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Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, September 2005, p. 3707-3714, Vol. 49, No. 9
0066-4804/05/$08.00+0 doi:10.1128/AAC.49.9.3707-3714.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Department of Biomedical Sciences, College of Veterinary Medicine, Department of Microbiology, College of Science, Oregon State University, Corvallis, Oregon 97331,1 Kuzell Institute for Arthritis and Infectious Diseases, California Pacific Medical Center Research Institute, San Francisco, California 941152
Received 29 December 2004/ Returned for modification 3 March 2005/ Accepted 18 June 2005
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A number of studies with other bacteria have employed DNA microarrays to narrow down the possible target(s) for the drug. Therefore, to examine the effect of mefloquine on the level of gene expression in M. tuberculosis and M. avium, we applied two different techniques, i.e., the commercially available M. tuberculosis DNA microarray and, because the M. avium array has not yet been annotated, a green fluorescent protein (GFP) promoter library of M. avium genes. Some of the genes identified using both approaches for the two mycobacterial species were then overexpressed in M. avium, identifying mechanisms of resistance and a putative mefloquine target.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(Stratagene) was grown on Luria-Bertani broth or agar
(Difco Laboratories), and transformants were selected on kanamycin at a
concentration of 50 µg/ml. DNA techniques. M. avium strain 104 was used as a source of genomic DNA for library constructions and for specific gene amplification by PCR. Chromosomal DNA was extracted and purified, as previously described (14). Plasmid DNA was isolated from E. coli using the QIAprep Miniprep kit (QIAGEN, Valencia, CA). M. smegmatis was resuspended in 50 mM glucose, 25 mM Tris (pH 8), 10 mM EDTA (GTE) solution with 10 µg/ml lysozyme and lysed overnight at 37°C in the shaking incubator (250 rpm). The next day, plasmid DNAs were isolated with the QIAprep Miniprep kit, according to the manufacturer's instructions.
GFP promoter library construction.
In order to
identify M. avium gene promoters expressed upon subinhibitory
concentrations of mefloquine, the green fluorescent protein reporter
system was used. The E. coli-Mycobacterium shuttle vector
pEMC1 was constructed from the reporter plasmid pMV261 by removing the
hps60 promoter and introducing the promoterless
GFPmut2 gene into XbaI and PstI restriction sites. The
GFPmut2 gene was obtained from Rafael Valdivia and Stanley
Falkow, Stanford University
(5). The trpA
transcriptional terminator was replaced upstream of the GFP gene,
between the EcoRI and HindIII sites, to ensure that GFP expression was
the result of the activation of bacterial promoters. Genomic DNA was
isolated from M. avium 104 and partially digested with Sau3A
enzyme. The digests were gel purified to generate DNA fragments with
300- to 1,000-bp fragments. The inserts were then ligated into the
dephosphorylated BamHI site of plasmid pEMC1 (Table
1). E. coli DH5-competent cells were transformed with the
genomic library and plated on Luria-Bertani agar containing kanamycin,
50 µg/ml. Plasmid DNA was recovered from a pool of greater than
2 x 104 E. coli clones. Screening of 1% of
E. coli transformants, from each of three separate ligations,
by restriction analysis of plasmids, showed that approximately 80% of
transformants had inserted DNA. Electrocompetent M. smegmatis
(mc2155) was electroporated with an M. avium
promoter GFP library. For preparation of M. smegmatis
competent cells, the bacterial pellet was incubated in an ice water
bath for 2 h and washed four times with cold washing buffer
(10% glycerol, 0.1% Tween 80), and then 100 to 200 µl of
competent cells was combined with 1 to 3 µl of library and
transferred to a prechilled 0.2-cm cuvette. Electroporation was carried
out under the following conditions: capacitance, 25 µF;
resistance, 1,000 
; voltage, 2.5 kV. Clones were recovered on
Middlebrook 7H11 agar plates containing kanamycin, 50 µg/ml.
Over 10,000 individual M. smegmatis clones were stored in
pools of five in 96-well plates containing Middlebrook 7H9 broth with
50% glycerol and stored at
–70°C.
