- Email: [email protected]
Efflux pumps and drug resistance in Gram-negative bacteria
OPINION
ment for their support of our work, and P.S. Langendijk, B.C. kleijer and G.J. lbleijer-Severs for allowing us to use their data in Figs 3 and 4. References 1 Wym-Williams, D.D. (1990) Rinary Co~nprt. hlicrobiol. 2, 15-20 2 Bjwnscn, P.K. (1986)Appl. bwiron. hlicrohiol. .i I, 1 199-l 204 3 Sicracki, .Cl.E.,Johnson, P.W. and Sicburth, J&l. (1985)Appl. hw~m~. hlicrohiol 49, 799- 810 4 Lawrence. J.K., Korbcr, D.R. and Caldwell, D.E. (1989)/. Miclobiol. Methods 10, 123-138 .5 Zanyk, I’&. et nl. (1991)Biwry Cornput. Microbial. 3, 24 -29 6 Evans-Hurrell, J.A. et al. (1993) FEMS !Microbio/. I.ett. 107, 77-82 7 Van der Waaij, I). (1982) 1. Antrmicroh. Chernother. 10, 263-270 8 Meijer, H.C., Kootstra, G.J. and Wilkinson, M.H.F. (1990)Rinar) Cornput. Microhiol. 2,2 I-3 1
9 Apperloo-Rcnkcma, H.Z. et al. ( 199 I) Med. Mcrobiol. Iwmunol. 180, 93-100 10 A&+, B.C. et al. ( 1991) Epidemiol. Infect. l&513-521 11 %leijcr, H.C., Kootstra, G.J. and Wilkinson, b1.H.F. (1991) Epidemiol. Infect. 107,383-391
12 Wilkinson, hl.H.F., Janscn, G.J. and Van der Waaij, D. (199.3) in Biotecbrlology Applications of Micloinjection, Microscopic Imngrq, a& Fhorescence (Bach, P.H. et al., cds), pp. 221-230, I’ergamon I’rcss 13 Janscn. G.1. et ~1. (1993) 1. Microbial. Metboffs 137-144
i7,
14 Wilkinson, .LI.H.F., Jansen, CJ. and Van dcr Waaij, D. Microem/. Thu. (in press) I.5 Gonzales, K.C. and Wintz, P. (1987) Digital Image Processing (2nd cdn), Addison-Wcslep 16 Kittler, J., Illingwurth, J. and Fdglein, J. (198.5)Compzft. Vision Crnph. Image Process. 30, 12.5-147
17 Sieracki, .CI.E., Reichenbach, S.E. and Webb, K.I.. (1989) Appl. Enuium. Microbd. 5.5, 2762-2772 18 Gctliff, J.M. and Fry, J.C. (lY?O)Bituq Covzprrt. Alicrobiol 2, 5.5-S 19 Shannon, C.E. and Weaver, W. (1949) The ,K&mtical Themy of Cowzmwhztion, University of Illinois
Press 20 .CIcijer-Severs.G.J., Van Santen, E. and blcijer, B.C. (1990) Sca~ci.1. Gastroenterol. 2.5, 698-704 21 Janscn. G.J. et al. ( 1993) Infection 21,
193-194 22 Janscn, G.J. et ~1.hfectim (in press) 23 Siskcn, J.E. (1989) in Fhresceme i%croscopy
of’Lwing
Cells in Cfdtfue
(Part H) (Methods IPICell /Gology, Vol. 30) (‘raylor, L.S. and Wang, Y.I.., eds), pp. 113-125, Academic Press 24 Wilkinson, 1M.H.F.(1994) Comp/lt. Methods Programs Biotned. 44, 61-67 2.5Beimfohr, C. et al. (1993) Syst. Appl. Microbml. 16, 4.50-4.56
l
.
Efflux pumps and drug resistance in Gram-negative bacteria Dzwokai Ma, David N. Cook, John E. Hearst and Hiroshi Nikaido rug-resistant strains of bacteria are an increasing threat to human health’ (see Ref. 2 and following articles for a review). Classical mechanisms of drug resistance involve enzymatic degradation and modification of drugs (for example, hydrolysis of /3-lactams and phosphorylation or acetylation of aminoglycosides), alteration of the drug target (for example, production of a modified penicillinbinding protein by methicillinresistant Staphylococctrs aurczfs) or active efflux of specific classes of drugs (for example, pumping out of tetracycline by the Tet protein)‘12. More recently, it has been recognized that active efflux proteins of surprisingly wide specificity are common in wild-type bacteria, and contribute significantly to the intrinsic (or background) resistance
D
The outer membrane of Gramnegative bacteria can only slow down the influx of lipophilic inhibitors, and so these bacteria need active cfflux pumps of broad specificity to survive. Pumps such as the Escherichia coli Acr system and its homologs make Gram-negative bacteria resistant to dyes, detergents and antibiotics. D. Mu, D. N. Cook awd J. FL Hearst me in the Dcpt of Chnistry, md H. Nikaido is 1,~th Dept of ~Molecrrlnr and Cell uiolugy, IJnwersity of California, Berkeley, CA ‘14720, IJSA.
of species such as Pseudomonas aeruginosa, which is notoriously resistant to most of the commonly used antimicrobials’. Multidrugefflux pumps have also been found in organisms, such as Escherichia
co/i, that are usually susceptible to common antimicrobial agents. In this article, we discuss the idcntity and possible functions of these pumps, with special emphasis on the Acr pump and its homologs. Acr and multiple-drug resistance It has been known for many years that mutations at the acr locus render E. co/i more susceptible to a broad range of inhibitors, such as basic dyes, detergents and hydrophobic antibioti4, but the nature of the defect in these mutants rcmained unknown. The diversity of the chemical structures of these agents makes putative mechanisms involving the alteration of drug targets or the specific modification or degradation of drugs unlikely. What appeared to hc most likely was a defect in the outer-membrane
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(a) acrRAB
E. coli
Fig. 1. Known homologs of acr genes on the Eschenchia co/i chromosome. (a) Shows chromosomal locatidns and (b) the structure of gene clusters. Arrows give the direction of transcription and percentages show the extent of amino acid identity between the gene products. Acre and AcrEF were previously called AcrE and EnvCD, respectively (see Ref. 6). It has not been established yet whether OrfR regulates the expresston of OrfAB. The sequence of OtfRAB was provided by Dr Guy Plunkett, University of Wisconsin, Madison, USA. GenBank Accession nos: acrAB. UO0734; acrEF, X57948; acrD, U10436; orfAB, UOO039 (locus EcoUW76).
