JAC Advance Access originally published online on November 12, 2005
Journal of Antimicrobial Chemotherapy 2006 57(1):79-84; doi:10.1093/jac/dki409
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Effects of clofazimine on potassium uptake by a Trk-deletion mutant of Mycobacterium tuberculosis
1 Tuberculosis Research Lead Programme, Medical Research Council, Pretoria, South Africa; 2 Tuberculosis Research Section, NIAID, National Institutes of Health, Rockville, MD, USA; 3 Medical Research Council Unit for Inflammation and Immunity, Department of Immunology, Faculty of Health Sciences, University of Pretoria and Tshwane Academic Division of the National Health Laboratory Service, South Africa; 4 MRC/NHLS/WITS Molecular Mycobacteriology Research Unit, DST-NRF Centre of Excellence for Biomedical Research, School of Pathology, University of the Witwatersrand and National Health Laboratory Service, Johannesburg, South Africa
* Corresponding author. Tel: +27-12-319-2425; Fax: +27-12-323-0732; E-mail: randerso{at}medic.up.ac.za
Received 27 June 2005; returned 8 September 2005; revised 23 September 2005; accepted 14 October 2005
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Abstract |
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Objectives: This study was designed to investigate the effects of the membrane-active, anti-mycobacterial agent, clofazimine, on potassium (K+)-uptake by a mutant of Mycobacterium tuberculosis (MTB), in which the Trk system, the major K+ transporter of this microbial pathogen, had been selectively inactivated.
Methods: The ceoB and ceoC genes of MTB, which encode the TrkA proteins, CeoB and CeoC, were deleted by homologous recombination, and the double-knockout mutant and wild-type strains compared with respect to K+ uptake and growth in the presence and absence of clofazimine (0.015–2.5 mg/L) using radioassay procedures.
Results: Surprisingly, the magnitudes of K+ uptake and rate of growth of the ceoBC-knockout mutant were significantly (P < 0.05) greater than those of the wild-type strain, due, presumably, to induction of a back-up transporter. Exposure of both the wild-type strain and ceoBC-knockout mutant of MTB to clofazimine was accompanied by dose-related decreases in K+ uptake, as well as growth, which were of comparable magnitude for both strains.
Conclusions: These observations demonstrate that the major K+ transporter of MTB, Trk, as well as an uncharacterized inducible back-up system, is equally sensitive to the inhibitory actions of clofazimine.
Keywords: ceoB and ceoC genes , growth rate , uptake of rubidium
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Introduction |
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The K+ transporters of bacteria have varying affinities for the cation, are operative over a wide range of extracellular K+ concentrations, and enable intracellular levels of K+ to be maintained at 0.1–1 M. These high intracellular concentrations of K+ are necessary to sustain diverse cellular processes such as maintenance of turgor pressure, regulation of cytoplasmic pH, activation of enzymes, gene expression and stress responses.1,2
Two major K+ uptake systems, namely Trk and Kdp, have been identified and sequenced in Mycobacterium tuberculosis (MTB).3–5 The Trk system is composed of two TrkA proteins, CeoB and CeoC, which are homologous to the single TrkA proteins of Escherichia coli and Streptomyces coelicolor. The genes encoding these proteins, ceoB (684 bp, Rv2691) and ceoC (663 bp, Rv2692), are highly homologous and are arranged in tandem as the ceoBC operon at position 3009.34 on the MTB chromosome, with the stop codon of ceoB overlapping the start codon of ceoC.3
The Kdp system of MTB is highly homologous to that of E. coli, and is encoded by the kdpFABC and regulatory kdpDE operons.3 In contrast to E. coli, these two operons are divergently transcribed and are separated by an intergenic promoter region of about 192 bp. Like the Kdp system of E. coli, the MTB Kdp system is inducible, and functions as an emergency scavenger system when extracellular concentrations of K+ are limiting.5,6
We have previously reported that decreased uptake of K+ is one of the earliest detectable changes following exposure of MTB to the riminophenazine group of anti-mycobacterial agents, of which clofazimine is the prototype.7–9 However, the relative susceptibilities of the Trk and Kdp systems of MTB to clofazimine have not been established. In an attempt to address this issue, we have constructed a ceoBC double-knockout mutant of MTB which is selectively deficient in the Trk system, and compared this mutant with the wild-type strain with respect to efficiency of uptake of K+ and rates of growth, measured in the presence and absence of clofazimine.
