ß-Lactam resistance modulated by the overexpression of response regulators of two-component signal transduction systems in Escherichia coli

Hidetada Hirakawa1,2,3, Kunihiko Nishino1,2,3, Junko Yamada1,2,3, Takahiro Hirata1,2,3 and Akihito Yamaguchi1,2,3,*

1 Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047; 2 Faculty of Pharmaceutical Science, Osaka University, Suita, Osaka 565-0871; 3 CREST, Japan Science and Technology Corporation, Osaka 567-0047, Japan

Received 27 March 2003; returned 18 June 2003; revised 4 July 2003; accepted 9 July 2003


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Objectives: In Escherichia coli, there are 32 open reading frames assumed, on the basis of sequence similarities, to be response regulator genes of two-component signal transduction systems. We cloned all 32 response regulators and examined whether or not response regulator-overexpressing cells confer resistance to ß-lactam antibiotics in E. coli.

Methods: E. coli KAM3 (acrB), a drug-hypersusceptible mutant, was used as a host strain for the overproduction of response regulators. MICs were determined by the agar dilution method.

Results: Thirteen response regulators out of 32 genes, namely baeR, cheY, cpxR, creB, evgA, fimZ, narL, ompR, rcsB, rstA, yedW, yehT and dcuR, conferred increased ß-lactam resistance. Among them, overexpression of baeR, evgA, rcsB and dcuR conferred high-level resistance. The baeR- and evgA-mediated resistance is due to up-regulation of the expression of multidrug exporter genes, acrD and mdtABC for baeR, and yhiUV for evgA, because baeR- and evgA-mediated resistance was completely absent in strains lacking these exporter genes. The fimZ-mediated cefalothin resistance is due to the chromosomal ampC gene, because the ampC deletion strain did not show fimZ-mediated resistance.

Conclusions: Two-component signal transduction systems contribute to ß-lactam resistance in E. coli. Multidrug exporters play roles in two-component signal transduction system-mediated ß-lactam resistance.

Keywords: ß-lactams, multidrug exporters, drug resistance, E. coli


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Two-component systems comprise signal transduction pathways in prokaryotic organisms that respond to environmental conditions.1 A typical two-component system consists of two types of signal transducers, a sensor kinase and its cognate response regulator. The sensor kinase monitors some environmental conditions and accordingly modulates the phosphorylation state of the response regulator. The response regulator controls gene expression and/or cell behaviour.2,3 Overproduction of response regulators is often thought to mimic the physiological phosphorylation response.4 In Escherichia coli, the existence of 32 response regulators and 30 sensor kinases has been assumed on the basis of the results of genome sequence analysis.5

We previously found that the overexpression of response regulators of two-component signal transduction systems confers resistance to a number of chemical compounds, such as detergents, basic dyes and bile salts, in addition to many antibiotics, such as kanamycin, fosfomycin, erythromycin and novobiocin, via regulated expression of some drug exporter genes, acrD, emrKY, mdtABC and yhiUV in E. coli.69

In this study, we systematically investigated the ß-lactam antibiotic resistance mediated by overexpression of the response regulators. The AcrAB multidrug exporter is constitutively expressed in E. coli and confers a wide range and moderate levels of ß-lactam antibiotic resistance.10 However, the expression of AcrAB was not affected by the overexpression of any response regulator.6 Therefore, in this study, we cloned all 32 open reading frames (ORFs) of a putative response regulator in E. coli into an expression vector carrying the kanamycin resistance marker, and response regulator-mediated ß-lactam resistance was then investigated in a strain lacking the acrB gene.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacterial strains and plasmids

The bacterial strains and plasmids used in this study are shown in Table 1. Chromosomal DNA of E. coli W310411 was used as a PCR template. E. coli KAM3,12 a derivative of TG113 that lacks a restriction system and acrB, was used as the host for the drug susceptibility test. KAM3{Delta}acrD, KAM3{Delta}mdtABC, KAM3{Delta}acrDmdtABC and KAM3{Delta}yhiUV6,9 were as constructed previously. The construction of an ampC deletion mutant of E. coli KAM3, KAM3{Delta}ampC, was performed by the gene replacement method as described previously using pKO3.14 E. coli cells were grown in 2x YT medium (tryptone 16 g/L, yeast extract 10 g/L, sodium chloride 5 g/L),15 supplemented with kanamycin (25 mg/L) when necessary, under aerobic conditions at 37°C.


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Table 1. Bacterial strains and plasmids used in this study
 
Construction of an expression vector, pTrc99K, for the cloning of response regulators

Expression vector pTrc99K, a derivative of pTrc99A, which has the kanamycin resistance marker instead of that for ampicillin, was constructed as follows. The kanamycin resistance gene was amplified from the pUC4K vector using primers that introduced ScaI and AviII sites at the ends of the amplified fragment. This PCR product was ligated into the same restriction sites of pTrc99A with most of the ampicillin resistance gene eliminated.

