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Antimicrobial Agents and Chemotherapy, November 2000, p. 3210-3212, Vol. 44, No. 11
Department of Biochemistry and Biophysics and
Center for Advanced Biomolecular Research, Texas A&M University,
College Station, Texas 77843-2128
Received 27 March 2000/Returned for modification 1 July
2000/Accepted 12 August 2000
Many laboratory strains of Escherichia coli are
resistant to methotrexate (MTX), a folate analogue that binds
dihydrofolate reductase (DHFR). Mutations that inactivate either
tolC or acrA confer MTX sensitivity.
Further, overexpression of a fusion protein with DHFR activity reverses
this sensitivity by titrating out intracellular MTX. These results
suggest that MTX accumulates in cells where mutations in
acrA or tolC have inactivated the TolC-dependent AcrAB multidrug resistance efflux pump.
Methotrexate (MTX) is a folate
analogue that inhibits the activity of dihydrofolate reductase
(DHFR) (16), which catalyzes the NADPH-dependent reduction
of dihydrofolate to tetrahydrofolate. Reduced folates are substrates in
a number of one-carbon transfers in purine, pyrimidine, and amino acid
biosynthesis (3). Inhibition of DHFR activity initially
results in the depletion of
N5,N10-methylene tetrahydrofolate,
followed by inhibition of DNA synthesis and ultimately cell death
(8). DHFR is thus a well-studied target of antibiotic and
antineoplastic therapy.
Although MTX binds both human and Escherichia coli DHFR very
tightly, with Ki values of 3.4 and 1.0 pM,
respectively (2), all of the E. coli isolates we
tested (genotypes of the strains used in this study are listed in Table
1), which included both common laboratory
strains (MG1655, MC4100, AG1688, and ZK126) and clinical isolates
(O157:H7, RM74A, STM1, LL, RM52B, DD, and RM33B), were resistant to MTX
added to solid medium at concentrations of up to 1 mM, the highest
concentration we tested (data not shown).
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Resistance to Methotrexate Due to
AcrAB-Dependent Export from Escherichia coli
ABSTRACT
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TABLE 1.
E. coli strains used
Antibiotic resistance can occur by a variety of mechanisms, including failure of the drug to bind its target, overexpression of the drug target, modification or degradation of the drug, creation of permeability barriers, or active export of the drug. It is increasingly recognized that active efflux plays a major role in the resistance of many organisms to a plethora of agents (11, 20). A wide variety of antibiotics are exported from E. coli by one of several active efflux systems (11, 12, 19, 20). At least two of these systems, the AcrAB and EmrAB efflux pumps, have been shown to depend on the outer membrane protein TolC (1, 7, 12, 19, 20).
To determine whether the MTX resistance was due to a TolC-dependent efflux pump, we examined the effect of a tolC::Tn10 mutation. LBB1175, in which tolC had been inactivated by the Tn10 insertion, was sensitive to 1 mM MTX, while W4573, the isogenic TolC+ control, was resistant. Similar results were obtained using the common laboratory strain MG1655, which is the reference wild-type E. coli K-12 strain used for the genome sequence (4), and AG1688 (see below). Strains carrying Tn10 at a different chromosomal location remained resistant to MTX. These results suggest that MTX resistance is mediated by a TolC-containing multidrug resistance efflux pump (MDR).
tolC mutants are pleiotropic (17, 26) and are hypersensitive to many hydrophobic agents (18). Thus, the loss of MTX resistance in the tolC mutant might not be due to the loss of function of an MDR. To address this possibility, we tested the effects of mutations that inactivate specific TolC-dependent MDRs. The AcrAB pump belongs to the RND (for resistance, nodulation, and division) family, and its substrates include sodium dodecyl sulfate, basic dyes, novobiocin, and tetracycline (19, 20); the EmrAB pump belongs to the MF (major facilitator) family, and its substrates include carbonyl cyanide m-chlorophenylhydrazone, nalidixic acid, and phenyl mercury acetate (19, 20). A strain containing the acrA1 mutation (N43) was sensitive to 1 mM MTX, while its isogenic parent (W4573) was resistant. In contrast, both the emrB mutant (OLS103) and its isogenic parent (AMS6) were MTX resistant. These results show that the MTX sensitivity of the tolC strains is at least partly due to inactivation of the AcrAB MDR, while the EmrAB pump does not have a major role in MTX export.
MICs of MTX were determined for a set of isogenic E. coli
strains containing combinations of acrA, emrB,
and tolC mutations (Table 2).