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Materials and methods |
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Antimicrobial agents and chemicals
Clofazimine [3-(p-chloroanilino)-10-(p-chlorophenyl)-2,10-dihydro-2-(isopropylimino)-phenazine], synthesized by the late Dr J. F. O'Sullivan (Department of Chemistry, University College, Dublin, Republic of Ireland), was dissolved in DMSO and dilutions were made in the solvent to final concentrations of 0.015–2.5 mg/L.
Chloramphenicol, kanamycin, hygromycin and ampicillin were used at 12.5, 10, 50 and 100 mg/L, respectively, for selection of plasmid-carrying clones, while isopropylthio-ß-D-galactoside, 5-bromo-4-chloro-3-indolyl-ß-D-galactoside (X-Gal) and sucrose, were used at 0.24 mg/L and 2%, for selection of blue colonies and sacB-expressing clones, respectively. Unless indicated, all other chemicals used were obtained from the Sigma Chemical Co. (St Louis, MO, USA).
Rubidium-86 chloride (86Rb+) was purchased from PerkinElmer Life and Analytical Sciences, Du Pont-NEN Research Products, Boston, MA, USA.
Bacterial strains, plasmids and growth conditions
All plasmids used are listed in Table 1. The E. coli DH5
and GM161 strains were used for preparation of competent cells in Psi broth, and the cells were grown in 2TY and Luria–Bertani broths or agar. MTB (H37Rv, ATCC 26518) competent cells used for electroporation of the suicide delivery vector (SDV) were prepared in 7H9 broth (Difco) supplemented with 10% bovine albumin fraction V, dextrose, NaCl, catalase (ADC) and 0.05% Tween 80. For 86Rb+ uptake assays, the ceoBC-knockout mutant and the wild-type MTB strains were grown in 7H9 broth supplemented with 10% oleic acid, albumin, NaCl, dextrose, catalase (OADC) and 0.05% Tween 80 with continuous stirring for 7 days at 37°C.
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Construction of mutant
The first stage in generating the ceoBC double-knockout strain of MTB involved the construction of a suicide plasmid for allelic exchange mutagenesis using procedures as described previously.10 The 7021 bp HindIII–PstI fragment carrying the ceoBC operon was excised from the bacterial artificial chromosome (BAC)-Rv353,11 and cloned in pGEM3Zf(+) to produce pGceocos (10 214 bp) as shown in Figure 1(a). The in-frame deletion in ceoB was constructed by producing the upstream and downstream fragments, ceoB-UP (881 bp) and ceoB-DOWN (845 bp), by PCR amplification of genomic DNA using the primers shown in Table 2. The 871 bp PstI–XbaI ceoB-UP and 835 bp XbaI–PstI ceoB-DOWN fragments were individually cloned in pGEM3Zf(+) to produce pGUP and pGDOWN, respectively. The 762 bp BclI–XbaI ceoB-UP and 427 bp XbaI–NheI ceoB-DOWN fragments, were excised from pGUP and pGDOWN, respectively, and cloned in BclI–NheI pGceocos to produce pGceoB
C.
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The 6679 bp HindIII–PstI ceoB
C fragment was excised from pGceoBC and cloned into p2NIL12 to produce p2ceoBC. Subsequently, the ceoC gene was insertionally inactivated by cloning the 1746 bp BamHI–BglII hyg resistance cassette, from the plJ963 vector,13 in the NheI site of p2ceoBC to produce p2ceoBC::hyg. This vector thus carried two mutations consisting of an in-frame deletion in ceoB and an insertional inactivation in ceoC. The lacZ-sacB marker gene cassette was excised as a PacI fragment from pGOAL1712 and was cloned in p2ceoBC::hyg to produce the SDV, p2ceoBC::hyg17. This vector was used for allelic exchange mutagenesis in MTB by two-step selection.12 Briefly, 
5 µg of ultraviolet pretreated SDV was electroporated into MTB14 and plated onto 7H10 agar supplemented with hygromycin, X-Gal and kanamycin, and incubated at 37°C for at least 3 weeks. One blue hygromycin- and kanamycin-resistant colony was spread on 7H10 agar supplemented with hygromycin and X-Gal and grown for 2 weeks at 37°C. A scraping of white hygromycin-resistant cells was serially diluted before plating on 7H10 agar containing hygromycin, sucrose and X-Gal. The white sucrose- and hygromycin-resistant colonies were patch-plated onto 7H10 medium containing hygromycin with and without kanamycin, and putative double-crossovers (DCOs; white, sucrose-resistant, kanamycin-susceptible clones) were selected for genotypic analysis. Genomic DNA samples were extracted as described15 and Southern-blot analysis was performed following standard procedures using [-32P]dCTP radiolabelled probes.16 Uptake of 86Rb+
86Rb+ was used as tracer to compare the uptake of K+ by the wild-type and mutant strains of MTB in the absence and presence of clofazimine (0.15–2.5 mg/L). Briefly, the bacteria were harvested and resuspended to 106 cfu/mL in K+-free buffer (KONO) containing 2 mCi/L 86Rb+, with and without clofazimine, and uptake of 86Rb+ determined as previously described.7
Measurement of bacterial growth
The growth curves for the wild-type and mutant strains were determined radiometrically using the BACTEC TB system (Becton Dickinson Diagnostic Instrument Systems, Towson, MD, USA). A seed culture of each strain was synchronized for 2–3 days in a BACTEC 12B vial until a growth index (GI) value of 400–500 equivalent to 104–105 cfu/mL was reached. These cultures (0.1 mL) were used for inoculation into BACTEC vials and growth was monitored daily until GI values of 999 were reached.