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Construction and overexpression of M. avium subclones under G13 promoter. The E. coli-Mycobacterium shuttle vector pLDG13 was constructed with a Mycobacterium marinum G13 promoter (1) inserted into the pFJS3 (Table 1) NheI restriction site. G13-upper (NheI) (5'-TTTGCTAGCGATCGCCACTAGCGCCGCGGT-3') and G13-lower (NheI) (5'-TTTGCTAGCTCGGTTACCAAGCGTGCATTT-3') primers were designed to amplify a 457-bp G13 fragment. Amplification was performed in a PCR Express thermal cycler (Hybaid) under the following conditions: heating at 95°C for 5 min and subjection to 30 cycles of denaturation at 95°C for 30 s, annealing at 65°C for 30 s, and extension at 72°C for 1 min. The PCR product was ligated directly into the pFJS3 plasmid and screened by digestion for the insert; samples were sequenced to determine the correct orientation of the promoter. Mycobacterium avium 104 genomic DNA was subjected to partial digestion with Sau3A restriction endonuclease. After electrophoresis on a 1% agarose gel, fragments with an 800- to 3,000-bp size were extracted from the gel and cloned into the BclI site in the pLDG13 plasmid (Table 1). The library was constructed with 70% efficiency, with the number of the clones representing twofold coverage of the M. avium genome. The resulting library was expanded in E. coli competent cells by electroporation and plated on Luria-Bertani agar containing kanamycin, 50 µg/ml. The pool of approximately 20,000 E. coli clones was used as the source of G13 library construction in M. smegmatis. Mycobacterial competent cells were prepared, as previously described (14), and electroporated as described above. Transformants were plated on 7H11 agar plates containing kanamycin (50 µg/ml) and mefloquine (16 µg/ml) (2x MIC). Over 8,000 individual M. smegmatis clones were stored in pools of 100 and used for screening for mefloquine resistance. M. smegmatis mutants with increased MICs were selected and further analyzed by susceptibility testing. Agar dilution testing was performed according to the proportion method as previously described (National Committee for Clinical Laboratory Standards, 1994). Twofold dilutions, ranging from 16 to 128 µg/ml of mefloquine, were used for identification of M. smegmatis clones resistant to mefloquine.
Transposon mutagenesis. Temperature-sensitive plasmid pTNGJC (Table 1) was created based on the pUC19 vector, where a temperature-sensitive mycobacterial origin of replication and a Tn5367-based transposon were cloned. The transposon plasmid was transformed into M. smegmatis mc2155, M. avium 104, and M. tuberculosis H37Rv. The presence of the transposon was confirmed by PCR amplification for the kanamycin gene. Single colonies from each mycobacterial transformant were grown at 30°C in the presence of kanamycin. The temperature was then switched to 42°C for 3 to 5 days. Screening of 25 colonies from each mycobacterial species showed that 100% of resulting clones contained the transposon in the chromosome. Library pools from each mycobacterial strain were plated on selective medium containing kanamycin at 50 µg/ml (M. smegmatis), kanamycin at 200 µg/ml (M. tuberculosis and M. avium), and mefloquine at 32 µg/ml for selection of mefloquine-resistant mutants.
Sequence analysis. Samples were sequenced at the Central Service Laboratory, Center for Gene Research and Biotechnology, Oregon State University, Corvallis. Database searches and alignments were performed using the BLAST server from the National Center for Biotechnology Information. The M. avium DNA sequence data from the NCBI database and the database at the TIGR Institute (www.tigr.org) were used to confirm and complete the sequences.
RNA preparation and probe synthesis. Total RNA was isolated from M. tuberculosis H37Rv in the following manner. Bacterial culture was grown in 7H9 broth at exponential phase and then divided into three parts. Mefloquine was added at a subinhibitory concentration (4 µg/ml) and 4x MIC (32 µg/ml) to two of the cultures, and a control culture of M. tuberculosis did not receive antibiotic. The next day, bacterial culture was resuspended in a guanidine thiocyanate-based buffer (Trizol) (GIBCO) and lysed with rapid mechanical agitation in a bead beater. Cells were centrifuged at 13,000 rpm for 5 min at 4°C. RNA was isolated from the supernatant, as previously described (11). RNA samples were treated with DNase I (RNase-free; Clontech) on RNeasy Midi columns for 15 min, according to the manufacturer's instructions (QIAGEN, Valencia, CA). The RNA concentration and quality were determined spectrophotometrically by absorption at an optical density at 260 to 280 nm, as well as verified on a 1% denaturing agarose gel.
