE multidrug transporter is unique in its small size and hydrophobic nature. Hydropathic analysis of the
EmrE sequence predicts four a-helical transmembrane segments. This model is experimentally supported by FTIR
studies that confirm the high a-helicity of the protein and by high-resolution heteronuclear NMR analysis of the protein
structure. The TMS of EmrE are tightly packed in the membrane without any continuous aqueous domain, as was shown
by Cysteine scanning experiments. These results suggest the existence of a hydrophobic pathway through which
the substrates are translocated. EmrE is functional as a homo-oligomer as suggested by several lines of evidence,
including co-reconstitution experiments of wild-type protein with inactive mutants in which negative dominance has been
observed. EmrE has only one membrane embedded charged residue, Glu-14, that is conserved in more than fifty
homologous proteins and it is a simple model system to study the role of carboxylic residues in ion-coupled transporters. We
have used mutagenesis and chemical modification to show that Glu-14 is part of the substrate-binding site. Its role in
proton binding and translocation was shown by a study of the effect of pH on ligand binding, uptake, efflux and
exchange reactions. We conclude that Glu-14 is an essential part of a binding site, common to substrates and protons.
The occupancy of this site is mutually exclusive and provides the basis of the simplest coupling of two fluxes. Because
of some of its properties and its size, EmrE provides a unique system to understand mechanisms of substrate
recognition and translocation.
Chapter 5: Structure, Function and Regulation of the Staphylococcal Multidrug Efflux Protein QacA
Melissa H. Brown, and Ronald A. Skurray
Abstract
The QacA multidrug exporter from Staphylococcus aureus mediates resistance to a wide array of monovalent or divalent cationic, lipophilic, antimicrobial compounds. QacA provides resistance to these various compounds via a proton motive force-dependent antiport mechanism that conforms to classical Michaelis-Menten kinetics. Fluorescent transport assays have demonstrated that this QacA:substrate interaction occurs with high affinity and competition studies have shown that QacA-mediated ethidium export is competitively inhibited by other monovalent cations, and non-competitively inhibited by divalent cations, suggesting that monovalent and divalent cations bind at distinct sites on the QacA protein. The closely related export protein QacB, mediates lower levels of resistance to divalent cations compared to QucA, presumably because it lacks a high affinity-binding site for them. The cell membrane has been identified as the origin of QacA-mediated efflux; substrates are bound and expelled from within this hydrophobic environment.
Regulation of qacA expression is achieved via the trans-acting repressor protein, QacR. QacR belongs to the TetR family of transcriptional repressor proteins, which all possess a helix-turn-helix DNA-binding domain at their N-terminal ends, and have highly divergent C-termini postulated to be involved in the binding of inducing compounds. QacR specifically binds to an inverted repeat, IR1, which has been identified as the qacA operator region, and overlaps the identified promoter sequence for qacA. QacR, like the multidrug export protein whose expression it regulates, has been shown to interact directly with a number of structurally-dissimilar compounds.
Chapter 6: MdfA, an Interesting Model Protein for Studying Multidrug Transport
Eitan Bibi, Julia Adler, Oded Lewinson, and Rotem Edgar
Abstract
The resistance of cells to many drugs simultaneously (multidrug resistance) often involves the expression of membrane transporters (Mdrs); each can recognize and expel a broad spectrum of chemically unrelated drugs from the cell. Despite extensive research for many years, the actual mechanism of multidrug transport is still largely unknown. In addition to general questions dealing with energy coupling, the molecular view of substrate recognition by Mdrs is generally obscure. This mini-review describes structural and functional properties of the Escherichia coli Mdr, MdfA, and discusses the possibility that this transporter may serve as a model for studying the multidrug recognition phenomenon and the mechanism of multidrug transport.