Construction of an expression plasmid library of the response regulator ORFs

The 32 ORFs assumed to be the response regulators of two-component systems were cloned from E. coli W3104 as described previously.5 Chromosomal DNA was isolated from E. coli W3104 as described previously.15 ORFs were amplified by PCR using primers that introduced an NcoI site in the forward primer and a BamHI site in the reverse one. The obtained DNA fragments were ligated into the pTrc99K vector digested with NcoI and BamHI. These plasmids are designated as pK plus the cloned gene name, such as pKarcA. Competent KAM3 cells were transformed with at least three of the constructed plasmids that had been extracted from independent colonies.

Drug susceptibility assay

The MIC was determined on YT agar (tryptone 8 g/L, yeast extract 5 g/L, sodium chloride 5 g/L, agar 15 g/L) containing various concentrations of ß-lactams (penicillin G, ampicillin, oxacillin, cloxacillin, carbenicillin, nafcillin, cefalothin, cefaloridine, cefmetazole, cefamandole, cefuroxime, cefotaxime, ceftazidime, flomoxef, aztreonam, carumonam, imipenem, biapenem, faropenem, meropenem and panipenem), by a two-fold serial dilution technique with the standard method of the Japanese Society of Chemotherapy.6 Various concentrations of isopropyl-ß-D-thiogalactopyranoside (IPTG) (1, 0.1 or 0.01 mM) were added to the agar plates as an inducer. Ten thousand cells were inoculated with the microplanter (MIT-60P; Sakuma Seisakusyo, Tokyo, Japan), a specially designed device for MIC determination, and incubated at 37°C for 16 h. The MIC was determined as the lowest concentration at which growth was largely prevented. The results were highly reproducible even in the case of a two-fold difference. MIC measurements were performed at least three times.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Identification of the response regulators conferring ß-lactam resistance

The AcrAB multidrug efflux pump is constitutively expressed in E. coli and confers intrinsic ß-lactam resistance.10,16 The acrB gene deletion mutant, KAM3, showed higher susceptibility to oxacillin, cloxacillin and nafcillin than the parent strain, TG1 (Table 2). We previously reported that response regulators do not up-regulate the expression of acrA and acrB. In this study, we investigated whether or not ß-lactam resistance determinants other than acrAB are modulated by response regulator overexpression. The 32 ORFs of putative response regulators were cloned under the trc promotor in the kanamycin resistance vector. The expression of the response regulators was induced with IPTG at a concentration of 0.01, 0.1 or 1 mM in cells lacking the acrB gene. Addition of IPTG did not affect the susceptibility of the host strain, KAM3 (Table 2). Thirteen response regulator genes conferred some ß-lactam resistance of various degrees, as shown in Table 2. Of these 13 genes, evgA- and baeR-overexpressing cells showed high-level oxacillin, cloxacillin and nafcillin resistance, with a 16- to 32-fold increase in MIC up to 50 or 25 mg/L (Table 2). In addition, baeR also conferred moderate or low-level resistance to carbenicillin (eight-fold), aztreonam (eight-fold), carumonam (eight-fold), cefamandole (four-fold), ceftazidime (four-fold), cefmetazole (two-fold), cefuroxime (two-fold) and cefotaxime (two-fold). The evgA gene also conferred low-level resistance to ampicillin, cefmetazole, ceftazidime, flomoxef, aztreonam and carbenicillin, with a two-fold increase in MIC. Overexpression of rcsB and dcuR conferred high-level or moderate resistance to some carbapenems. Among them, resistance to biapenem was the highest (32- or 16-fold). rcsB-expressing cells showed low-level resistance to six other ß-lactam antibiotics. The other nine of the above 13 regulator genes (cheY, cpxR, creB, fimZ, narL, ompR, rstA, yedW and yehT) conferred low-level ß-lactam resistance (two-fold). Even in cases where the MIC differences were small, this was highly reproducible. The remaining 19 regulator genes (arcA, atoC, b2381, basR, cheB, citB, glnG, hydG, kdpE, narP, phoB, phoP, rssB, torR, uhpA, uvrY, yfhA, ygiX and ylcA) conferred no ß-lactam resistance, irrespective of the IPTG concentration (data not shown). Among these 19 response regulators, we have already reported that citB-, kdpE-, narP- and torR- overexpressing cells conferred resistance to the other antibiotics and/or detergent.6 The remaining 15 response regulators conferred no resistance to any compounds tested under the conditions we employed.


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Table 2. Drug resistance of E. coli KAM3 cells harbouring pTrc plasmids carrying putative response regulator ORFsa
 
Contribution of multidrug exporters, AcrD, MdtABC, EmrKY and YhiUV, induced by overexpression of the BaeR and EvgA response regulators

