Multidrug resistance pumps in bacteria: variations on a theme

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MULTIDRUG RF~ISTANCE PUMPS 0VlDRs) are membrane translocases that have the surprising ability to extrude a variety of unrelated drugs from the cell. Although they have largely been studied in eukaryotic organisms, MDRs have recently been discovered in bacteria (Table I). The eukaryotic MDR extrudes anticancer agents from the cell and is largely responsible for the multidrug resistance of tumors L2. Similarly, bacterial MDRs can be amplified in resistant cells and can shift their Multidrug resistance pumps (MDRs) arise from three different gene famil'substrate' profiles, potentially making ies and are widespread in bacteria. For example, in Escherichia co/i alone, them a major menace to drug treatthere seem to be seven distinct MDRs. The most common belong to the ment. Apart from such practical conmajor facilitator family of membrane translocases; this type of MDR is siderations, there is a great deal of closely related to specific antibiotic extrusion pumps such as the tetraintrinsic appeal in the study of bacterial cycline/H* antiporter. This similarity in design, and the high incidence of MDRs, since they clearly violate the apparently independent evolution of MDRs, suggests that the property of rule of enzymes and translocases, being multidrug resistance might have resulted from a loss of specificity in a fairly nonspecific. This renew suggests specific hydrophobic-drug efflux pump. that an MDR is a variation on the theme of a regular transiocase that simply broadened its substrate spectrum, providing a cell with a simple defense ning the membrane, a design that is continuous channel between the inner against the ever-present toxins of the shared by some other efflux pumps and outer membranes. For example, in homologous to QacA, as we shall see the case of hemolysin extrusion, the en~ronment. actual pump is HIyB (note that HIyB is below. in the course of studying E. coil resist- homologous to the eukaryotic MDR, PThe MDR families The majorfacilitatorfamily.Most bacterial ance to uncouplers, we cloned a chromo- glycoprotein), while HlyD and a specialMDRs belong to the large major facili- sonml locus conferring resistance to ized porin, TolC, are required to pass the tator (MF) family of membrane trans- a widely used uncoupler, m-chloro- peptide all the way out of the cell Gig. 1; loca~es, which includes such well- metho~Jcarbonylcyanide phenyl hydra- renewed in Re~. 13). It was appealing to known members as the arabinose/H ÷ zone (CCCP)l°, as well as to nalidixic suggest that the general design of symporter of Escherichia coli, the glu- acid, thiolactomycin u and other anti- EmrA-EmrB is similar to that of the cose facilitator of eukaryotes, and microbial agents. The locus appeared to hemolysin pump, and that EmrA particibacterial tetracycline/H* antiporters encode two genes, emrA and emrB, and pates in forming a pathway for extrud(Tet)3,4. MF translocases have a trans- analysis of the sequence of EmrB ing drugs across the outer membrane I° membrane structure composed of 12 showed that it is homologous to QacA. Gig. 1). This arrangement would allow a.helices (an apparent result of a dupli- The action of uncouplers is based on the efficient extrusion of toxins from cation of a six-helix domains) and use a their ability to shuttle back and forth the cell, since the outer membrane of proton motive force (pmf) as a source across the c~oplasmic membrane. It Gram-negative bacteria is a rather good was thus unclear what might be barrier for hydrophobic compounds. of energy (Rg. 1). A homology search shows that EmrB The first report of an MDR in bac- achieved by extruding an uncoupler via teria, that of QacA, came from studies an MDR. The key to this puzzle might is fairly similar to an open reading of Staphylococcus resistance to quatern- be held by the second of the two pro- frame (OPal~) F475 that was identified ary ammonium compounds6,7 used as teins, EmrA, which is homologous to from the total E. coil chromosomal DNA antiseptics. QacA appeared to be a proteins participating in the outward sequencing project 15. ORF F475 also membrane translocase that pumps out transport of many bacterial peptide tox- carries a si~ature consensus, known ethidium bromide (and other drugs) ins and proteases. The closest relative as the drug extrusion (DE) consensus, from the cells in a pmf-dependent man- of EmrA is the CyaD protein of shared by those members of the MF ner8,9, suggesting that it acts as a Bordetella pertussis (28% identity and family that are drug extrusion pumps6: drug/H" antiporter. The nature of the 49% similarity), which participates in GPILGPVLGG. it seems quite likely that true 'substrates' of QacA remains un- the extrusion of cyclolysin. EmrA is ORF F475 encodes a new MDR. ,another gene participating in unclear, although its location on a plasmid homologous to a lesser degree to HIyD that contains genes conferring antibiotic (a component of the E. coli hemolysin coupler resistance that we identified also resistance suggests that the function of efflux pump) and CvaA (a component of turned out to encode an MDR~e. The QacA is to extrude some natural anti- the colicin V secretion system). These emrD gene is uncoupler inducible and microbial agents. The protein has an proteins, including EmrA, have an ident- encodes a 12-a-helix transmembrane unusual topology of 14 a-helices span- ical topology: a short amino-terminal protein with a DE consensus and cytoplasmic domain, a single trans- homology to MDRs. The homolog membrane a-helix and a large periplas- closest to EmrD is encoded by a recently K. Lewisis at the Departmentof Biology, mic domain l° (Fig. 1). The function of sequenced E. coil chromosomal locus, MassachusettsInstituteof Technology, these proteins is apparently to form a bcP 7, which confers resistance to Cambridge,MA02139, USA. 119 © 1994,ElsevierScienceLtd 0968-0004/94/$07.00

