Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands1
Department of Medical Microbiology, University of Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands2
Author for correspondence: Wil N. Konings. Tel: +31 50 3632158. Fax: +31 50 3632154. e-mail: w.n.konings{at}biol.rug.nl
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ABSTRACT |
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Keywords: multidrug resistance, multidrug transport, Lactococcus lactis, antibiotic resistance
Abbreviations: PMF, proton-motive force
a Present address: Hercules European Research Center, Postbus 252, 3770 AG Barneveld, The Netherlands.
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INTRODUCTION |
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One of the sophisticated biochemical mechanisms that bacteria have developed to evade the lethal effects of toxic drugs is the active efflux of these compounds. In contrast to specific drug-efflux systems, multidrug transporters can extrude a wide variety of structurally unrelated compounds. At present, four classes of multidrug efflux systems have been characterized in bacteria: (i) the ATP-binding cassette superfamily, (ii) the major facilitator superfamily, (iii) the resistance-nodulation-division family, and (iv) the small multidrug resistance family (Paulsen et al., 1996 ; van Veen & Konings, 1998
). Many organisms can express several multidrug transporters belonging to different classes.
In the Gram-positive bacterium Lactococcus lactis at least four drug-extrusion activities have been detected (Molenaar et al., 1992 ; Bolhuis et al., 1994
; Glaasker et al., 1996
). The PMF-driven extrusion of amphiphilic drugs from the inner leaflet of the membrane is mediated by the multidrug transporter LmrP (Bolhuis et al., 1995
, 1996
). LmrP is a 408-amino-acid integral membrane protein with 12 putative transmembrane segments, and belongs to the major facilitator superfamily. Extrusion of drugs by LmrP is driven by both the membrane potential and the transmembrane proton gradient, indicating that LmrP mediates an electrogenic proton/drug antiport reaction (Bolhuis et al., 1996
; Putman et al., 1999b
).
In this paper we describe the role of the secondary multidrug transporter LmrP in the resistance to a wide range of clinically important antibiotics that are used to fight infectious diseases. LmrP confers resistance to clindamycin, 14- and 15-membered macrolides, dalfopristin, RP 59500 and tetracycline antibiotics. Hence, LmrP and its endogenous and acquired homologues in pathogenic bacteria are a serious threat to the antibiotic-based treatment of patients with infectious diseases.
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METHODS |
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Bacterial strains and growth conditions.
The bacterial strains and plasmids used in this study are listed in Table 1. Lactococcus lactis was grown at 30 °C in M17 medium (Difco) supplemented with 0·5% glucose (w/v) plus 5 µg chloramphenicol ml-1 when appropriate. Escherichia coli was grown at 37 °C in Luria Broth containing 50 µg ampicillin ml-1 when appropriate.
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Hoechst 33342 transport.
Hoechst 33342 transport mediated by LmrP was studied as described previously (Putman et al., 1999a , b
). Inside-out membrane vesicles (0·5 mg protein ml-1) were resuspended in 50 mM potassium HEPES, pH 7·0, containing 2 mM MgSO4, 8·5 mM NaCl, 0·1 mg creatine kinase ml-1, plus 5 mM phosphocreatine. After 30 s incubation at 30 °C Hoechst 33342 was added to 1 µM final concentration. LmrP was energized by the generation of a proton-motive force (PMF) by the F0F1 H+-ATPase, upon the addition of 0·5 mM Mg2+-ATP. The amount of membrane-associated Hoechst 33342 was measured fluorimetrically (Perkin Elmer LS50B fluorometer), using excitation and emission wavelengths of 355 and 457 nm, respectively, and slit widths of 5 nm each. The initial rate of Hoechst 33342 transport was determined by linear regression of the fluorescence data obtained in the first 15 s after Mg2+-ATP addition.
Measurement of the transmembrane H+ gradient (pH) in membrane vesicles.
The pH (inside acid) in inside-out membrane vesicles was monitored by fluorescence quenching of acridine orange as described previously (Putman et al., 1999a
). The transmembrane potential (
, inside positive) in inside-out membrane vesicles was estimated from the increase in
pH upon dissipation of the
by the addition of the K+ ionophore valinomycin. To study the effect of antibiotics on the PMF, the drugs were added to a final concentration of 1 and 4 times the IC50 for inhibition of LmrP-mediated Hoechst 33342 transport.
Determination of growth rate.