In previous studies, we found that EvgA up-regulated the expression of drug exporter genes emrKY and yhiUV, while BaeR up-regulated acrD and mdtABC.69,17,18 When the yhiUV gene was deleted from the chromosome of E. coli KAM3, EvgA overexpression no longer caused any oxacillin, cloxacillin or nafcillin resistance (Table 3). MICs decreased from 25 or 50 mg/L in KAM3 cells to 3.13 mg/L in KAM3{Delta}yhiUV cells, while emrKY deletion did not affect the EvgA- overexpression-mediated drug resistance. These results indicate that YhiUV is the main cause of EvgA-overexpression-mediated oxacillin, cloxacillin and nafcillin resistance. The yhiUV deletion without EvgA overexpression had no effect on the oxacillin, cloxacillin and nafcillin susceptibility of the KAM3 strain, since the yhiUV gene is not expressed under normal growth conditions.6 However, EvgA overexpression maintains low-level ampicillin, oxacillin, carbenicillin, cefmetazole, ceftazidime, flomoxef and aztreonam resistance, even in the yhiUV deletion strain. The EvgA-overexpression-mediated low-level resistance to these drugs remained even when both the emrKY and yhiUV genes were deleted (Table 3). Therefore, the evgA-mediated low-level ß-lactam resistance may involve resistance determinants other than YhiUV and EmrKY. Regarding baeR-mediated resistance, single deletion mutants, KAM3{Delta}acrD and KAM3{Delta}mdtABC, showed lower ß-lactam resistance than that of KAM3, but moderate baeR-mediated resistance remained. The acrD and mdtABC double-deletion mutant, KAM3{Delta}acrDmdtABC, had completely lost baeR-mediated ß-lactam resistance, indicating that baeR-mediated ß-lactam resistance is a result of the induction of multidrug exporter AcrD and MdtABC gene expression.


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Table 3. Resistance of KAM3 transporter gene-deletion strains harbouring a plasmid carrying baeR or evgA
 
Contribution of AmpC chromosomal ß-lactamase induced by overexpression of the FimZ response regulator

The E. coli chromosome carries an intrinsic ß-lactamase gene, ampC, which mainly confers intrinsic resistance to cefalothin and cefaloridine. We suspected that fimZ-mediated cefalothin resistance might be a result of AmpC ß-lactamase. The ampC-deleted KAM3 strain exhibited cefalothin hypersusceptibility (MIC 0.78 mg/L) compared with the parental KAM3 strain (12.5 mg/L), similar to the results obtained by Mazzariol et al.10 In the KAM3{Delta}ampC strain, FimZ overexpression did not cause cefalothin resistance at all (0.78 mg/L), suggesting that fimZ-mediated cefalothin resistance is a result of AmpC ß-lactamase; however, FimZ-overexpressing cells did not confer resistance to cefaloridine. Mazzariol et al.10 reported that AmpC-mediated cefaloridine resistance is lower than cefalothin resistance. Thus, fimZ-mediated AmpC-dependent cefaloridine resistance might have been lower than the limit of detection in this experiment.

We also examined whether or not baeR- and evgA-mediated oxacillin resistance, and rcsB- and dcuR-mediated carbapenem resistance, is observed even in the ampC-deficient genetic background. As a result, these resistances were observed as well as ampC plus background, indicating that these response regulator-mediated ß-lactam resistances are independent of AmpC (data not shown).

Which determinants contribute to rcsB- and dcuR-mediated carbapenem resistance? A decrease in the OmpF outer membrane protein is known to cause resistance to carbapenems in addition to large lipophilic compounds.1921 If RcsB and DcuR repress ompF expression, they might confer resistance to carbapenems and lipophilic compounds. However, this was not the case, since rcsB- and dcuR-overexpressing cells did not confer resistance to lipophilic compounds.6 The resistance determinants responsible for rcsB- and dcuR-mediated resistance remain unknown.

Recently, it has been shown that some two-component systems regulate ß-lactam resistance in different bacterial species. In Streptococcus pneumoniae, a constitutively activated sensor kinase CiaH mutant caused cefotaxime and penicillin resistance.22,23 In Stenotrophomonas maltophilia, response regulator SmeR positively regulates multidrug exporter SmeABC, conferring intrinsic ß-lactam resistance.24 In Listeria monocytogenes, response regulator LisR overexpression suppresses the suceptibility to cephalosporins for sensor kinase LisK deletion mutant.25 However, to the best of our knowledge our study is the first to comprehensively investigate the putative response regulator gene-mediated ß-lactam resistance in total. In addition, it is the first case showing that some response regulators contribute to ß-lactam resistance in Gram-negative bacterial species.

Two-component system-mediated ß-lactam resistance may become a problem in future chemotherapy, and we hope that our comprehensive study will be informative in determining a strategy for overcoming possible multidrug resistance pathogens.

Note added in proof

After submitting this report, YLiU and YhiV have recently been renamed MdtE and MdtF by the systematic nomenclature indicated at http://bmb.med.miami.edu/ecogene/ecoweb/.


    Acknowledgements
 
We wish to thank Tomofusa Tsuchiya of Okayama University for providing us with the E. coli KAM3 strain, Takeshi Nishino of Kyoto Pharmaceutical University for most of the ß-lactam antibiotics examined in this study, and George M. Church for plasmid pKO3. K.N. was supported by a research fellowship from the Japan Society for the Promotion of Science for Young Scientists. This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Zoonosis Control Project of the Ministry of Agriculture, Forestry and Fisheries of Japan.


    Footnotes
 
* Correspondence address. Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki-shi, Osaka 567-0047, Japan. Tel: +81-6-6879-8545; Fax: +81-6-6879-8549; E-mail: akihito{at}sanken.osaka-u.ac.jp Back


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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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