commentary antibiotic efflux pumps · the drug efflux pumps in eucaryotic cells ( ; drug efflux...
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Antibiotic Efflux PumpsFrancoise Van Bambeke,*† Elisabetta Balzi‡§ and Paul M. Tulkens*
*UNITE DE PHARMACOLOGIE CELLULAIRE ET MOLECULAIRE, UNIVERSITE CATHOLIQUE DE LOUVAIN, BRUSSELS; AND
‡UNITE DE BIOCHIMIE PHYSIOLOGIQUE, UNIVERSITE CATHOLIQUE DE LOUVAIN, LOUVAIN-LA-NEUVE, BELGIUM
ABSTRACT. Active efflux from procaryotic as well as eucaryotic cells strongly modulates the activity of a largenumber of antibiotics. Effective antibiotic transport has now been observed for many classes of drug efflux pumps.Thus, within the group of primary active transporters, predominant in eucaryotes, six families belonging to theATP-binding cassette superfamily, and including the P-glycoprotein in the MDR (Multi Drug Resistance) groupand the MRP (Multidrug Resistance Protein), have been recognized as being responsible for antibiotic efflux.Within the class of secondary active transporters (antiports, symports, and uniports), ten families of antibioticefflux pumps have been described, distributed in five superfamilies [SMR (Small Multidrug Resistance), MET(Multidrug Endosomal Transporter), MAR (Multi Antimicrobial Resistance), RND (Resistance NodulationDivision), and MFS (Major Facilitator Superfamily)]. Nowadays antibiotic efflux pumps are believed tocontribute significantly to acquired bacterial resistance because of the very broad variety of substrates theyrecognize, their expression in important pathogens, and their cooperation with other mechanisms of resistance.Their presence also explains high-level intrinsic resistances found in specific organisms. Stable mutations inregulatory genes can produce phenotypes of irreversible multidrug resistance. In eucaryotes, antibiotic effluxpumps modulate the accumulation of antimicrobials in phagocytic cells and play major roles in theirtransepithelial transport. The existence of antibiotic efflux pumps, and their impact on therapy, must now betaken fully into account for the selection of novel antimicrobials. The design of specific, potent inhibitorsappears to be an important goal for the improved control of infectious diseases in the near future. BIOCHEM
PHARMACOL 60;4:457–470, 2000. © 2000 Elsevier Science Inc.
KEY WORDS. antibiotic; efflux pump; resistance; transport; chemotherapy; infection
Biological membranes most likely appeared very early onduring evolution to isolate hydrophilic microdomains fromthe surrounding medium, allowing catalyzed reactions tooccur in an efficient manner. Biomembranes, accordingly,constitute efficient barriers towards hydrophilic molecules,most of which can penetrate cells only by specific inwardtransport systems or find their entry restricted to theendocytic pathway. A contrario, biomembranes are easilycrossed by amphiphilic compounds, since these are able todiffuse through both the hydrophilic and the hydrophobicdomains of the bilayer. Therefore, it is not surprising thatmechanisms were devised, also very early, to protect cellsfrom the disordered invasion by amphiphilic molecules,many of which are endowed with biological activitiesleading to potentially harmful effects. A major mechanismin this respect is constituted by active outward transport.Although efflux systems have been known for many years,their importance, both in terms of number and variety ofsubstrates, has become clearly recognized only very re-cently. Drugs are often amphiphilic, whether by selectionor by design, ensuring their wide tissue distribution and/or
their penetration into membrane-protected compartments.Therefore, it comes as no surprise that many drugs shouldfall into this category of exogenous compounds for whichefflux mechanisms, globally referred to as ‘drug effluxpumps,’ are numerous and fairly active . Thus, more andmore membrane-spanning proteins involved in the outwardtransport of a surprisingly large variety of drugs have beenrecognized and characterized over the last years in almostall cell types, from procaryotes and archebacteria throughfungi and higher eucaryotes. This now raises the question ofwhich drug is not transported. For eucaryotic cells, drugefflux pumps have been viewed by many authors as com-plementing the cytochrome P450—or other enzyme-baseddetoxification systems (e.g. Ref. 2) to achieve efficientprotection against ‘chemical invaders’. Both systems, in-deed, show broad specificity and may even work in synergy(see, for example, the concept of a concerted barrier inenterocytes [3, 4]). Drug efflux, indeed, decreases the loadon enzyme-mediated detoxification systems, thereby avoid-ing their saturation, while chemical modifications by theenzyme-based systems, which usually increase the am-phiphilicity of drugs, provide drug pumps with bettersubstrates [2, 5]. Moreover, most drug efflux pumps have abroad substrate specificity and, therefore, may deal with awide range of drugs of completely unrelated pharmacolog-ical classes. The present commentary will focus on antibi-
† Corresponding author: F. Van Bambeke, Pharm.D., UCL 73.70 avenueE. Mounier 73, B-1200 Bruxelles, Belgium. Tel. (32) 2-764-73-78; FAX(32) 2-764-73-73; E-mail: [email protected]
§ Present affiliation: DG Research, European Commission, Brussels,Belgium.