For the determination of growth rates in the presence of various antibiotics, overnight E. coli cultures (DH5/pET302 and DH5
/pHLP1) were diluted into fresh medium and grown to mid-exponential phase. The cells were then diluted in fresh medium to an OD660 of 0·025 and 150 µl aliquots of the cell suspension were transferred to sterile low-protein-binding 96-well microplates (Greiner), containing 50 µl of various concentrations of the antibiotics in fresh medium. For the induction of LmrP expression isopropyl 1-thio-ß-D-galactopyranoside was added to a final concentration of 5 µM. Aliquots (50 µl) of sterile silicone oil were pipetted on top of the sample to prevent evaporation. The cells were grown semi-anaerobically at 30 °C, and the cell density was monitored by measuring the absorbance at 690 nm every 15 min for 23 h in a multiscan photometer (Titretek Multiscan MCC/340 MKII). Growth rates were determined by non-linear least-square fitting of the absorbance data to the Gompertz equation describing bacterial growth (Zwietering et al., 1990
). The antibiotic concentrations which inhibited the growth rate by 50% (IC50) were determined.
Tetracycline accumulation in whole cells.
L. lactis NZ9000 cells harbouring the control plasmid (pNZ8048) or the LmrP-expression plasmid (pHLP5) were grown at 30 °C to mid-exponential phase (OD660 0·5). Nisin A was added to a concentration of approximately 10 ng ml-1, to trigger the transcription of the lmrP gene. Subsequently, the cells were incubated for 1 h at 30 °C, harvested by centrifugation, washed three times in 50 mM potassium phosphate, pH 7·0, containing 0·2% glucose and 1 mM MgSO4, and resuspended in the same buffer to a protein concentration of approximately 0·75 mg ml-1. After a 10 min preincubation of 100 µl aliquots of the cell suspension at 30 °C, [3H]tetracycline was added to a final concentration of 5 µM. At the time points indicated in the legend to Fig. 4, the reaction was stopped by the addition of 2 ml ice-cold 100 mM potassium phosphate, pH 7·0, containing 100 mM LiCl. The samples were rapidly filtered through 0·45 µm pore-size cellulose acetate filters (Schleicher & Schuell). The filters were washed once with 2 ml ice-cold 100 mM potassium phosphate, pH 7·0, plus 100 mM LiCl. The radioactivity on the filters was determined by liquid scintillation counting.
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RESULTS |
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The growth rates of DH5/pET302 and DH5
/pHLP1 were determined in the presence of a variety of antibiotics. A typical result, obtained with erythromycin, is shown in Fig. 3
. The dose-response curve of the LmrP-expressing cells is shifted towards higher erythromycin concentrations, compared to the curve of the control cells, indicating that LmrP increases the resistance to this antibiotic, even at this low level of expression of LmrP. The concentration of the various antibiotics required to inhibit the growth rate by 50% (IC50) is shown in Table 3
. Because of the presence of an ampicillin-resistance marker on plasmids pET302 and pHLP1, ampicillin was not included in the cytotoxicity experiments. Since the sulfonamide antibiotic sulfamethoxazol did not significantly inhibit E. coli growth at a concentration of 400 µg ml-1 (data not shown), this compound was omitted from further studies.
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LmrP consistently increased the resistance to the macrolides azithromycin, clarithromycin, erythromycin and roxithromycin. These macrolide antibiotics contain a 14- or 15-membered lactone ring that has one or more deoxysugars attached (Woodward, 1957 ; Ballow & Amsden, 1992
). Interestingly, LmrP did not seem to confer resistance to the 16-membered macrolide spiramycin. LmrP expression increased the resistance to the streptogramins dalfopristin and RP 59500, but did not affect the resistance to quinupristin. The fourth-generation antibiotic RP 59500 is a combination of dalfopristin and quinupristin, which demonstrates synergistic and bactericidal activity against Gram-positive pathogens including methicillin-resistant Staphylococcus aureus (Leclerq & Courvalin, 1991
; Aumercier et al., 1992
; Kang & Ryback, 1995
). The LmrP-expressing strain showed higher resistance to all tetracycline antibiotics tested, indicating that substitutions on carbon atoms 5, 6 and 7 do not preclude the interaction between LmrP and the tetracycline antibiotics. LmrP expression did not increase the resistance to aminoglycosides, ß-lactams, glycopeptides, quinolones, and chloramphenicol.