Multidrug resistance pumps in bacteria: variations on a theme Kim Lewis

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MF (major facilitator)

Smr (Staphylococcus multidrug resistance)

RND13(resistance, nodulation, division)

MFP13(membrane fusion proteins)

ABC(ATP-binding cassette)

Properties

Drug/H+ antiporters and specific translocases

Drug/H+antiporters

Drug/H+ antiporters?

Auxilliaryproteins assisting the translocases

MDRs(eukaryotes) Specific translocases (prokaryotes)

Membrane topology

12-14 transmembrane helices

Fourtransmembrane helices

12 transmembrane o helices

One transmembrane helix, periplasmic location

12 transmembrane helices

Distribution

Gram-positiveand Gram-negative species

Gram-positiveand Gram-negative species

Gram-negative species. Metal efflux translocases found in the samefamily

Gram-negative species

Prokaryotes and eukaryotes

Family members (location)

QacAS. aureus (plasmid) QacB S. aureus (plasmid)

Smr S. aureus (plasmid)

AcrB E. coil

EmrA(emrAB locus)

P-glycoprotein (MDR1) H. sapiens

Acre E. coii

AcrA(acrAB locus)

MexB P. aeruginosa

AcrF (acrEFlocus)

EmrB E. coli EmrD E. coli Bcr E. coli

QacE(broad-hostrange plasmids]

PFMDR1

EmrE/MvrC E. coil

P. falciparum

MexA(mexAB, oprK operon)

Bmr B. subtiiis Nora S. aureus

bicyclomycin. The bcr gene is identical to a previously described sur gene whose amplification leads to increased resistance to sulfathiazole (B. Nichols, pers. commun.) la. Bicyclomycin and sulfathiazole are chemically unrelated

compounds, which makes a case for Bcr being a new MDR of E. coli. The Bmr pump of Bacillus subtilis was discovered in the only targeted search for a bacterial MDR19 performed so far. Bmr extrudes ethidium bromide in a

EmrAB

AcrAB

HlyBD

OUT

ill"-

pmf-dependent manner. It is a cnromosome-encoded protein, and was found by selecting for a stepwise increase of resistance to rhodamine 6G, which led to gene amplification. Interestingly, gene amplification is how eukaryotic