Chapter 7: Enterococcus faecalis Multi-Drug Resistance Transporters: Application for Antibiotic Discovery
Deborah R. Davis, James B. McAlpine, Christopher J. Pazoles, M. Kelly Talbot, Elisabeth A. Alder, Abbie C. White, Brandie M. Jonas, Barbara E. Murray, George M. Weinstock, and Bruce L. Rogers
Abstract
Using bioinformatics approaches, 34 potential multi-drug resistance (MDR) transporter sequences representing 4 different transporter families were identified in the unannotated Enterococcus faecalis database (TIGR). A functional genomics campaign generating single-gene insertional disruptions revealed several genes whose absence confers significant hypersensitivities to known antimicrobials. We constructed specific strains, disrupted in a variety of previously unpublished, putative MDR transporter genes, as tools to improve the success of whole-cell antimicrobial screening and discovery. Each of the potential transporters was inactivated at the gene level and then phenotypically characterized, both
with
single disruption mutants and with 2-gene mutants built upon a
D norA deleted strain background. <
Chapter 8: Molecular Basis of Multidrug Transport by ATP-Binding Cassette Transporters: A Proposed Two-Cylinder Engine Model
Hendrik W. van Veen, Christopher F. Higgins, and Wil N. Konings
Abstract
ATP-binding cassette multidrug transporters are probably present in all living cells, and are able to export a variety of structurally unrelated compounds at the expense of ATP hydrolysis. The elevated expression of these proteins in multidrug resistant cells interferes with the drug-based control of cancers and infectious pathogenic microorganisms. Multidrug transporters interact directly with the drug substrates. Insights into the structural elements in drug molecules and transport proteins that are required for this interaction are now beginning to emerge. However, much remains to be learned about the nature and number of drug binding sites in the transporters, and the mechanism(s) by which ATP hydrolysis is coupled to changes in affinity and/or accessibility of drug binding sites. This review summarizes recent advances in answering these questions for the human multidrug resistance P-glycoprotein and its prokaryotic homolog LmrA. The relevance of these findings for other ATP-binding cassette transporters will be discussed.
Chapter 9: Prokaryote Multidrug Efflux Proteins of the Major Facilitator Superfamily: Amplified Expression, Purification and Characterisation
Alison Ward, Chris Hoyle, Sarah Palmer, Scott Morrison, Kate Langton, John O'Reilly, Nick Rutherford, Jeff Griffith, Martin Pos, Bert Poolman, Mick Gwynne, Geoff Badman, Richard Herbert, and Peter Henderson
Abstract
In bacterial genomes 3-12% of open reading frames are predicted to encode membrane transport proteins. These proteins can be vital for antibiotic efflux, protein/toxin secretion, cell nutrition, environmental sensing, ATP synthesis, and other functions. Some, such as the multidrug efflux proteins, are potential targets for the development of new antibacterials and also for applications in biotechnology. In general membrane transport proteins are poorly understood, because of the technical difficulties involved in isolating sufficient protein for elucidation of their structure-activity relationships. We describe a general strategy for the amplified expression, purification and characterisation of prokaryotic multidrug efflux proteins of the 'Major facilitator superfamily' of transport proteins, using the Bacillus subtilis multidrug resistance protein, 'Bmr', and the Staphylococcus aureus norfloxacin resistance protein 'NorA' as examples.
Chapter 10: Multidrug Resistance and ABC Transporters in Parasitic Protozoa
Marc Ouellette, Danielle Légaré and Barbara Papadopoulou
Abstract
Drug resistance is an important problem in parasitic protozoa. We review here the role of ABC transporters in drug resistance in parasites. We have concentrated on gene and gene products for which there is a strong evidence for their role in resistance.
Chapter 11: The Pleiotropic Drug ABC Transporters from Saccharomyces cerevisiae
Anabelle Decottignies, Bruce Rogers, Marcin Kolaczkowski, Elvira Carvajal, Elisabetta Balzi, Gwenaelle Conseil, Kyoko Niimi, Attilio Di Pietro, Brian C. Monk and André Goffeau
Abstract
The Saccharomyces cerevisiae genome contains 16 genes encoding full-size ABC transporters. Each comprises two nucleotide binding folds (NBF) alternating with transmembrane domains (TM). We have studied in detail three plasma membrane multidrug exporters: Pdr5p (TC3.A.1.205.1) and Snq2p (TC3.A.1.205.2) which share NBF-TM-NBF-TM topology as well as Yor1p (TC3.A.1.208.3) which exhibits the reciprocal TM-NBF-TM-NBF topology. The substrate specificity of Pdr5p, Snq2p and Yor1p are largely, but not totally, overlapping as shown by screening the growth inhibition by 349 toxic compounds of combinatorial deletants of these three ABC genes. Multiple deletion of 7 ABC genes (YOR1, SNQ2, PDR5, YCF1, PDR10, PDR11 and PDR15) and of two transcription activation factors (PDR1 and PDR3) renders the cell from 2 to 200 times more sensitive to numerous toxic coumpounds including antifungals used in agriculture or medicine. The use of the pdr1-3 activating mutation and when necessary of the PDR5 promoter in appropriate multideleted hosts allow high levels of expression of Pdr5p, Snq2p or Yor1p. These overexpressed proteins exhibit ATPase activity in vitro and confer considerable multiple drug resistance in vivo. The latter property can be used for screening specific inhibitors of fungal and other ABC transporters.