Biochemical Pharmacology, Vol. 60, pp. 457–470, 2000. ISSN 0006-2952/00/$–see front matter© 2000 Elsevier Science Inc. All rights reserved. PII S0006-2952(00)00291-4
otics, since the role of drug efflux mechanisms, as a majorcause of bacterial resistance, has been recognized onlyrecently. It therefore needs to be stressed and examined indetail if one wishes to cope with this major challenge inanti-infective therapy. Quite interestingly, also, it nowappears that antibiotic transport mechanisms play impor-tant roles in eucaryotic cells by modulating the pharmaco-logical and toxicological profile of antibiotics. Thus, theidentification and characterization of the correspondinggenes and gene products, in bacteria as well as in eucaryoticcells, have not only an immediate, fundamental interest butalso open interesting perspectives for new and more ratio-nal developments in chemotherapy.
STRUCTURE AND PHYLOGENY
Transporters can be classified on the basis of three maincriteria, namely the energy source, the phylogenic relation-ship, and the substrate specificity. A 4-digit nomenclaturehas been proposed recently, constructed in analogy withenzyme nomenclature and in which the first group of digitsrefers to the mode of transport and energy source, thesecond and the third to the phylogeny (superfamilies andfamilies), and the fourth to the substrate ( [6, 7]; see also theweb site ,http://www.biology.ucsd.edu/;msaier/transport/titlepage.html.). The so-called primary active transport-ers use various forms of energy and constitute the bulk ofthe drug efflux pumps in eucaryotic cells ( ; drug effluxtransporters are classically energized by ATP). The second-ary active transporters, acting as symports and antiports(i.e. coupling the drug efflux to the downhill transport of anion along a concentration gradient), are predominant inbacteria . Within each of these two main classes, phylo-genic studies have led to the recognition of superfamilies,families, and clusters, in correlation with their substratespecificity . Yet, most drug efflux pumps confer a multi-drug resistance phenotype, corresponding to the largevariety of substrates they may recognize, including severalclasses of antibiotics as well as non-antibiotic drugs. Anti-biotic-specific efflux pumps appear to be restricted to thoseorganisms producing antibiotics and are often an integralpart of the corresponding biosynthetic pathway .
Table 1 lists the main drug efflux pumps acting onantibiotics described thus far, within the correspondingfamilies of drug transporters. We also mention in whichorganisms they are mainly found (common bacteria, thespecial category of antibiotic-producing organisms, com-mon fungi, and mammalian cells). The secondary activetransporters, being of a simpler structure, will be presentedfirst. In this group, pumps acting on antibiotics are found inthe so-called SMR* family, the MET family, the RND
family, the MFS, and the MAR family. SMR have beenfound thus far only in bacteria , whereas MET [10, 11]seem to be restricted to superior eucaryotes. Other familiesare widely distributed, but antibiotic efflux pumps have notbeen described in all organisms (viz. the RND ). TheSMR family can be divided into two groups of geneproducts; one of them is immediately related to drug efflux,whereas the other is not, which suggests that evolutionfrom the ancestor transporter towards a gene product witha drug efflux phenotype occurred only once [9, 13]. Highlyconserved motifs are involved in transport activities as wellas in binding of the substrates. MET members present ageneral organization similar to that of SMR members butare characterized by signal motifs rich in tyrosines at theirC-termini, which direct them to intracellular compart-ments of eucaryotic cells . Members of the RNDsuperfamily share common topological features, namely 2large extracytoplasmic loops and 12 transmembrane seg-ments resulting from an internal duplication of a geneencoding a 6-transmembrane segment (see Ref. 12 forreview). All the known members of this superfamily havethe function of efflux transporter, but proteins conferringmultidrug resistance are grouped in two of the sevenfamilies recognized in this superfamily. Thus, the HAE1family is largely predominant and includes the well knowndrug efflux pumps of Gram-negative bacteria with verybroad substrate specificity. They probably all derive from asingle ancestor [13, 14]. In contrast, there is only onemember characterized so far in the HAE2 family. Yet,Mycobacterium tuberculosis possesses 10 genes encodingproteins of this family, which could explain the poorsensitivity of this organism to many common antibiotics, ifall of them were to correspond to antibiotic efflux pumps. The situation is more complex for drug efflux pumpsbelonging to the MFS [15, 16]. Indeed, drug-specific andmultidrug efflux pumps appear to be randomly interspersedin families (see, for example, the DHA2 [also calledDHA14] family), indicating that narrowing and broadeningof specificity have occurred repeatedly during evolution[13, 14]. Yet, they are also thought to derive from acommon ancestor. Moreover, sequence analysis suggeststhat a simple duplication of a gene encoding a 6-transmem-brane segment protein led to the appearance of the 12-transmembrane segment family (DHA1 [also calledDHA12]). Then, the 14-transmembrane segment family(DHA2 [also called DHA14]) evolved from the insertion ofan increasingly hydrophobic central loop of the DHA12precursor into the membrane (the DHA12 family actuallydisplays a long intracytosolic peptide loop running betweenthe 6th and the 7th transmembrane segments, which mayhave provided the necessary scaffold for these two addi-tional membrane spanning segments seen in DHA14).
