JAC Advance Access originally published online on January 22, 2007
Journal of Antimicrobial Chemotherapy 2007 59(3):544-547; doi:10.1093/jac/dkl510
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Contribution of the Rv2333c efflux pump (the Stp protein) from Mycobacterium tuberculosis to intrinsic antibiotic resistance in Mycobacterium bovis BCG
1 Departamento de Microbiología, Medicina Preventiva y Salud Pública, Universidad de Zaragoza, 50009 Zaragoza, Spain 2 Dipartimento di Genetica e Microbiologia, Università degli Studi di Pavia, 27100 Pavia, Italy
* Corresponding author. Tel: +34-976-762420; Fax: +34-976-762604; E-mail: ainsa{at}unizar.es
Received 13 July 2006; returned 17 August 2006; revised 5 November 2006; accepted 21 November 2006
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Abstract |
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Objectives: To characterize the efflux pump encoded by the gene Rv2333c from Mycobacterium tuberculosis, and assess its contribution to intrinsic antibiotic resistance using Mycobacterium bovis BCG as a model organism.
Methods: Firstly, the Rv2333c gene was expressed from a multicopy plasmid in M. bovis BCG. Secondly, the gene was inactivated in the chromosome of M. bovis BCG. Antibiotic susceptibility tests and tetracycline uptake/efflux experiments were carried out with the strains mentioned above.
Results: When the Rv2333c gene was inactivated in the M. bovis BCG chromosome, there was a decrease in the MIC values of spectinomycin and tetracycline, and an increase in [3H]tetracycline accumulation. When the Rv2333c gene was cloned into a multicopy plasmid, there was an increase in the MIC values of spectinomycin and tetracycline, and a decrease in [3H]tetracycline accumulation. These results indicate that both antibiotics are substrates of the Rv2333c efflux pump, which has been named Stp, for Spectinomycin Tetracycline efflux Pump.
Conclusions: The Rv2333c efflux pump (Stp protein) of M. tuberculosis contributes to intrinsic spectinomycin and tetracycline resistance.
Keywords: mycobacteria , spectinomycin , tetracycline , efflux
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Introduction |
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Tuberculosis has re-emerged in recent decades, being now a leading cause of mortality worldwide, despite the availability of the BCG vaccine and effective drugs. Mycobacterium tuberculosis has developed resistance against all first-line anti-tuberculosis agents and many second-line agents. In addition, M. tuberculosis is intrinsically resistant to most antibiotics, generally due to its unusual multi-layer cell envelope that functions as an effective barrier to the penetration of antibiotics.1 Efflux pumps (energy-dependent membrane proteins capable of extruding either a single antibiotic or a wide variety of chemically and structurally unrelated substances) act in synergy with the cell wall permeability barrier to increase intrinsic drug resistance.2,3 In mycobacteria, several drug efflux pumps have been reported, but most of them are still not well characterized.1,4 Knowledge of the molecular mechanisms underlying antibiotic resistance will be of help for controlling drug-resistant and multidrug-resistant (MDR) bacteria.
In this study, we have characterized the efflux pump encoded by the Rv2333c gene from M. tuberculosis, which is identical to the Mb2361c gene from Mycobacterium bovis (here, we will refer to both genes as the stp gene), and assessed its contribution to intrinsic antibiotic resistance using M. bovis BCG as a model organism.
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Materials and methods |
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Bacterial strains, plasmids, growth conditions and chemicals
Mycobacteria were cultivated at 37°C in Middlebrook 7H9 broth supplemented with 10% ADC and 0.05% Tween 80 or on Middlebrook 7H10 agar plates supplemented with 10% OADC. Hygromycin (10 mg/L) and kanamycin (20 mg/L) were used when appropriate. Escherichia coli cells were grown at 37°C in Luria-Bertani (LB) broth or on LB agar plates. E. coli and M. bovis BCG Pasteur and derivatives were transformed by electroporation, and plasmids and bacterial artificial chromosomes (BACs) were maintained with appropriate antibiotics for selection. Acriflavine, amikacin, carbonyl cyanide m-chlorophenylhydrazone (CCCP), ciprofloxacin, clofazimine, chloramphenicol, chlorpromazine, econazole, ethambutol, ethionamide, fluconazole, gentamicin, isoniazid, ofloxacin, p-aminosalicylic acid, rifampicin, streptomycin, spectinomycin, tetracycline, triclosan and vancomycin were used in antimicrobial susceptibility tests.