TolC P-glycoprotein

Bmr H+

[ ~

rA

lyD

~rA

H+ E

I/i IN

ADF TPP +

B. subtilis

Thiolactomycin

ADI Hemolysin

Ac line

E. coil

TPP +

TPP +

H. sapiens

Figure 1 Membrane topology of representative MDRs. Homologous proteins are indicated by use of the same color. Bmr is found in B. subtilis and pumps out tetraphenyl phosphonium (TPP÷) and other amphiphilic cations. EmrAB protects from more hydrophobic substances. Note that EmrB and Bmr are homologous. The hypothetical topology of both EmrAB and another MDR from E. coil, AcrAB, is modeled on the structure of the hemolysin transporter HlyBD12, which forms a channel across both membranes of E. coil HlyBD consists of a membrane translocase, HlyB, and a 'membrane fusion' protein, HlyD, joined to an outer membrane porin, TolC. The putative membrane fusion proteins EmrA, AcrA and H!yD are homologous. Thcv consist of one transmembrane (x-helix and a large periplasmic domain (note the two green domains of ErnrA, for example). By analogy with the hemolysin transporter, EmrAB and AcrAB are shown to be joined to a porin, which might be TolC or another specialized porin. The MexAB MDR transporter from P. aeruginosa (not shown), which is closely homologous to AcrAB, does in fact have an outer-membrane protein encoded in the same operon as the translocase. The EmrE of E. coil is a tiny four-(x-helix MDR which is shown as a single peptide, but might in fact be oligomenc. P-glycoprotein is the human MDR1 pump and is homologous to HlyB.

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MDRI becomes overexpressed in drugresistant tumors 2. Bmr is highly homologous to the NorA protein of Staphylococcus aureus (44% identity), which was found to confer resistance to norfloxacin 2°, a synthetic inhibitor of DNA gyrase. A comparison of Bmr and NorA suggests that NorA is in fact an MDR21. This example leads to the following generalization: whenever a pump conferring resistance to a particular substance is found, suspect it of being an MDR. Small MDRs.Among the Staphylococcus plasmid-encoded pumps conferring resistance to quaternary ammonium compounds, QacC is functionally quite similar to QacA, but its structure is drastically different. QacC is by far the smallest peptide that can govern solute translocation of any sort, being only 107 amino acids long. The peptide forms four putative transmembrane domains and does not belong to the MF family. QacC (renamed Star for Staphylococcus multidrug resistance) is a member of a new family of tiny proteins scattered around the bacterial world. Star is a true MDR pump, extruding a fairly broad range of lipophilic cations in a pmf-dependent manner 8,9,22. A broad-host-range plasmid R751 (originally found in the Gram-negative bacterium Klebsiella aerogenes) carries transposon Tn402, which encodes trimethoprim-resistant dihydrofolate reductase and QacE 23, a protein closely related to Star. This finding suggests that the smallest MDRs might also be the ones most widely spread among different bacterial species, traveling on the R751 plasmid, which easily crosses interspecies barriers. A chromosomeencoded homolog of Smr, EmrE (formerly known as MvrC or EBr) is also found in E. coil 24,25. Accumulation of tetraphenylphosphonium (TPP ÷) is widely used as a method to measure the membrane potential in bacteria, but the discovery of MDR pumps, such as EmrE, that extrude TPP ÷just as well as other iipophilic cations 26, suggests it is time to re-evaluate this approach. 'Link' proteins: the Acr family. Genes encoding the last group of MDRs to be discovered in E. coli were first reported in the ]960s. These are the acr loci conferring resistance to acridine, a widely used DNA-intercalating mutagen. Mutations of acr make cells sensitive to acridine, and also to the anionic detergent sodium dodecyi sulphate, uncouplers, antibiotics, and substances in general whose permeability is restric-

A l i g n m e n t ratio I

I

I

0.2

i Ii

I

I

0,4

0,6

I I

I

I

0.8

1

1.0 E MNAT SVAT CGAT UNC- 17 Bmr

Nora Tet(A) Tet(C) Tet(B) --- Bcr --- Cm IA "-- E m r D Tet(K) Tet(L)

EmrB F475 Mmr TcmA Act I I

QecA ATR1

Figure 2 Relatedness between drug extrusion pumps of the MF family. The MDRs are shown in bold. Bmr19 and NorA2° are closely related MDRs from B. subtilis and S. aureus, respectively. The tetracycline (Tet) antiporters 14,36 have two different origins. Bcr17, EmrD16 and EmrB1° are E. coil MDRs. CmlA is a chloramphenicol pump from Pseudomonas. F475 is a likely new MDR from E. coil Mmr, TcmA and Actll encode antibiotic efflux pumps from antibiotic-producing Streptomyces species. QacA7 is an MDR from Staphylococcus. The chromaffin granule amine transporter CGAT34 (and related proteins of the same group) is a broad.specificity efflux pump. ATR133is the aminotriazole-resistance protein from the yeast S. cerevisiae. The figure is based on Ref. 4 and was kindly provided by M. Varela, J. Griffith and P. Henderson.