* Abbreviations: ABC, ATP Binding Cassette; CFTR, Cystic FibrosisTransmembrane conductance Regulator; CT, Conjugate Transporters;DHA, Drug:H1 Antiporters; HAE, Hydrophobic Amphiphilic Efflux;MAR, Multi Antimicrobial Resistance; MET, Multidrug EndosomalTransporters; MDR, Multi Drug Resistance; MFS, Major FacilitatorSuperfamily; (c)MOAT, (canalicular) Multispecific Organic Anion
Transporters; MRP, Multidrug Resistance Proteins; OAT, Organic AnionTransporters; OCT, Organic Cation Transporters; PgP, P-glycoprotein;PDR, Pleiotropic Drug Resistance; RND, Resistance Nodulation Division;SET, Sugar Efflux Transporters; and SMR, Small Multidrug Resistance.
458 F. Van Bambeke et al.
Some of the conserved motifs throughout the MFS havebeen shown to play a role in activity. The MAR familypresents no sequence homology with MFS, but a similartopological organization .
Within the group of primary active transporters, theABC drug efflux pumps have been classified extensively infamilies according to structural homology [18–22]. Morerecently, they have been distinguished phylogenetically onthe basis of their import or export activity . Whereasimport pumps are present only in procaryotes, efflux pumpsare maintained in both procaryotes and eucaryotes, suggest-ing that selection of the transport directionality occurredbefore divergence between procaryotes and eucaryotes .ABC domains generally present a high degree of homology,whereas transmembrane domains differ between transport-ers, and might contribute to defining their substrate speci-ficity . Considering efflux pumps only, two families, theMDR and the CT2, deserve special attention since theycorrespond to the most studied and pharmacologicallyimportant transporters. They also show functional inter-changeability between different types of organisms [24, 25].The MDR family, which includes the well-known P-glycoprotein in eucaryotes (PgP, a product of the MDR1gene as shown in Table 1), is responsible for the efflux of awide range of drugs besides antibiotics, including antican-cer agents. The CT2 family, comprising MRP in superioreucaryotes and Yor1 in Saccharomyces cerevisiae, also isinvolved in the efflux of many drugs, again includingantibiotics and anticancer agents [2, 21, 26–28]. Thisfamily is phylogenetically close to the CFTR family, achloride channel, which, however, does not transport drugs(its mutation causes cystic fibrosis). The PDR transportersshare several biochemical features with the human PgP[19–21] and constitute the major class of ABC drug effluxpumps in yeasts and fungi. Finally, proteins from theDrugE1 family are involved in drug-specific efflux in anti-biotic-producing organisms [13, 23].
ORGANIZATION AND FUNCTION
Figure 1 shows in a combined fashion the topologicalorganization of the main antibiotic-extruding pumps pre-sented in Table 1 together with a schematic view of theirmode of operation and the type of antibiotics transported.The mechanisms of transport and of substrate recognition,however, remain largely unknown in most instances, andmany of the current views are based on extrapolations fromdata obtained with transporters of physiological substrates(the assumption is that proteins deriving from a commonancestor have maintained sufficient similarity not only ofstructure but also of function throughout evolution). SMR,RND, and most MFS transporters (and probably also theMET and MATE transporters) use a proton gradient as thedriving force. A minimum of 12 transmembrane segmentsseems required for activity, so that SMR transporters areprobably organized in trimers [29, 30]. The putative mech-anism of drug transport, as established by site-directed
mutagenesis of a SMR transporter, could involve thefollowing steps: (i) exchange between the drug and a protonfixed on a charged residue; (ii) translocation of the drug bya series of conformational changes driving it through ahydrophobic pathway; and (iii) replacement of the drug bya proton in the external medium and return to the initialconformational state [14, 31]. The overall result of thetransport is therefore an exchange between the drug and aproton (antiport). The residue responsible for proton ex-change in SMR could be a conserved glutamate . Thesame mechanism probably applies to MFS transport, but theproton exchanger may be a conserved arginine  (again,a conserved acidic residue may be involved in the recogni-tion of positively charged substrates ). This mode ofextrusion explains why both SMR and MFS transporterspreferentially extrude cationic molecules (see below). Lessis known about transport by RND members. Like SMR andMFS transporters, however, RND transporters possesshighly conserved charged residues in their transmembranesegments, which may play an important role in substratebinding or proton transport . In Gram-negative bacte-ria, the 2 large extracellular loops of RND are thought tointeract with two other proteins , namely the ‘mem-brane fusion protein’ (connecting the inner membrane tothe outer membrane) and the ‘outer membrane protein’(crossing this outer membrane). This tripartite proteincomplex allows the bacteria to extrude the substrate di-rectly into the external medium, bypassing the periplasmand thereby amplifying the efficacy of the transporter. It isnow proposed to be a common feature for RND, MFS, andABC efflux pumps in Gram-negative bacteria, with the‘membrane fusion proteins’ differing between transportsystems, whereas the ‘outer membrane proteins’ are proba-bly common to all three transporters . ABC transport-ers, which contain two ATP binding cassettes (ABCdomains), derive their energy from the hydrolysis of ATP.An additional characteristic of MRP transporters is thattheir activity is strictly dependent on the presence ofglutathione. Whether glutathione directly activates thetransporter, or is itself co-transported with the drug, or evenforms a conjugate to the drug, is not clear, but its weakionic character is thought to be critical (see Ref. 26 forreview). As for proton antiporters, a conformational changeof the ABC protein is necessary for drug extrusion andprobably is triggered by drug binding and ATP hydrolysis[36, 37].