Electrogenic tetracycline transport by LmrP
To determine whether the observed resistance to tetracycline antibiotics in LmrP-expressing cells is indeed caused by an active extrusion process, the accumulation of tetracycline in LmrP-expressing and control L. lactis cells was measured. In the presence of an energy source the control cells (NZ9000/pNZ8048) accumulated sixfold more tetracycline than the LmrP-expressing NZ9000/pHLP5 cells (Fig. 4a). Dissipation of the membrane potential by the addition of valinomycin (Fig. 4b
) resulted in a slight increase in the amount of tetracycline in the control cells. This is most likely due to an increase in
pH, which is a driving force for the passive influx of tetracycline into the cell (Munske et al., 1984
). The accumulation of tetracycline in the LmrP-expressing cells was hardly affected by the addition of valinomycin, indicating that the
pH is a driving force for LmrP-mediated tetracycline extrusion. Dissipation of the
pH by the addition of nigericin slightly reduced the high level of tetracycline accumulation in control cells, but did not affect the low level of tetracycline accumulation in LmrP-expressing cells (Fig. 4c
). When both components of the PMF were dissipated by the addition of both valinomycin and nigericin (Fig. 4d
), equal tetracycline accumulation in both the control and LmrP-expressing cells was observed. These results demonstrate that the increased tetracycline resistance in LmrP-expressing cells is a result of the active extrusion of tetracycline by LmrP. Both the
pH and
can drive the LmrP-mediated tetracycline efflux, indicative of an electrogenic tetracycline/H+ antiport reaction.
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DISCUSSION |
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Although macrolides, lincosamides and streptogramins differ structurally, these antibiotics are often grouped together (MLS antibiotics) because of their similar mechanism of action, which involves the inhibition of protein synthesis through the binding to the 50S subunit of the bacterial ribosome (Contreras & Vásquez, 1977 ). Although the most widespread mechanism of resistance to MLS-type antibiotics is target-site alteration (Weisblum, 1995
), this work and other studies (Leclerq & Courvalin, 1991
; Clancy et al., 1996
) demonstrate that the resistance to MLS-type antibiotics can also result from enhanced antibiotic efflux. LmrP confers resistance only to the 14- and 15-membered macrolides. The macrolide specificity of LmrP resembles that of the multidrug transporter MefA from Streptococcus pyogenes, which confers resistance to 14- and 15- but not to 16-membered macrolide antibiotics (Clancy et al., 1996
). LmrP expression increased the resistance to dalfopristin and RP 59500, but not to quinupristin, suggesting that quinupristin is not transported by LmrP. However, the observed inhibition of LmrP-mediated Hoechst 33342 transport by quinupristin demonstrated that this streptogramin can bind to LmrP. The ability of quinupristin to modulate the activity of multidrug efflux systems may be one of the mechanisms responsible for the synergistic activity of the combination of dalfopristin and quinupristin in antibiotic-based therapy.
Cells expressing LmrP showed an increased resistance to the tetracycline antibiotics. Studies with radio-labelled tetracycline demonstrated that LmrP-expressing cells accumulate significantly less tetracycline than control cells, indicating that the increased tetracycline resistance is due to the active extrusion of tetracycline by LmrP. Dissipation of the pH or
only did not increase the accumulation of tetracycline in the LmrP-expressing cells, suggesting that the LmrP-mediated transport of tetracycline is an electrogenic process. This observation is similar to previous observations on the LmrP-mediated transport of TPP+ (Bolhuis et al., 1996
) and Hoechst 33342 (Putman et al., 1999b
) and the TetL-mediated transport of tetracycline (Guffanti & Krulwich, 1995
). Specific tetracycline transporters, such as TetB of E. coli and TetL of Bacillus subtilis, extrude a complex of tetracycline and a divalent cation in an exchange with one (TetB; Yamaguchi et al., 1990
, 1991
) or more protons (TetL; Guffanti & Krulwich, 1995
). The role of divalent cations in the LmrP-mediated tetracycline transport will be a subject of further research.
The antibiotic specificity of LmrP indicates that PMF-dependent multidrug transporters can affect the efficacy of a number of clinically relevant antibiotics. Although L. lactis is occasionally found as a pathogen in immunocompromised hosts, this bacterium is generally considered to be non-pathogenic and safe to use in starter cultures for cheese production (Gasser, 1994 ). Given the broad antibiotic specificity of the lactococcal secondary multidrug transporter LmrP and the ATP-binding cassette transporter LmrA (Putman et al., 2000
), additional studies are required to determine the probability of transfer of chromosomal resistance genes, such as lmrA and lmrP, to other bacteria in food or the digestive tract.
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ACKNOWLEDGEMENTS |
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Received 20 February 2001;
revised 1 July 2001;
accepted 5 July 2001.