ted by the outer membrane. Accordingly, act genes were thought to encode some structural component of the outer membrane. Unexpectedly, the sequence of acr genes from two loci27,28 showed that they are homologous to toxic-metal extrusion pumps CzcA and CnrA of Alcaligenes eutrophis 29. AcrB is a large, 113 kDa protein with a 12-o~-helixtransmembrane domain structure. The level of acriflavine accumulation was higher in an acrB mutant, but became the same as in the wild type in the presence of CCCP, suggesting an efflux activity for AcrB zS. The neighboring gene acrA~7,28 encodes a protein that is homologous to EmrA. By analogy with the possible function of EmrA, it was suggested that the function of AcrA is to link the inner and outer membranes ~3. This arrangement would allow the Acr proteins to transport substances across the outermembrane permeability barrier. Interestingly, AcrA is a lipoprotein that contains

palmitate ~7. This finding further strengthens the notion that proteins belonging to this family form a link between the outer and inner membranes. Indeed, acylated membrane proteins are used by enveloped virions to fuse membranes, it will be interesting to learn whether proteins such as EmrA and HlyD are also acylated. The second acr locus, acrEF (formerly envCD) is very similar to acrAB. Acre is 78% identical to AcrB, and AcrF is 69% identical to AcrA. Yet another locus at 53 rain encodes a gene homologous to AcrB (Ref. 28 and H. Nikaido, pets. commun.), and might have a similar function. The MexAB proteins (multiple efflux), recently described in Pseudomonas aeruginosa 3°, are highly homologous to AcrAB and confer resistance to a broad range of antimicrobial agents. The MexAB proteins are, quite remarkably, implicated in the efflux of pyoverdin, an iron chelator synthesized by the 121