acrD
(b) d-
-
-me
-
-
-
e
-
AcrA (397 aa)
AcrB (1049 aa)
AcrE (384 aa)
AcrF (1034 aa)
OrfA (395 aa)
OrfB (1037 aa)
I 1O-20%
4---e
AcrD (1037 aa)
4560%
barrier, but no change has been dcmonstratcd in lipopolysaccharides or major outer-membrane proteins’. Finally, the cloning and sequencing of the acr gcncs suggested that they are involved in an active efflux process6 [J. Xu, M.L. Nillcs and K.P. Bertrand (1993) I’roc. 93rd Annu. Meet. Am. Sot. Microbial.] because one of them appears to code for a transporter protein (see below). Measurement of accumulation of acriflavine in E. coli cells strongly suggested that the Acr proteins actively pump this dye out of the cell, using cncrgy from the proton-motive force”. At least three other loci on the I:. coli chromosome have a high dcgrec of similarity to the UCYsequence (Fig. 1). Each locus contains a gent
60-75%
that encodes a large (2100 kDa) protein that appears to be an innermembrane protein with 12 transmembrane c1 hclices, the putative cfflux-pump protein. These proWins belong to a family of putative efflux transporters, the rcsistanccnodulation-division (RND) family’. Except for acrD, each locus also contains one additional gene, which seems to form an opcron together with the pump-encoding gene. This second gene encodes a smaller protein (about 40 kDa) that appears to bc a lipoprotein; it is anchored to the inner membrane through its amino-terminal lipid moiety, but most of the protein is in the periplasm”. Pan and Sprat? idcntificd a third gene in these systems, which appears to encode a transcriptional
regulator with a helix-turn-h&x motif near the amino terminus. Structure of the Acr transporter complex Acr transporters arc thought to have a similar quaternary structure to some other cfflux systems that are discussed below. In these systems, which arc found in Gram-negative bacteria, the efflux pump, which spans the inner membrane, is thought to form a complex togcthcr with a periplasmic protein and an outer-membrane channel (Fig. 2). The cfflux proteins of the RND family, which include the pump proteins of the Acr system, contain two large periplasmic domains, each of about 300 amino acid residue&‘. This structural characteristic is not found in transporters of the major facilitator (MF) and ATP-binding cassette (ABC) families, which also contain 12 transmembrane cx helices. The lipoproteins in the Acr systems have sequences that are similar to HlyD, which is involved in exporting hemolysin directly into the medium; Dinh et al.’ propose calling these proteins the ‘mcmbrane fusion protein (IMFP) family’, as they are thought to bring an inner-membrane transporter into contact with an outer-membrane channel. (Some members of this family contain amino-terminal lipophilic domains instead of covalently attached lipids.) Finally, an outermembrane channel is needed to pump the drugs directly into the mcdium, bccausc cfflux into the pcriplasm would result in the rapid rcentry of drugs into the cytoplasm. This function is taken by TolC and its homologs in systems that are involved in the secretion of hemolysin and several other exoproteins from Gram-negative bacteria”‘.“. Intcrcstingly, &me gene clusters for drug-cfflux or protein-export systems contain gcncs encoding their own outer-membrane channel (AprF and OprK in I’. aer@nosa, and PrtF in Erwinia chrysanthemi; SW Table 1), but at least two proteinexport systems of b;. co[i use a common channel, TolC (Ref. 11). TolC may also act as the outcrmcmbranc channel that is involved in the direct cfflux of drugs, which is consistent with the hypersensitivity
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of to/C: mutants to many hydrophobic inhibitors, including detergents (sodium dodecyl sulfate, deoxycholate and Triton X-l 00) and antibiotics (novobiocin, fusidic acid and erythromycin)‘2.
Medium
_ Outer-membrane 1
Other efflux transporters organized like the Acr system There are now known to be many efflux transporters that appear to be organized as a complex spanning both the inner and outer membranes (as shown in Fig. 2), and that are mostly involved in multidrug cfflux or secretion of polypeptides in Gram-negative bacteria (Table 1). Some of these transporter complexes contain both a pump of the RND family and a periplasmic component of the MFP family (Table 1). The MtrAB complex, which gives resistance to dyes, detergents and antibiotics, is coded by chromosomal genes in Neisseria gonorrhoeae’. In P. aeruginosa, the genes encoding MexAB are clustcred with a gene for an outermembrane protein, OprK, which is the putative channel’“. The phenotypes of strains that overexpress these products and mutants that are defective in these products show that IMexAB-OprK (Ref. 13), as well as another similar system, MexCD-OprM (Ref. 14), is im-
Membranefusion protein Effluk pump prqtein
Inner membrtihe
Cytoplasm Fig. 2. Hypothetical structure of Acr-like transporters.
The efflux-pump protein is assumed to be associated with the periplasmic protein of the membrane fusion pro-
tein family. as described in the text. Furthermore, the genes encoding efflux-pump proteins and membrane-fusion proteins often occur together with the genes encoding outer-membrane channels (see Table 1). which, as well as the kinetics of drug efflux. suggests that most of these systems are associated with an outer-membrane channel. However, there are some apparent exceptions, for example, AcrD.
portant in the intrinsic resistance of P. aerzfginosa to tetracycline, chloramphenicol and fluoroquinolones. The Nol system of Rhizobium meliloti appears to secrete oligosaccharides that act as nodulation signals”. CzcAB (Ref. 16) and CnrAB (Ref. 17) are Acr homologs that are encoded by plasmid genes; they catalyze the active efflux of divalent heavy metal ions.