The exact mechanism of drug transport is still contro-versial. Among the different models that have been pro-posed, the two most likely ones present efflux pumps asacting either like hydrophobic ‘vacuum cleaners’ or likeflippases. In the first model, the drug is thought to movefreely into the lipid phase of the membrane, then reachingthe protein and its central channel, from where it is activelyexpelled outwardly. In the second model, the drug is alsothought to reach the protein from within the membrane,but then would be flipped to the outer layer (as proposed forphosphatidylcholine flip-flop under the action of flippases).
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460 F. Van Bambeke et al.
Antibiotic Efflux Pumps 461
In contrast to the previous, more conventional models ofsimple pore-like channels oriented towards export, whichpick up their substrate from the cytosol and orient ittowards the outside of the cell thanks to a regulator valve,the two models presented assume that the drug is extractedprimarily from the inner phase of the membrane. Indeed,strong membrane anchoring is probably a common require-
ment for substrates of drug efflux pumps. Moreover, trans-port has been suggested to occur not only from the cytosolicface of the membrane of eucaryotes and Gram-positiveorganisms (see data with the MFS transporter QacA ofStaphylococcus aureus; ), but also from both the innerand the outer leaflets of the inner membrane of Gram-negative organisms, ensuring clearance from the cytosol as
FIG. 1. Topology, mechanism of action, and typical substrates of the main classes of antibiotic efflux pumps (constructed from data andschemes adapted from Refs. 14, 117, and 118). The MET and MAR families are not represented since information on those pumpsis still scanty (MET pumps possess 4 transmembrane segments, like SMR pumps; MAR pumps present the same topologicalorganization as MFS). Topology: the membrane is represented in grey, with the extracellular milieu at the top of each scheme, andproteins as a chain of circles, the solid ones corresponding to conserved motifs (identified by letters). In ABC transporters, the locationsof the ATP binding cassettes are indicated by two black circles. The 5 transmembrane segments at the N-terminal part of MRP (in thediscontinuous line square) are present in MRP1, MRP2 (also called cMOAT), and MRP3 . Numerous other organizations ofABC and transmembrane domains have been proposed (for instance, a mirror image of that shown here for the Pgp, or ABCtransporters in which transmembrane segments and ATP binding domains are not fused ). Mechanism of action: SMR, RND, andMFS transporters are drug-H1 antiporters. H1 probably is exchanged from a conserved glutamate (E) of the ‘a’ conserved domain inSMR and from a conserved arginine (R) of the ‘b’ conserved domain in MFS. Recognition of cationic drugs may imply the sameconserved glutamate for SMR transporters and a conserved acid residue (glutamate or aspartate; E in the figure) in the ‘d’ domain forMFS transporters. No corresponding data are available so far for RND transporters. ABC transporters involved in drug efflux use ATPas an energy source. In all types of transporters, the drug seems to be extracted from the membrane rather than from the cytosol. Thetransporter then could act as a flippase (catalyzing the flip-flop of the drug from the inner to the outer face of the membrane) or as a‘vacuum cleaner’ (moving the drug from the membrane to the central domain of a channel closed to the cytosolic face of the membranebut open to its extracellular face, as already shown for MDR [also known as PgP]). MRP transporters also require the presence ofglutathione, which could be conjugated to the drug prior to or during extrusion. Antibiotics: classes of antibacterial agents for whichtransport has been described for at least one pump in each family are grouped according to their general physicochemical character (seeFig. 2).
