Department of Microbiology, Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama, 7008530, Japan
Received 30 April 2003; returned 25 May 2003; revised 19 June 2003; accepted 25 June 2003
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Abstract |
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Materials and methods: We isolated a mutant, YM442, which showed elevated resistance to several antimicrobial agents from P. aeruginosa YM44 lacking four major multidrug efflux pumps, MexAB, MexCDOprJ, MexEFOprN and MexXY. We cloned genes responsible for the resistance from chromosomal DNA of YM442 using YM44 as host.
Results: We designated the genes mexVW. Introduction of a recombinant plasmid pTAJ2 carrying the mexVW into YM44 cells conferred resistance to fluoroquinolones, tetracycline, chloramphenicol, erythromycin, ethidium bromide and acriflavine. Elevated ethidium bromide extrusion was observed with cells of YM442 and of YM44/pTAJ2. An outer membrane protein OprM was able to cooperate with MexVW. Elevated expression of the mexV gene was observed with YM442 compared with YM44.
Conclusions: MexV (membrane fusion protein)MexW (RND-type membrane protein)OprM is a tripartite multidrug efflux pump. It is suggested that other outer membrane component(s) could cooperate with MexVW.
Keywords: MexVW, multidrug efflux pump, RND-type, P. aeruginosa
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Introduction |
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Pseudomonas aeruginosa shows significant degrees of intrinsic and acquired resistance to a wide variety of antimicrobial agents, including most ß-lactams, fluoroquinolones, tetracycline, chloramphenicol, erythromycin and so on. This is a serious clinical problem because P. aeruginosa is a major opportunistic pathogen and a leading cause of nosocomial infections. At least six RND family drug efflux pumps are known to exist in cells of P. aeruginosa, MexABOprM,1 MexCDOprJ,2 MexEFOprN,3 MexXYOprM,4 MexJKOprM5 and MexHIOpmD6 (H. Sekiya and T. Tsuchiya, unpublished results). Among these Mex pumps, expression of MexABOprM is constitutive and is further enhanced in the presence of antimicrobial agents in the growth medium. Both MexCDOprJ and MexXYOprM are expressed only under conditions where inducing drugs are present. MexEFOprN and MexJK are silent in wild-type P. aeruginosa. Whole genome sequence information of P. aeruginosa is now available,7 and suggests the presence of about 10 RND family multidrug efflux pumps in this microorganism. Previously, we reported the functional cloning of multidrug resistance genes of P. aeruginosa using a drug-hypersensitive Escherichia coli mutant as a host, and obtained many candidate plasmids conferring multidrug resistance.4 We were able to clone mexAB, mexCD and mexXY, but not others.4 These three pumps are functional in wild-type P. aeruginosa. It seems possible that other genes for multidrug efflux pumps are silent, or expression or pump activity is very weak in wild-type P. aeruginosa. Thus, it is impossible to clone such gene(s) by functional cloning.
It is very likely that mutation in hitherto uncharacterized pumps would lead to other multidrug resistances. It is valuable to isolate multidrug-resistant mutants from a P. aeruginosa strain that lacks major multidrug efflux operons.8 It is expected that silent gene(s) for multidrug efflux pumps is(are) functional in such mutants. Here we report the isolation of such a mutant, the functional cloning of genes for a new member of an RND-type multidrug efflux pump (MexVW) from the mutant and properties of the pump.
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Materials and methods |
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P. aeruginosa PAO1, YM44 (mexAB,
mexCDoprJ,
mexEFoprN,
mexXY), YM442, a drug-resistant derivative of YM44, and YM64 (
mexABoprM,
mexCDoprJ,
mexEFoprN,
mexXY)8 were used in this study. YM44 was constructed by Flp-FRT recombination technology as described for YM64.8,9 Bacterial cells were grown in L medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl, pH 7) at 37°C under aerobic conditions.
Isolation of drug-resistant mutants
Cells of P. aeruginosa YM44 were grown in the L medium and were spread onto L agar plates containing four-fold higher concentrations (0.25 mg/L) than the MICs for norfloxacin. We obtained many colonies that appeared on the plates after incubation at 37°C for 20 h. After single colony isolation, we checked the drug resistance patterns of the mutants. One of the mutants, which showed resistance to several antimicrobial agents, was designated YM442, and used for further analysis.
Gene manipulation
Chromosomal DNA was prepared from cells of YM442. The DNA was partially digested with a restriction endonuclease Sau3AI, separated by sucrose density gradient centrifugation, and 410 kilobase pair (kbp) fragments were collected. A shuttle vector pUCP20T for P. aeruginosa and E. coli was used as a cloning vector. The chromosomal DNA fragments were ligated to the BamHI site of pUCP20T. The hybrid plasmids were introduced into cells of YM44 by electroporation. Transformants appeared on LB agar plates containing carbenicillin 50 mg/L and norfloxacin 0.5 mg/L. After single colony isolation, plasmid DNA was prepared from the cells, and re-introduced into cells of YM44. Drug resistance of the re-transformant was confirmed, and the plasmid harboured by the cells was designated pER2. The size of the DNA insert in pER2 was about 14 kbp. We sub-cloned a DNA region responsible for drug resistance from pER2, and obtained plasmid pTAJ2. The size of the DNA insert in pTAJ2 was about 5.1 kbp.
The nucleotide sequence was determined by the dideoxy chain termination method10 with an automated DNA sequencer (ALF Express, Pharmacia Biotech.).