Not all multidrug-efflux systems contain pumps of the RND family. In the first multidrug pump, EmrAB (Ref. 18), discove :d by Kim Lewis and coworkers i , E. coli, the periplasmic component is a member of the MFP family, but the pump protein belongs to the MF family. These transporters are known to pump out carbonyl cyanide m-chlorophenylhydraxone (an uncoupler),
Table 1. Export systems with presumed organization similar to that of the Acr system” Organism
Escherichia coli Neisseria gonorrhoeae Pseudomonas aeruginosa Rhizobium meliloti Alcaligenes eutrophus
E. co/i E. co/i Etwinia chrysanthemi P. aeruginosa
Pump
Perlplasmlc component
RND family
MFP family
AcrB MtrB MexB MexD NolG, NolH, Noll CzcA CnrA
AcrA MtrA MexA MexC NolF CzcB CnrB
MF family
MFP family
EmrB
EmrA
ABC family
MFP family
HlyB PrtD AprD
HlyD PttE AprE
OM channel
? ? OprK OprM ? ? ? ? TolC PrtF AprF
Substrates
Detergents, dyes, antibiotics Detergents, dyes, antibiotics Pyoverdine, antibiotics Antibiotics Nodulation signals? Heavy metal ions Heavy metal ions CCCP, nalidixic acid Hemolysin Proteases Alkaline protease
“Only Gram-negative export systems that are thought to be organized as shown in Fig. 2 are given and, therefore, for example, Gram-positive multidrug pumps are omitted. Sources are listed in the text in some cases; for others, see the recent reviews in Refs 3,7,11. Abbreviations: OM. outer membrane; RND, resistance-nodulation-division; MF, major facilitator; MFP, membrane fusion protein: ABC, ATP-binding cassette; CCCP, carbonyl cyanide m-chlorophenylhydrazone. -
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(4
Medium
OM
IM
Cytoplasm
\ .
.
Acr complex
.
Ll . . . . . .
, . . . .... ..4 . 4-f l
.
.
l
phenylmercury acetate and a few other inhibitors. Finally, the complex may contain a member of the MFP family as the periplasmic component and an ABC-type pump (Table 1). These systems are mainly protein-export machineries”. [Some ABC pumps in Gram-positive bacteria are involved in drug efflux, but these proteins are not associated with components of the MFP family nor with outer-membrane channels (reviewed in Ref. 3).]
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.
.
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.
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.
Fig. 3. Competition between the influx and efflux processes. (a) In both Pseudomonas aeruginosa and Escherichia co/i, lipophilic inhibitors diffuse across the outer membrane (OM) slowly (thin arrow), so that the efflux system can remove the inhibitor molecules efficiently from the cytoplasm. (b) Less-hydrophobic agents cross the outer membrane of E. co/i rapidly through porin channels, so that the efflux system is not effective at dealing with the influx. (c) If an agent penetrates slowly through the porin channels (either because the porin channel has a low permeability, as in P. aeruginosa, or because the agent itself is bulky or hydrophobic), then the efflux system again becomes very effective at lowering the concentration of the inhibitor in the cell. IM, inner membrane.
Regulation and function of Acr and its homologs Pan and SpratP showed that the MtrR protein acts as a repressor of mtyAB genes. Our recent results based on 1ucZ fusions have also shown that AcrR acts as a repressor for the acrA B operon (D. Ma, unpublished). Interestingly, the expression of the acrAB operon appears to bc regulated by several ‘global’ stress signals; it is enhanced by 4% ethanol, fatty acids (for example, 5 rnbt decanoate) or media of high osmolarity (for example, 0.4 M NaCl), and is also increased in the stationary phase during growth in I, broth (D. Ma, unpublished). Interestingly, expression of the acrAB operon is upregulated in mayK mutants (D. Ma, unpublished). The mayRAB genes respond primarily to the presence of antibiotics, and have been suspected to activate the expression of antibioticefflux pumps that have not been previously identified genetically”. The mayRAB system also activates oxygen-stress genes”. The expression of orfAB is regulated by oxygen stress through OxyS, a small regulatory RNA (G. Storz, pers. commun.). Some Acr-like efflux pumps have a physiological function in exporting some metabolites out of the cell. Data available suggest that the MexAB-OprK system exports the siderophore pyoverdine into the medium’“, and export of endogenous oligosaccharide signal molecules is the presumed function of the Nol system ‘j. It remains to be determined whether AcrAB also exports some endogenous metabolites. A major function of AcrAB, however, is likely to be pumping out exogen-