462 F. Van Bambeke et al.
well as from the periplasm (see the model of extrusion byRND transporters in Refs. 39 and 40). Several lines ofexperimental evidence, which, however, are based mostoften on studies with non-antibiotic substrates, support thisassumption. First, the only common feature of all pumpsubstrates is a liposolubility that must be sufficient to ensurea proper penetration into the bilayer . Second, substrateor inhibitor molecules can compete with lipophilic fluoro-phores for insertion into the membrane [41, 42]. Third,lipophilic fluorophores are themselves substrates of thepumps . Fourth, the rates and kinetics of efflux are notdirectly related to the cytosolic drug content [41, 44]. Thestructural characteristics of MDR proteins rather favor thehypothesis of a vacuum cleaner. Indeed, the 12 transmem-brane domains are organized in such a way that they forma large aqueous pore, open to the extracellular medium butclosed towards the cytosol (somewhat like an empty, openbottle or beaker floating on the surface of water [44, 45]). Inaddition, these transmembrane segments are rich in aro-matic amino acids, which could help the hydrophobicsubstrates to travel into this channel . Functionalstudies, in contrast, favor the flip-flop hypothesis, since thephysiological transporters of phospholipids and of glutathi-one conjugates, which belong to the MDR and CT2families, respectively, are known as flippases [47–49].
ANTIBIOTIC SUBSTRATE SPECIFICITY
A key characteristic of the antibiotic efflux pumps is thevariety of molecules they may transport, which actually canbe related directly to their well-known poor substratespecificity. Considering the pharmacochemical aspects first,it is clear that only very minimal common structuraldeterminants are necessary to obtain detectable transport.Nevertheless, for each class of transporters, investigatorshave tried to determine which substrate features are themost specific. Results available thus far are presented in asummarized fashion in Fig. 1. Through the use of simulta-neous multiple disruption of several transporter genes,however, it has become evident that the substrate specific-ity is very broad and overlapping across a large array ofdistinct transporters [28, 50, 51]. The substrate specificity ofthe MET and the MAR transporters, for instance, has notyet been established with sufficient details and, for theMAR transporters, appears dubious since the NorM trans-porter representative of this family is claimed to recognizeboth fluoroquinolones and aminoglycosides, two classes ofantibiotics with strikingly different physicochemical prop-erties . Although these substrate specificities may ap-pear difficult to establish, a unifying hypothesis is that most,if not all, transporters recognize molecules with a polar,often slightly charged head associated with a hydrophobicdomain (see Fig. 2). Unfortunately, the importance oflipophilia is often difficult to ascertain in the absence ofpublished studies with homogenous series of drug deriva-tives. It, nevertheless, appears striking for the RND multi-drug efflux pumps when considering the data available on
the b-lactams, for which a transport ranking of cloxacil-lin . nafcillin . penicillin G . carbenicillin . penicillinN (the latter being almost not transported) has beendemonstrated clearly in close relationship with their corre-sponding octanol/water partition coefficients . Gener-ally speaking, also, the physicochemical properties of theantibiotics transported by a given class of pumps correspondto those of the non-antibiotic drugs as well as of those ofthe putative physiological substrates. A major discrepancy,however, concerns chloramphenicol, which is extruded byMFS despite its neutral character [e.g. Ref. 53]. Site-directed mutagenesis studies, however, suggest that therecognition of this drug is probably mediated by interac-tions different from those observed for other antibiotics. Also remarkable is the rarity of pumps for aminogly-cosides [54, 55], but this can probably be explained by thehigh hydrophilicity of these antibiotics, which preventstheir entry into cells by nonspecific diffusion (aminoglyco-side antibiotics largely mimic polyamines, an essentialsubstrate for many types of cells, and use their inwardtransport system for entering both bacteria [for activity] andspecific eucaryotic cells [causing toxicity; see Refs. 56 and57 for reviews]; aminoglycoside-producing organisms gen-erally tend to protect themselves not by efflux pumps, butby the production of aminoglycoside-inactivating enzymes). Similarly, the absence of efflux pumps acting onglycopeptide antibiotics has been explained, at least inbacteria, by the fact that these bulky, largely hydrophilicdrugs act in the outer space of Gram-positive bacteria, andnever cross the bacterial membrane (glycopeptides areinactive against Gram-negative bacteria precisely becausethey cannot cross the outer membrane of these organisms). But the situation has evolved so quickly for the otherclasses of antibiotics that we cannot exclude the possibilitythat efflux will eventually be demonstrated for glycopep-tides also if appropriate studies are undertaken. Beyondthese specific considerations, it must also be emphasizedthat a given antibiotic may be a substrate for different typesof pumps, so that (i) it may be expelled by differentorganisms for which no common transporter has beenidentified so far (giving the false impression that thetransporter is ubiquitous), and (ii) modulation of theactivity of a given transporter may be compensated for by amodulation in the opposite direction of another trans-porter, with, therefore, no or little change in the cellularaccumulation of the drug (giving the false impression thatthe drug is not transported). Moreover, a given pump mayextrude not only different antibiotics within the same classbut also different classes of antibiotics [28, 39, 40, 55, 60].Finally, a single cell may possess a vast and complex arsenalof efflux pumps allowing for the extrusion of a very broadspectrum of drugs (viz. Escherichia coli and S. cerevisiae, thecomplete genome sequencing of which has revealed theexistence of more than 250 putative transporters monopo-lizing 10% of the total genetic material [6, 7]).