A variation of this procedure was used to determine the susceptibility of the wild-type and mutant strains of MTB to clofazimine (0.015–2.5 mg/L). Briefly, synchronized cultures with GI values of 400–500 were inoculated in 0.1 mL volumes into BACTEC vials with and without clofazimine, while a control system was inoculated with 0.1 mL of a 1 : 100 dilution of inoculum. The vials were incubated at 37°C and GI values recorded daily until the value of the diluted control system reached 30, at which time the experiment was terminated. The GI values were calculated by subtracting the values for each clofazimine-treated system measured on the day before the diluted control system attained a value of 30 from the corresponding values recorded on that day. The MIC value was taken as the lowest concentration of clofazimine with a GI value less than that of the diluted control system.
Statistical analysis
The results are expressed as the means ± SEM. All statistical analyses were carried out with the INSTAT program using the paired Student's t-test. The significance levels were taken at a P-value <0.05.
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Results |
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Construction of the ceoBC double-knockout mutant strain
The Trk system of MTB was functionally inactivated by allelic exchange mutagenesis using a suicide vector carrying mutations in both the ceoB (ceoB) and ceoC (ceoC::hyg) genes (Figure 1a). The mutations were designed to avoid the possible creation of polar effects. The deletion removed 348 bp of an internal region of ceoB. The ceoC gene was inactivated by the insertion of a hyg gene 249 bp downstream of its start codon and in an orientation opposite to that of ceoC (Figure 1a). Three single-crossover (SCO) clones were recovered from the electroporation of MTB with SDV. Twelve putative DCOs were obtained from one of the SCOs by counter-selection against the sacB gene. Of these, clone BC5 was genotypically confirmed by Southern-blot analysis (Figure 1b). The viability of the ceoB ceoC::hyg double mutant confirmed that the Trk system is dispensable for growth of MTB in vitro.
Effect of inactivation of the Trk system on 86Rb+-uptake, growth and susceptibility to clofazimine of MTB
The mutant and the wild-type strains were compared for efficiency of uptake of K+ using 86Rb+ as tracer, and these results are shown in Table 3. Unexpectedly, the uptake of 86Rb+ by the mutant strain was considerably higher than that of the wild-type control.
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The effects of clofazimine on the uptake of 86Rb+ by the mutant and wild-type strains of MTB are shown in Figure 2. The Trk-deletion mutant and wild-type strains of MTB were more or less equally susceptible to the inhibitory effects of clofazimine on the uptake of 86Rb+, with maximum inhibition of influx observed at 1.25–2.5 mg/L for both strains.
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The growth curves for the mutant and wild-type strains of MTB are shown in Figure 3. The rates of growth of the two strains were equivalent over the first 2 days, but thereafter the growth rate of the mutant was slightly, but nevertheless significantly faster (P < 0.05) than that of the wild-type strain. In the presence of clofazimine, the MIC values for both the wild-type and the ceoBC-knockout mutant strains of MTB were identical (0.06 mg/L for both).
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We have previously reported that increased phospholipase activity and inhibition of uptake of K+ are the earliest detectable effects following exposure of MTB to the riminophenazine class of anti-mycobacterial agents, occurring within minutes and preceding decreases in microbial ATP levels.8,9 Although these observations implicate mycobacterial K+ transporters as being possible primary targets of the riminophenazine, clofazimine, the identities of the K+ transporters affected by this antimicrobial agent have not been established. In the current study, we have constructed a ceoBC-knockout mutant of MTB in which the Trk system has been selectively disabled, and compared the efficiencies of K+ uptake by the mutant and wild-type strains in the absence and presence of clofazimine.