ous inhibitors, including dyes and antibiotics. Escherichia coli lives in an environment that is rich in hydrophobic growth inhibitors: bile salts inhibit the growth of most bacteria except for enterics, and the contents of the lower intestinal tract contain up to 1% free fatty acidP, which inhibit bacterial growth2’. Free-living relatives of E. coli are constantly exposed to antibiotics, the vast majority of which are lipophilic molecules; more than 90% of newly discovered natural antibiotics are active against Gram-positive bacteria, but inactive against E. coli22. The outer membrane slows down the influx of these lipophilic inhibitors, but dots not provide sufficient protection against highly hydrophobic molecules, for which the half-time for equilibration across the outer membrane is less than a few seconds2’. It is therefore not unreasonable to assume that E. coli and its relatives produce efflux pumps to protect themselves against lipophilic inhibitors. This idea is also consistent with observations that the expression of the AcrAB and OrfAB systems is regulated by stress conditions, and that null mutants of the acrAB, acrEE‘ or acyD genes do not show any obvious growth defects in laboratory media (D. Ma, unpublished; see also below). The sensitivities of mutants and strains that overexpress transporter systems suggest that the specificity of pumps varies. Thus, AcrAR seems to pump out almost any amphiphilic compound, regardless of whether it is cationic (basic dyes, erythromycin and mitomycin C) or anionic (novobiocin and fusidic acid). The original mutant in UCYEF (also called envCD), PM6 1, was hypersusceptible to drugs and defective in cell division24, which is why the family of such pumps was named the RND family’. However, the scptation defect in PM61 could not be corrected by the unaltered wild-type alleles of acrEF (Ref. 25), suggesting that the defect is not the direct consequence of a mutation in acyEF. Indeed, null mutants of acrE and acrF that have been constructed recently by interposon mutagenesis are ncithcr defective in septation nor hypersensitive to drugs, at
OPINION
least against the background of the E. coli K12 strains used (D. Ma, unpublished); it is likely that the acrEF genes are only expressed weakly under the usual laboratory conditions. Nevertheless, they can function as a drug-efflux system when overcxprcssed because clones that express acrEF strongly can rescue the drug-sensitive phenotype of an acrA mutant [j. Xu, M.L. Nilles and K.1’. Bertrand (1993) Proc. 93rd Annu. Meet. Am. Sot. lMicrobiol.1. A null mutant of acrD is not hypersusceptible to those drugs that have been tested so far (D. IMa, unpublished). EmrAB, the associated transporter of which does not belong to the RND family, seems to have a very different substrate specificity from the AcrAB system (Table 1). One striking difference between the drug-efflux systems of E. co/i and those of P. aeruginosa is that only the latter produce clinically significant levels of resistance to common antibiotics. However, comparison between different spccics is complicated by potential differences in outer-membrane permeability. As shown in Fig. 3, the steady-state concentration of any drug in the cytoplasm of Gramnegative bacteria is determined by the balance between efflux and influx, which is determined largely by the rate of penetration through the outer membrane. Thus, highly hydrophobic agents (detergents, many dyes and hydrophobic antibiotics, such as fusidic acid, rifampin and novobiocin) penetrate the outer membranes at a rate that is much slower than their rate of cfflux, so that both organisms are resistant to these agents (Fig. 3a). Common broad-spectrum antibiotics, such as tetracycline, chloramphenicol and norfloxacin, which are either smaller or less hydrophobic than the above agents, penetrate through porin channels of the E. co/i outer membrane rapidly enough to overwhelm the efflux process (Fig. 3b), but their penetration rate across the P. aeruginosa outer membrane, which does not contain the archetypal trimeric porins’“, may be too slow ro compete against their cfflux (Fig. 3~).
These considerations suggest that a slow rate of penetration through the outer membrane is essential for significant drug resistance by the multidrug-efflux mechanism to occur. Interestingly, some environmental signals that stimulate the efflux pumps in E. cd, such as antibiotics, salicylate or oxygen radicals, also downregulate the larger porin channel, OmpF, which lowers the outer-membrane permeability (reviewed in Ref. 3). Because these signals are those that bacteria are likely to encounter when they invade the tissues of plants and animals3, in such an environment, the decreased influx and increased cfflux caused by these signals may produce significant intrinsic drug resistance, even in those bacteria that arc usually considered susceptible, such as E. cofi and its close relatives. Acknowledgements We thank Gisela Storz (National Institutes of Health) for information on OrfAB and Marie Alberti for contributions to the original studies mentioned in this article. This work was supporred by National Instirutes of Health grants GM 41911 and AI 09644. References 1 Davies, J. (1994) Science 264,375-382 2 Levy, S.B. (1994) Trends Microbial. 2, 34 l-342 3 Nikaido, H. (1994) Science 264, 382-388 4 Nakamura, H. (1965) 1. Bacterwl. 90, 8-14 5 Sukupolvi, S. and Vaara, M. (1989) Biochim. Biophp. Acta988, 377-387