The wild-type strain (W4573) was resistant to 1,024 µM MTX, the
highest concentration tested. The emrB mutation did not
affect the MIC, either alone (compare W4573 to SK636) or in combination
with acrA1 (compare N43 to SK627) or
tolC::Tn10 (compare SK642 to SK660).
Inactivation of either acrA or tolC resulted in a
decrease of the MTX MIC to 256 or 64 µM, respectively. Since the
acrA1 allele is an IS2 insertion in the second
codon of acrA (14), it is unlikely that the
remaining MTX resistance in the acrA1 mutant is due to
residual activity of the acrA gene product. The
tolC gene product seems to have more than one role in MTX resistance. It is unclear if this is due to the loss of function of
another, unidentified TolC-containing MDR or the highly pleiotropic effects of tolC mutations on outer membrane structure
(17, 26). Similar alterations have not been found in the
outer membrane of acr mutants (19, 21, 24).
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The additional role of TolC is not related to the EmrAB MDR, since the acrA1 emrB double mutation (in SK627) yielded an MIC identical to that yielded by the acrA1 single mutation. Similar combinations of mutations in a different background (AG1688) yielded identical MICs (data not shown). This further demonstrates that the observed effects are not strain specific.
To determine whether inhibition of DHFR was sufficient to explain
the MTX sensitivity of tolC strains, we examined
whether the MTX sensitivity of AG1688
tolC::Tn10 (SK037) could be suppressed by overexpression of DHFR activity. In the course of other
(unpublished) studies, we had constructed a plasmid, pSK029, which
expresses a fusion protein, cI-DHFR, in which the N-terminal DNA
binding domain of the bacteriophage 
repressor is fused to E. coli DHFR; the fusion protein is expressed under the control of
the lacUV5 promoter. Neither pSK029 nor pXZ020, a control
plasmid expressing cI-GCN4 (a fusion to the leucine zipper of GCN4),
affected the MTX resistance of wild-type AG1688 whether or not the
fusion proteins were overexpressed (Table
3, lines 3 and 5). AG1688
tolC::Tn10 strains containing either
plasmid were sensitive to MTX under conditions in which the fusion
proteins were uninduced (Table 3, lines 4 and 6). However, in the
presence of isopropyl-
-D-thiogalactopyranoside (IPTG),
which induces the overexpression of cI-DHFR, SK029
tolC::Tn10 was resistant to high
concentrations of MTX (Table 3, line 4). In contrast,
IPTG-induced overexpression of the control protein cI-GCN4
had no protective effect on the tolC strain (Table 3, line
6).
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These results show that MTX sensitivity in the tolC strain is due to the inhibition of endogenous DHFR by the drug. When cI-DHFR is overexpressed, the DHFR activity provided by the DHFR domain in the fusion protein cannot be titrated out, which strongly suggests that the plasmid-coded DHFR acts to sequester MTX that is added to the medium. Increasing the level of DHFR should not relieve sensitivity due to mechanisms that do not involve uptake of MTX.
The results of this study can be summarized as follows. (i) All of the TolC+ AcrA+ strains of E. coli we tested were resistant to at least 1 mM MTX when grown on solid medium containing the drug. (ii) MTX resistance is decreased by mutations that disrupt tolC or acrA, genes that code for integral components of the AcrAB MDR, suggesting that resistance is due to active export of MTX via the AcrAB MDR. (iii) Mutation of the emrB gene does not decrease MTX resistance, suggesting that MTX is not a substrate of this MDR. (iv) The difference between the MICs for tolC::Tn10 and acrA::IS2 strains suggests the possibility of another mechanism for low-level TolC-dependent MTX resistance.
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ACKNOWLEDGMENTS |
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We thank David Weiss for useful discussions and Ry Young for providing strain KH803. We thank Debby Siegele for help with the transductions and for providing many of the strains used in this study and Joe A. Fralick for both helpful comments and for providing strains W4573, N43, AMS6, LBB1175, and OLS103.
This work was supported by funding from Robert A. Welch Foundation grant A-1354 to J.C.H.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843-2128. Phone: (979) 862-4054. Fax: (979) 845-4946. E-mail: jimhu{at}tamu.edu.
Present address: Columbia University, Department of Chemistry, New
York, NY 10027.
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Antimicrobial Agents and Chemotherapy, November 2000, p. 3210-3212, Vol. 44, No. 11
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