Antimicrobial susceptibility was determined by inoculating spots of 103 cells (diluted from log-phase cultures) onto 7H10 agar plates containing 2-fold antibiotic serial dilutions. Visible growth was scored after 21 days of incubation at 37°C. MIC determinations were carried out in triplicate and repeated at least three times.
Tetracycline accumulation experiments
M. bovis BCG, SUM36, KO2333 and SAN17 strains were grown to the exponential phase in 7H9 broth, washed twice with 0.1 M potassium phosphate (pH 7.0) and resuspended in 0.1 M potassium phosphate (pH 7.0)/1 mM MgCl2. Cells were incubated at 37°C for 15 min before the addition of [3H]tetracycline to a final concentration of 5 µM. Uptake experiments were performed essentially as previously described.5 Experiments were done at least three times to ensure reproducibility.
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Results |
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Construction of M. bovis strains overexpressing or lacking the stp gene
Two complementary approaches were followed. Firstly, a 2.4 kb fragment obtained from BAC Rv42 containing the stp gene and 581 bp upstream of its start codon [which is mostly a non-coding region of DNA where the promoter of stp and any putative regulatory sequence(s) should be located] was cloned in vector pSUM36,6 yielding plasmid pSAN17 that was electroporated in M. bovis BCG, resulting in M. bovis SAN17 strain. Using RT-PCR, we detected that in M. bovis SAN17 strain the transcript corresponding to the stp gene was between 2 and 3 times more abundant than in wild-type cells, as revealed by densitometric analysis (data not shown).
Secondly, we constructed a suicide delivery vector in which the stp gene was interrupted by the insertion of a hygromycin resistance cassette flanked by transcription and translation termination signals.7 After electroporation in M. bovis BCG, candidates for single (SXO) and double cross-over (DXO) were selected as described previously,8 and were analysed phenotypically, by PCR and by Southern blot (data not shown). Once the inactivation of the stp gene in the chromosome of M. bovis BCG was confirmed, the strain was named M. bovis KO2333.
Susceptibility profiles of M. bovis KO2333 and M. bovis SAN17 strains
We assayed the susceptibilities of M. bovis BCG and strains KO2333 (carrying the disrupted stp gene), SAN17 (containing the stp gene cloned in pSUM36) and SUM36 (containing vector pSUM36) to a variety of antimicrobial agents (including first- and second-line anti-tuberculosis drugs, broad-spectrum antibiotics and other molecules). M. bovis KO2333 strain was 2- and 4-fold more susceptible to spectinomycin and tetracycline, respectively, than M. bovis BCG (Table 1). The MICs for M. bovis SAN17 of these two antibiotics were 2-fold higher than those for the SUM36 strain (Table 1). No significant changes in the MICs of the other drugs tested were observed for any of the strains (data not shown). All together, these data indicate that the Stp pump contributes to the intrinsic resistance of M. bovis BCG to spectinomycin and tetracycline.
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Accumulation experiments
[3H]Tetracycline efflux experiments showed that SAN17 cells (carrying the stp gene cloned into the pSUM36 vector) accumulated approximately 25% less tetracycline (Student's t-test: 3.168; P = 0.016) than SUM36 cells (which harbour the cloning vector pSUM36), indicating that the tetracycline resistance phenotype mediated by the Stp protein was due to an efflux mechanism (Figure 1b). Similarly, KO2333 cells, due to the inactivation of the stp gene, accumulated approximately 25% more [3H]tetracycline (Student's t-test: –6.356; P < 0.05) than M. bovis BCG achieving a steady-state level of accumulation within about 3 min of incubation (Figure 1a). This suggests that the Stp pump has a role in determining the baseline MIC for M. bovis BCG of this compound.
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Discussion |
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We have characterized the contribution of the Stp efflux pump from M. tuberculosis to intrinsic antibiotic resistance, using M. bovis BCG as a model organism.