6 Ma, 1). et al. (1993) /. Bacteria/. 175, 6299-6313 7 Saier, M.H. et al. ( 1994) Mol. Microbial. 11,841-847 8 Pan, W. and Spratt, B. (1993) Mol. Microhiol. 11, 769-77.5 9 Dinh, T., Paulsen, LT. and Saier, M.H., Jr (1994)_/. Bacterial. 176,3825-3831 10 Wandersman, C. and Delepelaire, P. (1990) hoc. Nat/ Acad. Sci. USA 87, 4776-4780 11 Fath, b1.J. and Koltcr, R. (1993) Microhiol. Rev. 57,995-1017 12 Davies, J.K. and Reeves, P. (1975) /. Bncteriol. 123, 102-l 17 13 Poole, K. et al. (1993) /. Bacterial. 175, 7363-7372 14 Li, X-Z., Livermore, D. and Nikaido, H. (1994) Antirnicrob. Agents Chemother. 38,1732-1741 15 Baev, S. et al. (1991) Mol. Gen. Genet. 228,113-124 16 Nies, I). et al. (1989) Proc. Nat/ Acad. Sci. USA 86,7351-7355 17 Liesegang, H. et al. (1993) /. Racteriol. 175767-778 18 Lomovskaya, 0. and Lewis, K. (1992) Pm. Nat1Acad. Sci. USA89, 8938-8942 19 Ariia, R.F. et al. (1994) 1. Bacterial. 176, 143-148 20 Lenmcr, C. (ed.) (1981) Geigy Sciejrtific Tuhles (8th edn) (Vol. I), p. 155, C&a-Geigy 21 Sheu, C.W. and Frccsc, E. (1973) 1. Bacterial. 11.5, 869-875 22 Vaara, IM. (1993) Antimicroh. Agents Chemother. 37,22SS-2260 23 Plesiat, P. and Nikaido, H. (1992) Mol. Microbial. 6, 1323-1333 24 Klein, J.R., Henrich, B. and Plapp, R. (1991) Mol. Gen. Genet. 230,230-240 25 Klein, J.R., Hcnrich, B. and Plapp, R. (1990) Curr. Microhiol. 21,341-347 26 Nikaido, H. (1994)1. Biol. Chnn. 269, 3905-3908
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*Does the l-k/~1 coat’brbtein gp120 produce h&n’ dar&ge?by G. Bag&k, _ T.M. Corasanitl, A. Fin&i-Agro: and G. Nistico - T’ds in I?‘1ar?~+Ok!giCi3~, Sciences 15,362-363 *Parasites and T helpe; cell development: some insights, by S.1. Relner’ - Parasitology Today 10,485-488
ment for their support of our work, and P.S. Langendijk, B.C. kleijer and G.J. lbleijer-Severs for allowing us to use their data in Figs 3 and 4. References 1 Wym-Williams, D.D. (1990) Rinary Co~nprt. hlicrobiol. 2, 15-20 2 Bjwnscn, P.K. (1986)Appl. bwiron. hlicrohiol. .i I, 1 199-l 204 3 Sicracki, .Cl.E.,Johnson, P.W. and Sicburth, J&l. (1985)Appl. hw~m~. hlicrohiol 49, 799- 810 4 Lawrence. J.K., Korbcr, D.R. and Caldwell, D.E. (1989)/. Miclobiol. Methods 10, 123-138 .5 Zanyk, I’&. et nl. (1991)Biwry Cornput. Microbial. 3, 24 -29 6 Evans-Hurrell, J.A. et al. (1993) FEMS !Microbio/. I.ett. 107, 77-82 7 Van der Waaij, I). (1982) 1. Antrmicroh. Chernother. 10, 263-270 8 Meijer, H.C., Kootstra, G.J. and Wilkinson, M.H.F. (1990)Rinar) Cornput. Microhiol. 2,2 I-3 1
9 Apperloo-Rcnkcma, H.Z. et al. ( 199 I) Med. Mcrobiol. Iwmunol. 180, 93-100 10 A&+, B.C. et al. ( 1991) Epidemiol. Infect. l&513-521 11 %leijcr, H.C., Kootstra, G.J. and Wilkinson, b1.H.F. (1991) Epidemiol. Infect. 107,383-391
12 Wilkinson, hl.H.F., Janscn, G.J. and Van der Waaij, D. (199.3) in Biotecbrlology Applications of Micloinjection, Microscopic Imngrq, a& Fhorescence (Bach, P.H. et al., cds), pp. 221-230, I’ergamon I’rcss 13 Janscn. G.1. et ~1. (1993) 1. Microbial. Metboffs 137-144
i7,
14 Wilkinson, .LI.H.F., Jansen, CJ. and Van dcr Waaij, D. Microem/. Thu. (in press) I.5 Gonzales, K.C. and Wintz, P. (1987) Digital Image Processing (2nd cdn), Addison-Wcslep 16 Kittler, J., Illingwurth, J. and Fdglein, J. (198.5)Compzft. Vision Crnph. Image Process. 30, 12.5-147
17 Sieracki, .CI.E., Reichenbach, S.E. and Webb, K.I.. (1989) Appl. Enuium. Microbd. 5.5, 2762-2772 18 Gctliff, J.M. and Fry, J.C. (lY?O)Bituq Covzprrt. Alicrobiol 2, 5.5-S 19 Shannon, C.E. and Weaver, W. (1949) The ,K&mtical Themy of Cowzmwhztion, University of Illinois
Press 20 .CIcijer-Severs.G.J., Van Santen, E. and blcijer, B.C. (1990) Sca~ci.1. Gastroenterol. 2.5, 698-704 21 Janscn. G.J. et al. ( 1993) Infection 21,
193-194 22 Janscn, G.J. et ~1.hfectim (in press) 23 Siskcn, J.E. (1989) in Fhresceme i%croscopy
of’Lwing
Cells in Cfdtfue
(Part H) (Methods IPICell /Gology, Vol. 30) (‘raylor, L.S. and Wang, Y.I.., eds), pp. 113-125, Academic Press 24 Wilkinson, 1M.H.F.(1994) Comp/lt. Methods Programs Biotned. 44, 61-67 2.5Beimfohr, C. et al. (1993) Syst. Appl. Microbml. 16, 4.50-4.56
l
.
Efflux pumps and drug resistance in Gram-negative bacteria Dzwokai Ma, David N. Cook, John E. Hearst and Hiroshi Nikaido rug-resistant strains of bacteria are an increasing threat to human health’ (see Ref. 2 and following articles for a review). Classical mechanisms of drug resistance involve enzymatic degradation and modification of drugs (for example, hydrolysis of /3-lactams and phosphorylation or acetylation of aminoglycosides), alteration of the drug target (for example, production of a modified penicillinbinding protein by methicillinresistant Staphylococctrs aurczfs) or active efflux of specific classes of drugs (for example, pumping out of tetracycline by the Tet protein)‘12. More recently, it has been recognized that active efflux proteins of surprisingly wide specificity are common in wild-type bacteria, and contribute significantly to the intrinsic (or background) resistance
D
The outer membrane of Gramnegative bacteria can only slow down the influx of lipophilic inhibitors, and so these bacteria need active cfflux pumps of broad specificity to survive. Pumps such as the Escherichia coli Acr system and its homologs make Gram-negative bacteria resistant to dyes, detergents and antibiotics. D. Mu, D. N. Cook awd J. FL Hearst me in the Dcpt of Chnistry, md H. Nikaido is 1,~th Dept of ~Molecrrlnr and Cell uiolugy, IJnwersity of California, Berkeley, CA ‘14720, IJSA.