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MICROBIOLOGICAL AND THERAPEUTICSIGNIFICANCE
Considered clinically important only for tetracyclines untila few years ago, antibiotic efflux pumps appear nowadays asa major component of microbial resistance to many classesof major antibiotics [39, 40, 60]. For at least three of them,namely the tetracyclines [61, 62], the macrolides , andthe fluoroquinolones  (which are interesting to analyzein this context since they are totally synthetic, amphiphiliccompounds with no known ‘natural’ counterpart), antibi-otic efflux appears sufficient per se to confer a medium orhigh level of resistance, defeating medically applicabletreatments of the corresponding infections with theseantibiotics. Typical examples include Streptococcus pyogenes and to some extent S. pneumoniae [65, 66]. Antibioticefflux may also contribute to the decreased susceptibility ofS. aureus to fluoroquinolones [64, 67] and of Pseudomonas
aeruginosa to many classes of antibiotics . Most insidi-ously, antibiotic efflux may be found in association withother mechanisms, such as antibiotic inactivation, to con-fer high-level resistance on bacteria. In some respects, thisphenomenon bears similarities with the cooperation ofdrug-extruding pumps and the cytochrome P450-based deg-radation pathways in enterocytes, which we presented inthe introduction of this review. A typical example is givenby the cooperation between the penicillin efflux pumps andthe b-lactamases, both of which may effectively decreasethe concentration of b-lactams in the periplasmic space ofGram-negative bacteria to the point where penicillin-binding proteins are no longer saturated. These bacteriathen display the surprising phenotype of high-level resis-tance without being high-level producers of b-lactamase[69–71]. The situation is more subtle for fluoroquinolones,but illustrates quite well the cooperation between two
FIG. 2. Structural formulae of the main antibiotics for which efflux has been demonstrated through at least one type of the transportersdescribed in Table 1 and Fig. 1. The molecules are presented so as to show their amphipathic character when appropriate (the zonesconsidered more lipophilic are surrounded by a solid line, and those considered more hydrophilic, by a dotted line). The charged groupsare systematically oriented upwards (fluoroquinolones are represented twice, since these can act as cations as well as anions, andaccordingly are transported by RND, MFS, MDR, and MRP pumps). The local pH, which may vary widely from one type of organismto another and from the precise location of the pumps, strongly influences the ionization of these groups and their role in recognitionby the transporters. The structures shown emphasize the common characters of each class of antibiotics, since structural variationswithin each class (denoted by the existence of variable substituents [R]) do not appear to alter their recognition properties markedly,systematically, or specifically.
464 F. Van Bambeke et al.
apparently unrelated mechanisms of resistance. Resistanceto these antibiotics may result from point mutations at thelevel of the drug targets (DNA gyrase/topoisomerase IV). Asingle mutation most usually gives rise to only low- ormedium-level resistance, and the bacteria may still beconsidered as sensitive in routine microbiological testing.The combination of two or more mutations, however, willconfer high levels of resistance [72, 73]. Because thesemutations are easily obtained in many bacterial species invitro, they were considered as being primarily responsiblefor the resistance seen in the clinic. It now appears,however, that many, if not most, of the organisms with thephenotype of low- to medium-level resistance to fluoro-quinolones harbor one or several efflux mechanisms [73–75]. Recent data also show that single point mutations andthe existence of fluoroquinolone efflux pumps producesynergistic effects [74, 76]. We speculate that efflux pumps,by decreasing the cellular concentration of fluoroquino-lones, may facilitate the selection of mutants with two ormore mutations, thereby increasing the risk of emergence ofhighly resistant organisms. A further striking demonstra-tion of the key role of antibiotic efflux pumps in bacterialresistance is given by the recent observation that thedisruption of one or several of their genes, or their directpharmacological inhibition, results in a major increase oftheir intrinsic susceptibility to the corresponding antibiot-ics. It may also decrease the frequency of appearance ofresistant mutants [51, 76]. Moving to so-called non-suscep-tible organisms, it now appears that, in many cases, thisphenotype is not due to an intrinsic lack of susceptibility(absence of target or impermeability) as was thought for along time, but is rather caused by the presence of effluxpumps that are constitutionally very active against the drugunder study [40, 54, 76–78]. It is also important tounderscore the role of stable mutations at the level of theregulatory genes controlling the expression of multidrugpumps . An example of this multidrug regulation circuitis well described in yeast, where it has been shown thatexposure to a given single drug could lead to mutations inregulatory genes provoking the constitutive and simulta-neous overexpression of several multidrug efflux pumps (ofdifferent types). This results in the