For this purpose, we first expressed the stp gene from its own promoter, cloned in vector pSUM36. pSUM vectors are present at 5–10 copies per cell allowing a moderate increase in the expression levels of the genes cloned in them; in fact, we have detected that in strain SAN17 (expressing the pSUM-cloned stp gene) there is a two-to-three times increase in the transcription of the stp gene. This allows the detection of the specific phenotype associated with such a moderate increase in the number of units of the Stp efflux pump, therefore avoiding an excessive overproduction of the efflux pump, which would compromise the structure and non-specific permeability to antibiotics of the membrane and would have deleterious effects for the bacteria. Also, the gene was inactivated in the chromosome of M. bovis BCG using homologous recombination. As the stp gene does not seem to belong to any operon (following the information on the M. tuberculosis H37Rv genome), we assume that the observed phenotype for the mutant strain will be due only to the non-production of the Stp protein.
We have found that Stp transports spectinomycin and tetracycline out from M. bovis BCG, since the strain carrying the stp gene on a multicopy plasmid showed increased MICs of these two antibiotics and accumulated less tetracycline than wild-type cells. In agreement, the strain carrying the stp gene inactivated in the chromosome showed decreased MICs of these two antibiotics and accumulated more tetracycline than wild-type cells. In both cases, the variation in the MIC values is low. In general, efflux pumps are involved in low-level antibiotic resistance, but they are thought to be necessary for the development of higher resistance levels.
Moreover, efflux pumps are proposed to have a primary physiological role within the cell, the transport of antibiotics being a secondary function. For example, the multidrug-efflux transporter MdfA from E. coli confers alkali tolerance.9 In this way, in a set of preliminary experiments, we have found that M. bovis KO2333 grew slower than the wild-type strain in liquid media from the late exponential phase and through the stationary phase (data not shown). Thus, Stp could also be implicated in transport processes related to cellular growth.
Several studies have described the implication of drug efflux pumps in the pathogenicity of several bacterial species.2 Interestingly, a microarray analysis revealed an overexpression of the stp gene from M. tuberculosis in a macrophage infection model,10 indicating that this efflux pump may also be important for intracellular survival of mycobacteria. Altogether, these results suggest that the Stp protein would have the extrusion of another unknown metabolite(s) as its primary not-yet elucidated physiological function, and, simultaneously, could extrude spectinomycin and tetracycline from cells as a secondary function. Also, since both spectinomycin and tetracycline are antibiotics produced by environmental bacteria, we can speculate that efflux pumps transporting such substrates would allow mycobacteria (for which an environmental origin has also been suggested) to survive in the environment.
The stp gene seems to be present in all strains of the M. tuberculosis complex. It is found in those strains whose genomes have been sequenced (such as the laboratory strain M. tuberculosis H37Rv, the clinical isolate M. tuberculosis CDC1551 strain, M. bovis BCG and others), and in addition, using PCR we have detected it in ten out of ten DNA samples from our collection of M. tuberculosis complex clinical isolates (drug-resistant and drug-susceptible isolates of M. tuberculosis, M. bovis and Myobacterium africanum from different geographic origins; data not shown). Thus, this universal presence regardless of the drug resistance levels would suggest an implication of the Stp efflux pump in other cellular processes.
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None to declare.
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Acknowledgements |
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We thank Carmen Lafoz, Begoña Gracia and Ana B. Gómez (University of Zaragoza) and Silvia Buroni (University of Pavia) for technical assistance, and Virginie Mick for helping in cloning procedures. We also thank S. Cole for supplying M. tuberculosis H37Rv BAC Rv42. We acknowledge Dr R. Manganelli for critical reading of the manuscript.
This work was supported by the European Union research project "New strategies for treatment and prevention of mycobacterial diseases" (contract no. QLK2-2000-01761) (E. D. R. and C. M.), by CIBER grant CB06/06/0020 from the Spanish Ministerio de Sanidad y Consumo (C. M.), by the Ministerio de Ciencia y Tecnología BIO-2002-01287 and BIO-2005-01801 (J. A. A.), by FAR 2004 of the University of Pavia (E. D. R.) and by the Integrated Action Italy-Spain (E. D. R. and J. A. A.). S. R. G. holds a grant from the Ministerio de Educación y Ciencia (ref: AP2001-1114).
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References |
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6 Ainsa JA, Martin C, Cabeza M, et al. (1996) Construction of a family of Mycobacterium/Escherichia coli shuttle vectors derived from pAL5000 and pACYC184: their use for cloning an antibiotic-resistance gene from Mycobacterium fortuitum. Gene 176:23–6.[CrossRef][Web of Science][Medline]
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