Chapter 12: AcrAB and Related Multidrug Efflux Pumps of Escherichia coli
Hiroshi Nikaido and Helen I. Zgurskaya
Abstract
The AcrAB system of Escherichia coli is a multidrug efflux system composed of an RND-type transporter AcrB and a periplasmic accessory protein AcrA, and pumps out a wide variety of lipophilic and amphiphilic inhibitors directly into the medium, presumably through the TolC outer membrane channel. AcrA, a highly elongated protein, is thought to bring the outer and inner membranes closer. It forms a trimer that interacts with a monomeric AcrB, which was shown by in vitro reconstitution to be a proton antiporter. Details of interaction between the AcrAB complex and TolC remain a major topic for study.
Chapter 13: Antimicrobial Efflux Systems Possessed by Neisseria gonorrhoeae and Neisseria meningitidis Viewed as Virulence Factors
Corinne Rouquette-Loughlin, Wendy L. Veal, Eun-Hee Lee, Leticia Zarantonelli, Jacqueline T. Balthazar, and William M. Shafer
Abstract
Efflux pumps can make a significant contribution to the capacity of bacteria to resist the action of antibiotics. Certain efflux pumps also recognize antimicrobial agents that are present in their respective hosts and their ability to export toxic agents could enhance bacterial survival during infection prior to appearance of cellular or humoral host defensive systems. This review is concerned with the principal efflux pumps possessed by two closely related strict human pathogens, Neisseria gonorrhoeae and Neisseria meningitidis. Specific emphasis is placed on the organization of the structural genes encoding the mtr and far efflux pumps, the substrates (often host-derived) recognized by these pumps, and the cis- and trans-acting transcriptional factors that regulate efflux pump gene expression in gonococci and meningococci. The overriding theme of this review is that the efflux pumps possessed by these pathogens likely contribute to their pathogenic mechanisms by providing a means to escape a number of antimicrobial compounds that bathe mucosal surfaces.
Chapter 14: Inhibition of Efflux Pumps as a Novel Approach to Combat Drug Resistance in Bacteria
Olga Lomovskaya and Will Watkins
Abstract
Efflux mechanisms have become broadly recognized as major components of resistance to many classes of antibiotics. Some efflux pumps selectively extrude specific antibiotics, while others, referred to as multidrug resistance (MDR) pumps, expel a variety of structurally diverse compounds with differing antibacterial modes of action. There are numerous potentially beneficial consequences of the inhibition of efflux pumps in improving the clinical performance of various antibiotics, and several companies and research laboratories have initiated programs to discover and develop efflux pump inhibitors. This review will summarize recent achievements in this new, very exciting and equally challenging field.
Chapter 15: Functions of Tetracycline Efflux Proteins That Do Not Involve Tetracycline
Terry A. Krulwich, Jie Jin, Arthur A. Guffanti, and David H. Bechhofer
Abstract
Tet(L) and Tet(K) are specific antibiotic-resistance determinants. They catalyze efflux of a tetracycline(Tc)-divalent metal complex in exchange for protons, as do other Tet efflux proteins. These Tet proteins also catalyze Na+ and K+ exchange for protons. Each of the "cytoplasmic substrates",
Na+, K+ and the Tc-metal ion complex, can also be exchanged for
K+, a catalytic mode that accounts for the long-recognized
K+ uptake capacity conferred by some Tet proteins. The
multiple catalytic modes of Tet(L) and Tet(K) provide potential new avenues for development of inhibitors of these efflux
systems as well as avenues for exploration of structure-function relationships. The multiple catalytic modes of Tet(L), which
is chromosomally encoded in Bacillus subtilis, also correspond to diverse physiolog-ical roles, including roles in antibiotic-, Na+-, and alkali-resistance as well as K+ acquisition. The use of K+ as an external coupling ion may contribute not
only to the organism's K+ uptake capacity but also to its ability to exclude
Na+ and Tc at elevated pH values. Regulation of
the
chromosomal tetL gene by Tc has been proposed to involve a translational re-initiation mechanism that is novel for an antibiotic-resistance gene and increases Tet expression seven-fold. Other elements of tetL expression and its regulation are already evident, including gene amplification and use of multiple promoters. However, further studies are required to clarify the full panoply of regulatory mechanisms, and their integration to ensure different levels of tetL expression that are optimal for its different functions. It will also be of interest to investigate the implications of Tet(L) and Tet(K) multifunctionality on the emergence and persistence of these antibiotic-resistance genes.