cDNA probe synthesis and array hybridization were performed
using the Sigma-Genosys Panorama cDNA labeling and hybridization kit
according to the manufacturer's protocol. cDNA was synthesized using 1
µg total RNA from M. tuberculosis and 4 µl of
Sigma-Genosys labeling primers. The cDNA labeling primers were annealed
to the RNA template by placing samples in a thermal cycler (Hybaid) at
90°C for 2 min and then cooling to 42°C for an
additional 20 min. The following master mix was made for cDNA labeling:
50 U avian myeloblastosis virus reverse transcriptase (Perkin-Elmer),
1x reverse transcriptase buffer, 333 µM dATP, 333
µM dGTP, 333 µM dTTP, 20 Ci
[-33P]dCTP (2,000 to 3,000 Ci/mmol; Amersham
Pharmacia Biotech), and RNase-free water. Labeling mix was added to
each reaction mixture and incubated at 42°C for 2 h.
Unincorporated-radiolabeled nucleotides were removed from labeled cDNA
probe by purification over Spandex G-25 gel filtration columns
according to the manufacturer's instructions
(Sigma).
Hybridization and data analysis. Prior to use of the probe solutions, Panorama M. tuberculosis Gene Arrays were rinsed in 50 ml 2x SSPE (1x SSPE is 0.18 M NaCl, 10 mM NaH2PO4, and 1 mM EDTA [pH 7.7]) for 5 min and drained. The blots were prehybridized in hybridization solution containing heat-denatured salmon testes DNA at a final concentration of 100 µg/ml. Labeled cDNA was heated at 95°C for 10 min to denature the cDNA, added to the hybridization solution, and hybridized to the arrays at 65°C overnight. The membranes were washed three times with microbial array wash solution (Sigma) containing 0.5x SSPE and 0.2% sodium dodecyl sulfate solution at 65°C for 3 min and agitated. The membranes were wrapped in plastic wrap, exposed to a phosphoimaging screen for 24 h, and then scanned with a phosphoimager (Molecular Dynamics, Amersham Pharmacia Biotech). The data were analyzed by Sigma-Genosys staff using ArrayVision software developed by Imaging Research, Inc. (St. Catharines, Ontario, Canada). Raw fluorescence intensity values were corrected for background by using the Array Vision Software and subsequently normalized by using the Lowess algorithm. The data were filtered for low spot signal intensity by using the threshold of the mean plus 1 standard deviation of the intensity observed from all empty and control spots on the array. Results were analyzed using the Genespring version 6 software, based on the normalized signal intensities. The fold changes were calculated by division of test (mefloquine treatment) spot signal data by the control data. Microarray experiments were repeated two times, and averages of the fold changes are reported in Table 3.
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RESULTS |
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Identification of M. avium-expressed promoters after mefloquine treatment using the GFP promoter library. To determine the bacterial genes expressed upon exposure to mefloquine, M. smegmatis containing an M. avium GFP promoter library was incubated with subinhibitory concentrations of mefloquine, and the increase of GFP expression was monitored. Screening of 2,200 clones of the M. avium promoter library in M. smegmatis for significantly increased GFP expression identified 26 clones. Promoters were activated at the 24-h time point. No significant changes in GFP expression were observed at 4 h after M. avium treatment with mefloquine, compared with the GFP expression at baseline. The promoters upregulated are displayed in Table 2. Twenty-four sequenced clones contained M. avium DNA sequences that agree with a definition of a promoter of the predicted open reading frames and were considered natural promoters. The sequences of two clones were apparently located within the gene, indicating the potential for cryptic promoters. These sequences were not further analyzed. Among the identified M. avium sequences are promoters for genes associated with lipid metabolism (accD3, fadD19, and fadA2), intermediary metabolism (guaB2), information pathways (rpsT, serS, and infB), regulatory proteins (phoR), cellular differentiation (Rv3661), and 12 hypothetical integral membrane proteins or transporters from functional classification of cell wall and cell processes.
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A total of 133 genes were identified as responsive after treatment with subinhibitory concentrations of mefloquine, including the majority of genes from functional classification of cell wall and cell processes, intermediary respiration and metabolism pathways, PE/PPE family proteins, and proteins with unknown function. One hundred eight genes showed more than a twofold increase in expression upon mefloquine treatment, while expression of 25 genes was downregulated. The microarray data obtained from M. tuberculosis treatment with 4x MIC of mefloquine revealed changes in many genes compared with untreated control. Exposure to high concentrations of mefloquine resulted in significant stress response and expression of genes encoding heat shock proteins. Therefore, comparing the experiments examining subinhibitory and high concentrations of the antimicrobial revealed that much of the response upon exposure to high concentrations of mefloquine was nonspecific. Genes identified by M. tuberculosis DNA array in response to different concentrations of mefloquine after 24 h of treatment are shown in Table 3.