irreversible acquisitionof a phenotype of multidrug resistance (see Ref. 79 forreview), a situation commonly observed in pathogenicfungi resistant to multiple drugs, which indeed often over-express multidrug efflux pumps [80, 81]. Moving to bacte-ria, we now know that patients infected by P. aeruginosaand treated by a b-lactam alone (or in combination) maybecome colonized rapidly by strains with a mutation in theregulator of the genes encoding the MexA–MexB–OprMpump . These strains are resistant not only to b-lactams,but also to fluoroquinolones, tetracyclines, chlorampheni-col, and trimethoprim. Each drug, presumably, can beexpected to also be the inducer of regulatory mechanismsresponding to cytotoxic insult by overexpression of drugefflux pumps. Therefore, it is likely that ecological pressurethrough an inappropriate use of antibiotics will sooner or
later lead to the selection of strains showing stable resis-tance to a wide range of unrelated drugs (this may imply notonly bacteria but also other organisms that have beenexposed incidentally to the same type of drug). A second,but so far unproven, risk is related to the apparent plasticityand promiscuity of the drug transporters with respect totheir potential substrates. This could lead to the selection oftransporters with increased efficacy. This self-adaptation ofbacteria has been well documented with b-lactamases andaminoglycoside-inactivating enzymes. Each introduction ofmolecules designed to resist the action of these enzymes hasbeen followed rapidly by the emergence of apparently newenzymes acting against these ‘new’ antibiotics. Yet, thegenetic analysis showed that the new enzymes sometimesdiffered from the old ones only by single amino acidsubstitutions . Likewise, in the case of drug effluxpumps, it is known that single amino acid substitutions mayaffect substrate specificity drastically [6, 33].
The clinical impact of antibiotic efflux pumps on resis-tance in clinics remains difficult to establish, since we lacklarge-scale and international statistics comparing theirprevalence with that of the other resistance mechanisms.Yet, very recent surveys already published point to alarmingfigures of 40–90% of some bacterial pathogens (S. pneu-moniae, S. pyogenes, and P. aeruginosa) bearing effluxmechanisms for the major classes of clinically availableantibiotics ( [66, 78, 83–85]; see also abstracts 1211, 1212,1216–1218, 1220–1223, 1225, and 1228 of the 39thInterscience Conference on Antimicrobial Agents andChemotherapy [ICAAC], San Francisco, CA, 1999). Theabundance of research papers describing antibiotic effluxmechanisms contrasts, however, with the rarity of data fromclinical microbiology laboratories. This raises the questionof the adequacy of the routine procedures to detect thesestrains, which most often may be classified as moderatelyresistant and erroneously assigned to a conventional mech-anism of resistance in the absence of further detailedinvestigation. Multiresistant organisms will need to bescreened critically in this context.
Moving now to eucaryotes, it becomes very clear nowa-days that the transepithelial movement of several drugs,including antibiotics, implies transport systems. The con-certed action of different pumps located both on thebasolateral and the apical membranes of epithelial cells hasbeen proposed to account for the preferential transfer ofcertain antibiotics from the blood to the excretory pathway.This cooperation is best evidenced in the liver, where OATand cMOAT (also called MRP2, see Table 1) ensure theunidirectional transfer of drugs to the bile (see Ref. 86 forreview). It is also suspected to occur in the kidney proximaltubules , and secretory transepithelial transport ofantibiotics has been demonstrated in the intestine andairway epithelia [88, 89]. Practically speaking, the activityof pumps explains the poor intestinal resorption of severalantibiotics [88, 90, 91] and gives a rational explanation forthe behavior of the so-called orally available penicillins orcephalosporins. The recognition of the existence of antibi-
Antibiotic Efflux Pumps 465
otic transporters now may be put into use for more rationaldesign in the future . Similarly, antibiotic transportmechanisms operating in the liver and kidney explain someof the elimination features of b-lactams and fluoroquino-lones [93, 94]. The recognition of the existence of antibi-otic-extruding pumps in macrophages, which act on b-lac-tams and fluoroquinolones [95, 96], and, for MDR-expres-sing cells, on macrolides, tetracyclines, lincosamides, andrifamycins as well [97, 98], has shed new light on the lackof, or potentially reduced activity of, these antibioticsagainst intracellular bacteria. Antibiotic efflux pumps will,indeed, reduce the amount of drug present in phagocytes toa point where it may no longer exceed the minimalinhibitory concentration at the site of infection. This wasclearly demonstrated in the case of Listeria monocytogenes,which causes cytosolic infections (addition of an inhibitorof the fluoroquinolone efflux pump markedly increases theactivity of these antibiotics ). The situation may bemore complex for infections affecting the vacuolar appara-tus, because efflux pumps in this case could promote theaccumulation of antibiotics from the cytosol into thesevacuoles, since their membranes partly derive from invagi-nations of the pericellular membrane [2, 99].