Somewhat surprisingly, the efficiency of uptake of K+ by the ceoBC-knockout strain of MTB in the absence of clofazimine was significantly higher than that of the wild-type strain, and was associated with a small, but nevertheless significant increase in the rate of growth. These observations are compatible with the induction in the mutant of a high-affinity, back-up K+ transporter, possibly the Kdp system, which is operative in the absence of the Trk system. Interestingly, the wild-type and mutant strains of MTB were equally susceptible to the inhibitory effects of clofazimine on uptake of K+, as well as growth. These novel observations are of potential importance, because they reveal that it is not only the Trk system of MTB which is susceptible to clofazimine (wild-type strain), but also an inducible, back-up transporter, possibly the Kdp system, which is likely to be the major system operative in the Trk-deletion mutant.
Although the molecular/biochemical mechanism of clofazimine-mediated inhibition of mycobacterial K+ transporters has not been established, two possibilities exist. Clofazimine may interact directly with, and inactivate K+ uptake systems, or, alternatively, this agent may simply function as a membrane-destabilizing agent, dismantling membrane architecture, with consequent secondary dysfunction of membrane transporters. The range of bacterial K+ transporters which are sensitive to clofazimine, including Trk and an inducible, back-up system in MTB (present study) and Kup of E. coli,17 suggests that a membrane disruptive mechanism as recently proposed by us9 and others,18,19 appears most likely. In support of this contention, exposure to clofazimine is accompanied by a rapid increase in phospholipase activity in MTB;8,9 although increased phospholipase activity is not primarily linked to inhibition of uptake of K+, it is, nevertheless, compatible with membrane destabilization.9 Given the differences in phospholipid composition between the outer membranes of eukaryotic and prokaryotic cells, the development of novel membrane-destabilizing agents for antimicrobial chemotherapy may be a viable strategy. Possible approaches include the design of membrane-disruptive agents which selectively target prokaryotes, or agents which selectively inhibit the synthesis of fatty acids in bacteria.20,21
The essential requirement for K+ for bacterial metabolism and growth is underscored by the presence of two distinct K+ transporters in MTB. However, validation of these as potential, selective targets for anti-mycobacterial chemotherapy will require construction of a dual Trk/Kdp-knockout mutant of MTB and demonstration of lethality. Such studies are currently in progress. Even if target validation is achieved, successful pharmacotherapy based on the selective inactivation of a single K+ transporter is unlikely because of the existence of compensatory, back-up systems. Nevertheless, selective targeting of the Trk system may represent an adjunctive pharmacological strategy to complement chemotherapy with conventional anti-mycobacterial agents. Notwithstanding the comparatively sluggish uptake of K+ by the Trk system of MTB, which may favour virulence by slowing microbial growth, this contention is based on the possible involvement of the Trk transporter in subverting vacuolar acidification. The presence of NAD+-binding motifs on the CeoB and CeoC proteins,4 as well as the degree of sequence homology with the Trk proteins of E. coli which require proton motive force,22 suggest that the Trk system of MTB may transport K+ and H+ in a symport manner, possibly interfering with vacuolar acidification. This, in turn, may contribute to delayed phagosome maturation, preventing phagosome/lysosome fusion, favouring intracellular survival of MTB.23–25 In contrast, the system that is most likely to operate in the ceoBC-knockout mutant, the Kdp, transports K+ in exchange for the proton.26 Although speculative, utilization of the Kdp system by MTB may favour host-mediated eradication of the microbial pathogen. In this context it is noteworthy that Rengarajan et al.,27 using transposon site hybridization technology have recently reported that ceoB (Rv2691) is required for survival of MTB in macrophages.
In conclusion, although definitive target validation is awaited, clofazimine has proved to be a useful probe in identifying the K+ transporters of MTB as being potential, albeit complex, targets for anti-mycobacterial chemotherapy.
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None to declare.
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Present address. Schering-Plough (Pty) Ltd, Isando, Johannesburg, South Africa 
Present address. Department of Molecular and Cell Biology, University of Cape Town, Rondebosch, South Africa
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Acknowledgements |
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We gratefully acknowledge Dr Stewart Cole for providing the BAC-Rv35 clone, as well as the following members of the MRC/NHLS/WITS Molecular Mycobacteriology Research Unit for their assistance and advice: Drs Stephanie Dawes, Digby Warner, Limenako Matsoso, Bhavna Gordhan, Bavesh Kana and Edith Machowski. This work was supported by grants from the Medical Research Council of South Africa and the Department of Science and Technology of the South African Government.
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