Chapter 16: In Search of Natural Substrates and Inhibitors of MDR Pumps
Kim Lewis
Abstract
The function of microbial MDRs remains a hotly debated subject. Given the very broad substrate specificities of some MDRs, like the RND pumps that can extrude all classes of amphipathic compounds (cationic, neutral, and anionic), it seems difficult to develop a rationale for pinpointing possible natural substrates of these translocases. At the same time, several clues can be used to guide our search for natural MDR substrates. One is the fact that amphipathic cations appear to be the preferred substrates of MDRs. These substances are extruded by MDRs of all 5 known families and are the almost exclusive substrates of SMR and MF family MDRs. The universal nature of amphipathic cations as MDR substrates suggests that these were the substances that fueled the evolution of MDR pumps. Two factors apparently favored this particular class of molecules for the role of original MDR substrates need and opportunity. Unlike other substances, amphipathic cations accumulate in the cell driven by the membrane potential, which makes cations potentially the most dangerous toxins. At the same time, amphipathic cations are highly hydrated and do not permeate the membrane as readily as neutral compounds, making it feasible to design a defense based on an efflux pump. The paucity of known cationic (non-basic) antimicrobials might be a result of using MDR-expressing microbial cells for antibiotic discovery. Plant amphipathic cations, the berberine alkaloids, are good MDR substrates. The Berberis plants produce 5'-methoxyhydnocarpin-D, an MDR inhibitor that potentiates the action of berberine. It is suggested that the further evolution of MDR pumps was determined largely by the barrier function of the membrane they reside in. Thus Gram negative bacteria have an outer membrane barrier that slows the penetration of virtually all amphipathic molecules, and transenvelope MDRs of the RND and EmrAB-type extrude their substrates across this barrier. A low permeability of the cytoplasmic membrane of yeast similarly allows for the operation of broad-specificity ABC and MF MDRs. The presence of MDR sensors that regulate the expression of some MDR pumps strongly suggests that defense against external toxins is the function of these MDRs. The BmrR transcriptional activator of the MerR family induces expression of the Bmr pump in B. subtilis and is a sensor specifically designed to recognize amphipathic cations. Similarly, the QacR repressor binds chemically unrelated cations, which leads to the expression of the QacA pump in S. aureus. In E. coli, the EmrR sensor of the MarR repressor family binds unrelated neutral molecules, allowing for expression of the transenvelope EmrAB pump.
Chapter 17: Multidrug Efflux Pumps and Antimicrobial Resistance in Pseudomonas aeruginosa and Related Organisms
Keith Poole
Abstract
Pseudomonas aeruginosa is an opportunistic human pathogen characterized by an innate resistance to multiple antimicrobial agents. A major contribution to this intrinsic multidrug resistance is provided by a number of broadly-specific multidrug efflux systems, including MexAB-OprM and MexXY-OprM. In addition, these and two additional tripartite efflux systems, MexCD-OprJ and MexEF-OprN, promote acquired multidrug resistance as a result of mutational hyperexpression of the efflux genes. In addition to antibiotics, these pumps promote export of numerous dyes, detergents, inhibitors, disinfectants, organic solvents and homoserine lactones involved in quorum sensing. The efflux pump proteins are highly homologous and consist of a cytoplasmic membrane-associated drug-proton antiporter of the Resistance-Nodulation-Division (RND) family, an outer membrane channel-forming protein [sometimes called outer membrane factor (OMF)] and a periplasmic membrane fusion protein (MFP). Homologues of these systems have been described in Stenotrophomonas maltophilia, Burkholderia cepacia, Burkholderia pseudomallei and the non-pathogen Pseudomonas putida, where they play a role in export of and resistance to multiple antimicrobial agents and/or organic solvents. Although the natural function of these multidrug efflux systems is largely unknown, their contribution to antibiotic resistance and their conservation in a number of important human pathogens makes them logical targets for therapeutic intervention.
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