of species such as Pseudomonas aeruginosa, which is notoriously resistant to most of the commonly used antimicrobials’. Multidrugefflux pumps have also been found in organisms, such as Escherichia
co/i, that are usually susceptible to common antimicrobial agents. In this article, we discuss the idcntity and possible functions of these pumps, with special emphasis on the Acr pump and its homologs. Acr and multiple-drug resistance It has been known for many years that mutations at the acr locus render E. co/i more susceptible to a broad range of inhibitors, such as basic dyes, detergents and hydrophobic antibioti4, but the nature of the defect in these mutants rcmained unknown. The diversity of the chemical structures of these agents makes putative mechanisms involving the alteration of drug targets or the specific modification or degradation of drugs unlikely. What appeared to hc most likely was a defect in the outer-membrane
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(a) acrRAB
E. coli
Fig. 1. Known homologs of acr genes on the Eschenchia co/i chromosome. (a) Shows chromosomal locatidns and (b) the structure of gene clusters. Arrows give the direction of transcription and percentages show the extent of amino acid identity between the gene products. Acre and AcrEF were previously called AcrE and EnvCD, respectively (see Ref. 6). It has not been established yet whether OrfR regulates the expresston of OrfAB. The sequence of OtfRAB was provided by Dr Guy Plunkett, University of Wisconsin, Madison, USA. GenBank Accession nos: acrAB. UO0734; acrEF, X57948; acrD, U10436; orfAB, UOO039 (locus EcoUW76).
acrD
(b) d-
-
-me
-
-
-
e
-
AcrA (397 aa)
AcrB (1049 aa)
AcrE (384 aa)
AcrF (1034 aa)
OrfA (395 aa)
OrfB (1037 aa)
I 1O-20%
4---e
AcrD (1037 aa)
4560%
barrier, but no change has been dcmonstratcd in lipopolysaccharides or major outer-membrane proteins’. Finally, the cloning and sequencing of the acr gcncs suggested that they are involved in an active efflux process6 [J. Xu, M.L. Nillcs and K.P. Bertrand (1993) I’roc. 93rd Annu. Meet. Am. Sot. Microbial.] because one of them appears to code for a transporter protein (see below). Measurement of accumulation of acriflavine in E. coli cells strongly suggested that the Acr proteins actively pump this dye out of the cell, using cncrgy from the proton-motive force”. At least three other loci on the I:. coli chromosome have a high dcgrec of similarity to the UCYsequence (Fig. 1). Each locus contains a gent
60-75%
that encodes a large (2100 kDa) protein that appears to be an innermembrane protein with 12 transmembrane c1 hclices, the putative cfflux-pump protein. These proWins belong to a family of putative efflux transporters, the rcsistanccnodulation-division (RND) family’. Except for acrD, each locus also contains one additional gene, which seems to form an opcron together with the pump-encoding gene. This second gene encodes a smaller protein (about 40 kDa) that appears to bc a lipoprotein; it is anchored to the inner membrane through its amino-terminal lipid moiety, but most of the protein is in the periplasm”. Pan and Sprat? idcntificd a third gene in these systems, which appears to encode a transcriptional
regulator with a helix-turn-h&x motif near the amino terminus. Structure of the Acr transporter complex Acr transporters arc thought to have a similar quaternary structure to some other cfflux systems that are discussed below. In these systems, which arc found in Gram-negative bacteria, the efflux pump, which spans the inner membrane, is thought to form a complex togcthcr with a periplasmic protein and an outer-membrane channel (Fig. 2). The cfflux proteins of the RND family, which include the pump proteins of the Acr system, contain two large periplasmic domains, each of about 300 amino acid residue&‘. This structural characteristic is not found in transporters of the major facilitator (MF) and ATP-binding cassette (ABC) families, which also contain 12 transmembrane cx helices. The lipoproteins in the Acr systems have sequences that are similar to HlyD, which is involved in exporting hemolysin directly into the medium; Dinh et al.’ propose calling these proteins the ‘mcmbrane fusion protein (IMFP) family’, as they are thought to bring an inner-membrane transporter into contact with an outer-membrane channel. (Some members of this family contain amino-terminal lipophilic domains instead of covalently attached lipids.) Finally, an outermembrane channel is needed to pump the drugs directly into the mcdium, bccausc cfflux into the pcriplasm would result in the rapid rcentry of drugs into the cytoplasm. This function is taken by TolC and its homologs in systems that are involved in the secretion of hemolysin and several other exoproteins from Gram-negative bacteria”‘.“. Intcrcstingly, &me gene clusters for drug-cfflux or protein-export systems contain gcncs encoding their own outer-membrane channel (AprF and OprK in I’. aer@nosa, and PrtF in Erwinia chrysanthemi; SW Table 1), but at least two proteinexport systems of b;. co[i use a common channel, TolC (Ref. 11). TolC may also act as the outcrmcmbranc channel that is involved in the direct cfflux of drugs, which is consistent with the hypersensitivity
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of to/C: mutants to many hydrophobic inhibitors, including detergents (sodium dodecyl sulfate, deoxycholate and Triton X-l 00) and antibiotics (novobiocin, fusidic acid and erythromycin)‘2.
Medium
_ Outer-membrane 1
Other efflux transporters organized like the Acr system There are now known to be many efflux transporters that appear to be organized as a complex spanning both the inner and outer membranes (as shown in Fig. 2), and that are mostly involved in multidrug cfflux or secretion of polypeptides in Gram-negative bacteria (Table 1). Some of these transporter complexes contain both a pump of the RND family and a periplasmic component of the MFP family (Table 1). The MtrAB complex, which gives resistance to dyes, detergents and antibiotics, is coded by chromosomal genes in Neisseria gonorrhoeae’. In P. aeruginosa, the genes encoding MexAB are clustcred with a gene for an outermembrane protein, OprK, which is the putative channel’“. The phenotypes of strains that overexpress these products and mutants that are defective in these products show that IMexAB-OprK (Ref. 13), as well as another similar system, MexCD-OprM (Ref. 14), is im-
Membranefusion protein Effluk pump prqtein
Inner membrtihe
Cytoplasm Fig. 2. Hypothetical structure of Acr-like transporters.
The efflux-pump protein is assumed to be associated with the periplasmic protein of the membrane fusion pro-
tein family. as described in the text. Furthermore, the genes encoding efflux-pump proteins and membrane-fusion proteins often occur together with the genes encoding outer-membrane channels (see Table 1). which, as well as the kinetics of drug efflux. suggests that most of these systems are associated with an outer-membrane channel. However, there are some apparent exceptions, for example, AcrD.
portant in the intrinsic resistance of P. aerzfginosa to tetracycline, chloramphenicol and fluoroquinolones. The Nol system of Rhizobium meliloti appears to secrete oligosaccharides that act as nodulation signals”. CzcAB (Ref. 16) and CnrAB (Ref. 17) are Acr homologs that are encoded by plasmid genes; they catalyze the active efflux of divalent heavy metal ions.