RTPCR
Cells of P. aeruginosa (PAO1, YM44 and YM442) were harvested at the exponential phase of growth (3 mL culture). Total cellular RNA was isolated from the cells using the Qiagen RNeasy Mini Kit (Qiagen Inc., USA), treated with RNase-free DNase (Promega, USA) (1 U of enzyme/µg RNA for 2 h at 37°C) and re-purified using the same kit. A 1 µg sample of DNase-treated RNA was used as template for RTPCR with the Qiagen OneStep RTPCR Kit (Qiagen) according to the manufacturers protocol. Primer pairs specific for and internal to both mexV and the rpsL genes (encoding a constitutively expressed ribosomal protein that was used as a control) were used for RTPCR. Primers used for mexV were: CGTCAGCAGATCGCCCTTTTCAGC (forward) and CGCTTTTCGAGATGGCCTTGCTGC (reverse); and for rpsL: GCAACTATCAACCAGCTGGTG (forward) and GCTGTGCTCTTGCAGGTTGTG (reverse). A 6 µL sample of each reaction product was analysed by agarose X (3% w/v) gel electrophoresis for presence of the expected RTPCR products (for mexV, 331 bp; for rpsL, 220 bp).
Drug susceptibility
The MICs of various drugs were determined by the broth microdilution method with MuellerHinton broth (Difco) as reported previously.8 MICs were read after 24 h at 37°C.
Ethidium bromide efflux
Cells were grown in LB medium at 37°C and harvested at the middle exponential phase of growth. Ethidium bromide efflux was measured as reported previously.4
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Results and discussion |
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A mutant P. aeruginosa YM44 lacking mexAB, mexCDoprJ, mexEFoprN and mexXY was constructed. This strain, which possesses the oprM gene, is useful for isolation of mutants showing elevated activity of the RND-type pump, which can utilize OprM as an outer membrane component of the tripartite pump. The mutant we isolated, YM442, showed elevated resistance to norfloxacin, ofloxacin, chloramphenicol, cefpirome, tetracycline, ethidium bromide and acriflavine (Table 1). No detectable changes in the resistance levels to other ß-lactams and aminoglycosides were observed compared with the parental YM44 cells. Thus, it seems that one of the hitherto uncharacterized multidrug efflux pumps became functional in the YM442 strain.
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We tried to clone genes responsible for the multidrug resistance mentioned above from the chromosomal DNA of the mutant YM442. After cloning and sub-cloning using YM44 as a host, we obtained a hybrid plasmid pTAJ2. Cells of YM44/pTAJ2 showed elevated resistance to norfloxacin, ofloxacin, chloramphenicol, cefpirome, tetracycline, ethidium bromide and acriflavine, but not to gentamicin (Table 1). Thus, the drug resistance patterns in YM442 and YM44/pTAJ2 are the same. These results support the idea that the cloned gene(s) is(are) responsible for the elevated drug resistances observed with YM442. The level of drug resistance in YM44/pTAJ2 was higher than that of YM442 to some extent. This may be because of a gene dosage effect since the cloning vector pUCP20T is a multi-copy plasmid.
We determined the nucleotide sequence of several portions of plasmid pTAJ2. We have shown that pTAJ2 carries a DNA region encompassing the open reading frames (ORFs) from PA4374 to a portion of PA4376 (Figure 1), which have been reported in the whole genome sequence of P. aeruginosa.7 Complete ORFs included in pTAJ2 are PA4374 and PA4375. It has been suggested that these two ORFs code for a membrane fusion protein and an RND-type drug efflux protein.7 We designated the ORFs PA4374 and PA4375 as mexV and mexW, respectively, because deduced primary structures of these proteins are similar to those of other Mex proteins. The MexV deduced amino acid sequence showed the highest identity and similarity to MexH, 33% and 48%, respectively, and the MexW to MexI, 46% and 64%, respectively. The mexV (PA4374) gene is preceded by a promoter-like sequence. The lactose promoter of E. coli exists in the vector, but in the opposite direction in the downstream region of the part of PA4376 (Figure 1). Thus, it is very likely that the promoter-like sequence in the pTAJ2 functions as a promoter. A terminator-like sequence is present downstream from the mexW gene.7 We could not find an ORF near the MexVW genes that might encode a regulatory protein.
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Drug efflux
We confirmed that MexVW is a drug efflux pump by measuring ethidium bromide efflux. Cells of YM44 showed a higher level of intracellular ethidium bromide, suggesting no or very low efflux of ethidium bromide from cells (Figure 2). On the other hand, the intracellular ethidium bromide level in YM442 and YM44/pTAJ2 was lower than that in YM44, suggesting efflux of ethidium bromide. The intracellular ethidium bromide level increased when a H+ conductor carbonylcyanide m-chlorophenylhydrazone (CCCP) was added to the assay mixture. Thus, energy-dependent efflux of ethidium bromide occurred in cells of YM442 and YM44/pTAJ2. Addition of CCCP to a cell suspension of YM44 gave only a slight effect on the intracellular ethidium bromide level, suggesting that energy-dependent efflux of ethidium bromide is very low in YM44. We observed a similar intracellular ethidium bromide level with cells of YM442 compared to that with YM44/pTAJ2 (Figure 2). Thus, the energy-dependent efflux of ethidium bromide is high in these two types of cells. These results support the idea that genes on plasmid pTAJ2, namely mexV and mexW, code for a drug efflux pump.
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It seemed that the mexVW operon was silent or expression was weak in wild-type P. aeruginosa and in YM44, whereas the operon was over-expressed in the mutant YM442. We confirmed by the RTPCR method that expression of the mexV gene was very weak in wild-type PAO1 and parental YM44, and the gene was over-expressed in the mutant YM442 (roughly 4.4-fold over-expression) (Figure 3).
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Acknowledgements |
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Footnotes |
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References |
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