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out the drugs from the hydrophilic metabolites of the cytoplasm. However, (c) (a) (b) since MDRs easily change their specificity profiles, it H+ H+ would seem unlikely that Glucose Arabinose Drug the membrane would determine specificity. For example, a mutant in the emrAB locus with a specific increase in thiolactomycin resistance, but not nalidixic acid resistance, was described n, while a substiIN Arabinose Drug Glucose tution of Leu for Val2s6 in Bmr caused a dramatic decrease in binding of reRgure 3 A model for MF transporters. (a) The binding domain (red) of the arabinose symporter is linked to a serpine without notable nonspecific channel and rotates if both the H+ (green) and the arabinose (yellow) binding sites are. changes in resistance to either occupied, or empty. (b) In an efflux pump, which can be either drug-specific or an MDR, the other drugs 35. These obserdrug-binding site (yellow) is shifted to the opposite side of the binding domain to the H+-b!nding site vations rather suggest the (green). (©) The glucose facilitator lacks the proton channel and H+-bindingsites, apparently due to a presence of a conventional, secondary loss of energy-couplingfunction. albeit largely hydrophobic, binding site that can alter bacterium. This finding might be the first after one of its members, the human its broad specificity. Such a binding site report of a natural function for an MDR. glucose facilitator, which is essentially a is found in albumin, which binds a The list of 'substrates' for the glucose channel. Interestingly, the over- plethora of amphiphilic drugs (inAcr-Mex proteins is surprisingly, if not all topography of the facilitator i.~ cluding CCCP) at the 'short-chain fattysuspiciously, long, and future research sharer ~ by both the inward H÷ sym- acid-binding site'. MDRs share their will show whether these translocases porter translocases, such as H'/arabi- ability to bind amphiphUic substances do indeed, in their versatility, surpass nose, and by antlporters, such as the with some of the specific translocases: anything we presently know of. Tet proteins and MDRs. Relatively for example, Mmr and Tet bind, and minor changes are therefore respon- extrude, the fairly apolar comHow differentare the MDRsfrom other sible for what appear to be fundamental pounds methylenomycln and tetramembersof the samefamily? differences, regarding the direction of cycline, respectively. MDRs do not appear to be directly transport, presence of energy coupling, Let us see if the idea of an MDR being derived from some primordial MDR, and specificity (Fig. 3). If an MDR Is a variant of a conventional efflux pump simply because MDRs have arisen inde- basically a translocase that lost its sub- for an amphlphllic substance is applipendently many times in the course of strate specificity, then such a change cable to proteins that do not belong to evolution. For example, an E. cell MDR, will, of course, have a smaller effect the MF family (Table I). The eukaryotlc EmrB, is much closer to the methyleno- upon the structure of the protein than, MDR, P-glycoprotein, is a member of mycin extrusion pump, Mmr, from anti- for example, a shift from an uptake the large family of ABC (ATP-binding biotic-producing Streptomyces than it is translocase (such as an arabinose/H ÷ cassette) translocases, which use ATP to the Bacillus multidrug resistance symporter) to an efflux pump (such as a source of energy and have 12 pump Bmr or to EmrD from E. cell (Fig. as a tetracycline/H" antiporter). The transmembrane a-helices. This family, 2). Rather, it seems that MDRs were sequences of MDRs do not point to any similarly to MF, spans the eukaryote-derived from specific drug extrusion particular 'maltidrug consensus'. The prokaryote border, which indicates its pumps (such as Mmr) of the antibiotic- DE consensus, which MDRs share with ancient origin. MDRs of this family have producing bacteria 14. Selecting for a the specific efflux pumps of the same not yet been found in bacteria (but see mutation that allows an enzyme or a family, apparently codes for a domain Ref. 32 for a possible candidate). Some translocase to recognize a new sub- that determines the direction of trans- of the ABC proteins are uptake transstrate usually leads to a broader spec- port of a llgand, and is a further indi- locases, such as the well-studied histitrum of specificity, rather than to a cation of ~imilarity between MDRs dine (His) transporter of E. cell, while switch of specificity from one com- and conventional translocases. others are efflux pumps'. (the HlyB pound to another. This would suggest translocase of hemolysin, Fig. 1). The that MDRs were intermediates in the The MDR mechanism P-glycoprotein extrudes, among other evolution of one specific drug-extrusion ;One model for the eukaryotic MDR things, doxorubicin (adriamycin), an pump from another. Some of these suggested that it acts as a flippase and otherwise efficient anticancer drug. intermediates might have taken on a life is thus quite different from other mem- Doxorubicin is a natural antibiotic proof their own. bers of its family ~. It was proposed that duced by Streptomyces peucetius, and is There do not appear to be any struc- the 'binding site' of the protein was hid- exported by an/U3C-type pump 31. This tural features that set the MDRs apart den in the hydrophobic core of the example shows that, in the ,ed]C family from other members of the sanle family. membrane, thus allowing the membrane as well, there is a specific extrusion The major facilitator family was named to naturally perform the task of sorting pump with a binding site for an 122 OUT

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amphiphilic substance; such a pump will have the potential for turning into an MDR if it loses its substrate specificity. The RND family, to which the Acr proteins belong (Table !), is not as well characterized as the MF or ABC groups, but the fact that both MDRs and specific efflux pumps are among its members is consistent with the idea of an MDR being derived from a specific pump through a loss of specificity. It is the Smr family that provides the exception to this MDR origination hypothesis: Smr membership seems to be exclusive, with no specific pumps reported in this family so far. Perhaps a more significant feature of the Smr family is that its members are indeed the smallest of the known translocases, being around 100 amino acids long. Intuitively, it seems that a primordial founder of a family of translocases (or enzymes) would be fairly nonspecific, with speciation producing new family members, is it simpler to build an MDR than a specific pump, and are thus the smallest translocases basically living fossils of a primordial transiocase? Some questions are bound to remain unanswered, at least for the time being. Acknowledgements I wish to thank Milton Saier, Leo Grinius, Alex Neyfakh and Tom Haines