Not all multidrug-efflux systems contain pumps of the RND family. In the first multidrug pump, EmrAB (Ref. 18), discove :d by Kim Lewis and coworkers i , E. coli, the periplasmic component is a member of the MFP family, but the pump protein belongs to the MF family. These transporters are known to pump out carbonyl cyanide m-chlorophenylhydraxone (an uncoupler),
Table 1. Export systems with presumed organization similar to that of the Acr system” Organism
Escherichia coli Neisseria gonorrhoeae Pseudomonas aeruginosa Rhizobium meliloti Alcaligenes eutrophus
E. co/i E. co/i Etwinia chrysanthemi P. aeruginosa
Pump
Perlplasmlc component
RND family
MFP family
AcrB MtrB MexB MexD NolG, NolH, Noll CzcA CnrA
AcrA MtrA MexA MexC NolF CzcB CnrB
MF family
MFP family
EmrB
EmrA
ABC family
MFP family
HlyB PrtD AprD
HlyD PttE AprE
OM channel
? ? OprK OprM ? ? ? ? TolC PrtF AprF
Substrates
Detergents, dyes, antibiotics Detergents, dyes, antibiotics Pyoverdine, antibiotics Antibiotics Nodulation signals? Heavy metal ions Heavy metal ions CCCP, nalidixic acid Hemolysin Proteases Alkaline protease
“Only Gram-negative export systems that are thought to be organized as shown in Fig. 2 are given and, therefore, for example, Gram-positive multidrug pumps are omitted. Sources are listed in the text in some cases; for others, see the recent reviews in Refs 3,7,11. Abbreviations: OM. outer membrane; RND, resistance-nodulation-division; MF, major facilitator; MFP, membrane fusion protein: ABC, ATP-binding cassette; CCCP, carbonyl cyanide m-chlorophenylhydrazone. -
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(4
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OM
IM
Cytoplasm
\ .
.
Acr complex
.
Ll . . . . . .
, . . . .... ..4 . 4-f l
.
.
l
phenylmercury acetate and a few other inhibitors. Finally, the complex may contain a member of the MFP family as the periplasmic component and an ABC-type pump (Table 1). These systems are mainly protein-export machineries”. [Some ABC pumps in Gram-positive bacteria are involved in drug efflux, but these proteins are not associated with components of the MFP family nor with outer-membrane channels (reviewed in Ref. 3).]
l
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.
.
.
.
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.
Fig. 3. Competition between the influx and efflux processes. (a) In both Pseudomonas aeruginosa and Escherichia co/i, lipophilic inhibitors diffuse across the outer membrane (OM) slowly (thin arrow), so that the efflux system can remove the inhibitor molecules efficiently from the cytoplasm. (b) Less-hydrophobic agents cross the outer membrane of E. co/i rapidly through porin channels, so that the efflux system is not effective at dealing with the influx. (c) If an agent penetrates slowly through the porin channels (either because the porin channel has a low permeability, as in P. aeruginosa, or because the agent itself is bulky or hydrophobic), then the efflux system again becomes very effective at lowering the concentration of the inhibitor in the cell. IM, inner membrane.
Regulation and function of Acr and its homologs Pan and SpratP showed that the MtrR protein acts as a repressor of mtyAB genes. Our recent results based on 1ucZ fusions have also shown that AcrR acts as a repressor for the acrA B operon (D. Ma, unpublished). Interestingly, the expression of the acrAB operon appears to bc regulated by several ‘global’ stress signals; it is enhanced by 4% ethanol, fatty acids (for example, 5 rnbt decanoate) or media of high osmolarity (for example, 0.4 M NaCl), and is also increased in the stationary phase during growth in I, broth (D. Ma, unpublished). Interestingly, expression of the acrAB operon is upregulated in mayK mutants (D. Ma, unpublished). The mayRAB genes respond primarily to the presence of antibiotics, and have been suspected to activate the expression of antibioticefflux pumps that have not been previously identified genetically”. The mayRAB system also activates oxygen-stress genes”. The expression of orfAB is regulated by oxygen stress through OxyS, a small regulatory RNA (G. Storz, pers. commun.). Some Acr-like efflux pumps have a physiological function in exporting some metabolites out of the cell. Data available suggest that the MexAB-OprK system exports the siderophore pyoverdine into the medium’“, and export of endogenous oligosaccharide signal molecules is the presumed function of the Nol system ‘j. It remains to be determined whether AcrAB also exports some endogenous metabolites. A major function of AcrAB, however, is likely to be pumping out exogen-
ous inhibitors, including dyes and antibiotics. Escherichia coli lives in an environment that is rich in hydrophobic growth inhibitors: bile salts inhibit the growth of most bacteria except for enterics, and the contents of the lower intestinal tract contain up to 1% free fatty acidP, which inhibit bacterial growth2’. Free-living relatives of E. coli are constantly exposed to antibiotics, the vast majority of which are lipophilic molecules; more than 90% of newly discovered natural antibiotics are active against Gram-positive bacteria, but inactive against E. coli22. The outer membrane slows down the influx of these lipophilic inhibitors, but dots not provide sufficient protection against highly hydrophobic molecules, for which the half-time for equilibration across the outer membrane is less than a few seconds2’. It is therefore not unreasonable to assume that E. coli and its relatives produce efflux pumps to protect themselves against lipophilic inhibitors. This idea is also consistent with observations that the expression of the AcrAB and OrfAB systems is regulated by stress conditions, and that null mutants of the acrAB, acrEE‘ or acyD genes do not show any obvious