The search for new anti-infective agents now must takeinto account the growing problem of resistance. In thiscontext, glycylcyclines and ketolides were designed and/orscreened specifically for action against strains displaying theantibiotic efflux pumps recognizing their parent compounds(tetracyclines and macrolides ([100, 101]; see also abstracts2133, 2137, and 2140 in the 39th ICAAC). The failuresencountered with antibiotics designed to specifically resistinactivating enzymes (b-lactamases, aminoglycoside-modi-fying enzymes, and so forth) show, however, that chemical‘improvements’ are likely to be overcome quickly by bac-teria. Obtaining specific and potent inhibitors of antibiotictransporters, therefore, appears today as an important ob-jective in anti-infective chemotherapy. Although basicknowledge is still scanty in many areas, several lines ofresearch can be followed safely using either ligand-based ortarget-based design approaches. Ligand-based approacheshave long been the main source of new chemotherapeuticagents and, therefore, could be followed usefully in thiscase. Yet, they may fall short of an accurate definition of astarting pharmacophore. Indeed, we have seen that there isactually little stringent structural requirement for a drug tobe transported. Target-based approaches, on their side, mayoffer more opportunities for defining a specific ligand,especially since we now have a growing capacity to under-take precise molecular modeling allowing one to defineligands. In this context, it is important to emphasize thateffective inhibitors do not necessarily have to be directedagainst the binding site of the natural substrate. Forexample, effective inhibitors acting on proteins of thepicornavirus capsid have been designed to bind to func-
tional groups situated out of the site of interaction of theprotein with its target . Moreover, for members of theABC transporter superfamily, it also appears possible toinhibit the ATPase activity of the transporter rather thanits efflux capacity [44, 103]. Several groups of both aca-demic and industry-based researchers are heavily engagedin the search for pump inhibitors [104–107]. A majordifficulty, however, may arise from the fact that antibiotic-extruding pumps could be proteins with important physio-logical functions, the manipulation of which may causeunexpected toxicities. In this context, efforts directed tospecifically inhibit antibiotic-extruding pumps operatingonly in procaryotes may offer significantly greater chancesof effective therapeutic success (; promising com-pounds have also been presented in this respect at the 39thICAAC [viz. abstracts 1264–1272]). An indirect therapeu-tic application of our present knowledge of the antibiotic-extruding pumps could be the use of antibiotic moleculesthemselves to inhibit the transport of other chemothera-peutic agents such as anticancer drugs [109–113]. Therationale of this approach is that we already possess effec-tive pharmacophores with low levels of toxicity that arebacked by long clinical experience . Although thisapproach may seem attractive at first glance, it will, in ouropinion, quickly stumble on the problem of the overuse ofantibiotics, which is one of the major causes of worldwideantibacterial resistance. This non-antibiotic use of antibi-otics, therefore, may create an uncontrollable problem, notonly at the level of the patient but also at that of thecommunity, as clearly exemplified by the use of antibioticsas food additives . We therefore suggest that there isnot only room but also a necessity to design truly effectiveand finely tuned specific inhibitors of antibiotic effluxtransporters, which will usefully complement our presentanti-infective armamentarium. This could be achieved byconcerted genomic and proteomic studies targeted towardsthe discovery of specific genes and gene products, withcomplete biochemical and functional characterization inpurified systems.
We thank Dr. O. Lomovskaya (Microcide Pharmaceuticals, Inc.,Mountain View, CA), Prof. M. H. Saier (Department of Biology,University of California at San Diego, La Jolla, CA), Prof. A.Goffeau (Unite de Biochimie Physiologique, Universite Catholique deLouvain, Louvain-La-Neuve, Belgium), Prof. Y. Glupczynski (Cli-niques Universitaires de l’Universite Catholique de Louvain a MontGodinne, Belgium), and Dr. M. P. Mingeot-Leclercq (Unite dePharmacologie Cellulaire et Moleculaire, Universite Catholique deLouvain, Bruxelles, Belgium) for critical reading of our manuscript andvaluable suggestions. F. V. B is Charge de Recherches of the BelgianFonds National de la Recherche Scientifique. This work was supportedby the Fonds Special de Recherches (FSR) of the Universite Catholiquede Louvain, the Belgian Fonds de la Recherche Scientifique Medicale(Grant 3.4516.94), the Belgian Fonds National de la RechercheScientifique, and the Belgian Service de la Politique Scientifique ‘Polesd’Attractions Interuniversitaires’.
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