for helpful discussions, and Stuart Levy and Hiroshl Nikaido for critical reading of the manuscript. Thanks are due to Milton Saier and Brian Nichols fe~ providing information before publication. References 1 Higgins, C. F. and Gottesman, M. M. (1992) Trends Biochem. Sci. 17, 18-21 2 Roninso~., ¢. B., ed. (1991) Molecular and Cellular Biology of Multidrug Resistance in Tumor Cells, Plenum 3 Marger, M. D. and Saier, M. H., Jr (1993) Trends Biochem. ScL 18, 13-20 4 Griffith, J. K. et al. (1992) Curr. Opin. Cell Biol. 4, 684-695 5 Rubin, R. A., Levy, S. B., Heinrikson, R. L. and Kezdy, F. S. (1990) Gene 87, 7-13 6 Tennent, J. M. et aL (1989) J. Gen. Microbiol. 135, 1-10 7 Rouch, D. A. et al. (1990) MoL MicrobioL 4, 2051-2062 8 Littlejohn, T. G. et aL (1991) Gene 101, 59-66 9 Littlejohn, T. G. et aL (1992) FEMS Microbiol. Lett. 95, 259-266 10 Lomovskaya, O. and Lewis, K. (1992) Proc. Nat/ Acad. Sci. USA 89, 8938-8942 11 Furukawa, H. et al. (1993) J. BacterioL 175, 3723-3729 12 Schulein, R., Gentschev, I., Mollenkopf, H. J. and Goebel, W. (1992) MoL Gen. Genet. 234, 155-163 13 Saier, M. H., Jr, Tam, R., Reizer, A. and Reizer, J. Mol. Microbiol. (in press) 14 Levy, S. B. (1992) Antimicrob. Agents Chemother. 36, 695-703 15 Burland, V., Plunkett, G., III, Daniels, D. L. and Blattner, F. R. (1994) Genomics 16, 551-561 16 Naroditskaya, V., Schlosser, M. J., Fang, N. Y.

and Lewis, K. (1994) Biechem. Biophys. Res. Commun. 196, 803-809 17 Bentley, J. et aL (1993) Gene 127, 117-120 18 Nichols, B. P. and Guay, G. G. (1989) Antimicrob. Agents Chemother. 33, 2042-2048 19 Neyfakh, A. A., Bidnenko, V. and Chen, L. B. (1991) Proc. Nat/Acad. ScL U~A 88, 4781-4785 20 Yoshida, H. et al. (1990) J. Bacteriol. 172, 6942-6949 21 Neyfakh, A. A., Borsch, C. M. and Koatz, G. W. (1992) Antimicrob. Agent~ Chemother. 37, 128-129 22 Grinius, L. et aL (1992) P/asmid 27, 119-129 23 Paulsen, I. T. et aL (1993) Antimicrob. Agents Chemother. 37, 761-768 24 Morimyo, M., Hongo, E., Hama-lnaba, H. and Machida, I. (1992) Nucleic Acids Res. 20, 3159-3165 25 Purewall, A. S. (1991) FEMS Microbiol. Lett. 82, 229-232 26 Midgley, M. (1987) MicrobioL ScL 4, 125-127 27 Xu, J., Nilles, M. L. and Bertrand, K. P. (1993) "Abstracts of the 93rd ASM Meeting, 299 28 Dzwokai, M. et al. (1993) J. BacterioL 175, 6299-6313 29 Nies, D. H. (1992) P/asmid 27, 17-28 30 Hiller, G. and Weber, J. (1985) J. ViroL 55, 651-659 31 Poole, K., Krebes, K., McNally, C. and Neshat, S. (1993) J. Bacteriol. 175, 7353-7372 32 Molenaar, D. et al. (1992) J. Bacteriol. 174, 3118-3124 33 Kanazawa, S., Driscoll, M. and Struhl, K. (1988) Mol. Cell. BioL 8, 664-673 34 Liu, Y. et aL (1992) Cel/ 70, 539-551 35 Ahmed, M., Borsch, C. M., Neyfakh, A. A. and Schuldiner, S. (1993) J. Biol. Chem. 268, 11086-11_089 36 Sawai, T., Hirano, S. and Yamaguchi, A. (1987) FEMS Microbiol. Lett. 40, 233-237

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