growth defects in laboratory media (D. Ma, unpublished; see also below). The sensitivities of mutants and strains that overexpress transporter systems suggest that the specificity of pumps varies. Thus, AcrAR seems to pump out almost any amphiphilic compound, regardless of whether it is cationic (basic dyes, erythromycin and mitomycin C) or anionic (novobiocin and fusidic acid). The original mutant in UCYEF (also called envCD), PM6 1, was hypersusceptible to drugs and defective in cell division24, which is why the family of such pumps was named the RND family’. However, the scptation defect in PM61 could not be corrected by the unaltered wild-type alleles of acrEF (Ref. 25), suggesting that the defect is not the direct consequence of a mutation in acyEF. Indeed, null mutants of acrE and acrF that have been constructed recently by interposon mutagenesis are ncithcr defective in septation nor hypersensitive to drugs, at
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least against the background of the E. coli K12 strains used (D. Ma, unpublished); it is likely that the acrEF genes are only expressed weakly under the usual laboratory conditions. Nevertheless, they can function as a drug-efflux system when overcxprcssed because clones that express acrEF strongly can rescue the drug-sensitive phenotype of an acrA mutant [j. Xu, M.L. Nilles and K.1’. Bertrand (1993) Proc. 93rd Annu. Meet. Am. Sot. lMicrobiol.1. A null mutant of acrD is not hypersusceptible to those drugs that have been tested so far (D. IMa, unpublished). EmrAB, the associated transporter of which does not belong to the RND family, seems to have a very different substrate specificity from the AcrAB system (Table 1). One striking difference between the drug-efflux systems of E. co/i and those of P. aeruginosa is that only the latter produce clinically significant levels of resistance to common antibiotics. However, comparison between different spccics is complicated by potential differences in outer-membrane permeability. As shown in Fig. 3, the steady-state concentration of any drug in the cytoplasm of Gramnegative bacteria is determined by the balance between efflux and influx, which is determined largely by the rate of penetration through the outer membrane. Thus, highly hydrophobic agents (detergents, many dyes and hydrophobic antibiotics, such as fusidic acid, rifampin and novobiocin) penetrate the outer membranes at a rate that is much slower than their rate of cfflux, so that both organisms are resistant to these agents (Fig. 3a). Common broad-spectrum antibiotics, such as tetracycline, chloramphenicol and norfloxacin, which are either smaller or less hydrophobic than the above agents, penetrate through porin channels of the E. co/i outer membrane rapidly enough to overwhelm the efflux process (Fig. 3b), but their penetration rate across the P. aeruginosa outer membrane, which does not contain the archetypal trimeric porins’“, may be too slow ro compete against their cfflux (Fig. 3~).
These considerations suggest that a slow rate of penetration through the outer membrane is essential for significant drug resistance by the multidrug-efflux mechanism to occur. Interestingly, some environmental signals that stimulate the efflux pumps in E. cd, such as antibiotics, salicylate or oxygen radicals, also downregulate the larger porin channel, OmpF, which lowers the outer-membrane permeability (reviewed in Ref. 3). Because these signals are those that bacteria are likely to encounter when they invade the tissues of plants and animals3, in such an environment, the decreased influx and increased cfflux caused by these signals may produce significant intrinsic drug resistance, even in those bacteria that arc usually considered susceptible, such as E. cofi and its close relatives. Acknowledgements We thank Gisela Storz (National Institutes of Health) for information on OrfAB and Marie Alberti for contributions to the original studies mentioned in this article. This work was supporred by National Instirutes of Health grants GM 41911 and AI 09644. References 1 Davies, J. (1994) Science 264,375-382 2 Levy, S.B. (1994) Trends Microbial. 2, 34 l-342 3 Nikaido, H. (1994) Science 264, 382-388 4 Nakamura, H. (1965) 1. Bacterwl. 90, 8-14 5 Sukupolvi, S. and Vaara, M. (1989) Biochim. Biophp. Acta988, 377-387
6 Ma, 1). et al. (1993) /. Bacteria/. 175, 6299-6313 7 Saier, M.H. et al. ( 1994) Mol. Microbial. 11,841-847 8 Pan, W. and Spratt, B. (1993) Mol. Microhiol. 11, 769-77.5 9 Dinh, T., Paulsen, LT. and Saier, M.H., Jr (1994)_/. Bacterial. 176,3825-3831 10 Wandersman, C. and Delepelaire, P. (1990) hoc. Nat/ Acad. Sci. USA 87, 4776-4780 11 Fath, b1.J. and Koltcr, R. (1993) Microhiol. Rev. 57,995-1017 12 Davies, J.K. and Reeves, P. (1975) /. Bncteriol. 123, 102-l 17 13 Poole, K. et al. (1993) /. Bacterial. 175, 7363-7372 14 Li, X-Z., Livermore, D. and Nikaido, H. (1994) Antirnicrob. Agents Chemother. 38,1732-1741 15 Baev, S. et al. (1991) Mol. Gen. Genet. 228,113-124 16 Nies, I). et al. (1989) Proc. Nat/ Acad. Sci. USA 86,7351-7355 17 Liesegang, H. et al. (1993) /. Racteriol. 175767-778 18 Lomovskaya, 0. and Lewis, K. (1992) Pm. Nat1Acad. Sci. USA89, 8938-8942 19 Ariia, R.F. et al. (1994) 1. Bacterial. 176, 143-148 20 Lenmcr, C. (ed.) (1981) Geigy Sciejrtific Tuhles (8th edn) (Vol. I), p. 155, C&a-Geigy 21 Sheu, C.W. and Frccsc, E. (1973) 1. Bacterial. 11.5, 869-875 22 Vaara, IM. (1993) Antimicroh. Agents Chemother. 37,22SS-2260 23 Plesiat, P. and Nikaido, H. (1992) Mol. Microbial. 6, 1323-1333 24 Klein, J.R., Henrich, B. and Plapp, R. (1991) Mol. Gen. Genet. 230,230-240 25 Klein, J.R., Hcnrich, B. and Plapp, R. (1990) Curr. Microhiol. 21,341-347 26 Nikaido, H. (1994)1. Biol. Chnn. 269, 3905-3908
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