Overexpression of M. avium proteins using G13 promoter library. Based on the suggestion that the target gene(s) could be essential, we then decided to overexpress the mycobacterial proteins under strong sigma-70 (G13) promoter and select for the mefloquine resistance phenotype. To test if overexpression of genomic library caused increased resistance to mefloquine, pools of 100 colonies were plated on 7H11 plates containing kanamycin (50 µg/ml) and mefloquine (16 µg/ml). Selected colonies were sequenced. Sixteen isolated clones contained M. avium genomic fragments with complete open reading frames. To determine if the overexpression of these gene products resulted in increased resistance to mefloquine, the clones were assayed for the MIC of mefloquine (3). Genes identified using M. smegmatis expressing the M. avium G13 library are shown in Table 4. Although the MIC for wild-type M. smegmatis is 8 µg/ml for all overexpressed isolates studied, decreased susceptibilities to mefloquine were detected with increases in the MICs from two- to eightfold dilutions. Susceptibility of M. smegmatis clones containing overexpressed M. avium genes is summarized in Table 4.
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DISCUSSION |
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Identification of the target for mefloquine might facilitate identification of other compounds that would have a novel mechanism of action against mycobacteria. The target for mefloquine in Plasmodium is still unknown, although export pumps have been identified as mechanisms of resistance (10). M. avium and M. tuberculosis are quite different pathogens. However, the MIC of mefloquine for both bacteria is the same, which may imply that the targets and mechanisms of resistance are probably similar. Previous studies have already determined that M. avium strains resistant to quinolones, macrolides, rifamycins, and ethambutol were still fully susceptible to mefloquine (3). Our initial attempt to clone the resistant phenotype by identifying a resistant strain was unsuccessful. The fact that we could not isolate a resistant clone using in vitro and in vivo selection suggested that either the mutation of the target was lethal or mefloquine had more than a single target.
Using a couple of strategies, GFP expression and DNA microarray, we determined that, upon exposure to subinhibitory concentrations of mefloquine, both M. avium and M. tuberculosis upregulate a number of genes, the majority of which belong to metabolic pathways and cell wall synthesis. Some of the upregulated genes were membrane proteins of unknown function. It is assumed that the reason that genes are upregulated upon bacterial exposure to antimicrobials is that the target gets inhibited, and upstream genes in the pathway undergo compensatory regulation. Accordingly, the target should not be upregulated but inhibited. If this hypothesis is correct, it is possible that we cannot identify the target of a number of compounds by using the DNA microarray technique as the only tool. Another interesting observation of this study was that both the DNA microarray and the screening of a GFP library identified similar genes, although the results with the GFP library were more limited. The clones of several of the M. avium genes identified using the GFP library, under the control of the constitutive promoter, resulted in a twofold-increased MIC (data not shown), which indicates that they are unlikely to be the drug target.
Recently, Wilson and colleagues (15) performed a DNA microarray with RNA from M. tuberculosis exposed to isoniazid. Their findings showed that exposure to isoniazid induced several genes that encode proteins physiologically relevant to the drug's mode of action, including several genes encoding type II fatty acid synthase enzymes and tbpC (trehalose dimycolyl transferase).
Our results of DNA microarray demonstrated that, when M. tuberculosis is exposed to subinhibitory concentrations of mefloquine, a few genes involved in fatty acid metabolism and many in the intermediary metabolism were upregulated. The response to the exposure to 4x MIC resembles a stress response. One of the responses of the bacterium was to upregulate the expression of genes coding for membrane proteins, many of them involved in transport mechanisms. These results can suggest that either the target for mefloquine is on the cell wall or membrane or many of these proteins are associated with the export of the compound.
The screening for resistant clones using the overexpression library was the employed strategy with more relevant results. All the genes identified were pumps or transport proteins except for the Rv3661 gene, a gene involved in cell replication in Streptomyces. The finding that the overexpression of several pumps or transport-related genes can be associated with increased MIC is obvious. However, the fact that many were identified suggests that it is a nonspecific mechanism. In addition, the overexpression of the majority of the transport proteins was associated with a twofold increase in MIC, which cannot be considered significant.
The upregulation of arsA and arsC and Rv1819c, however, was associated with a fourfold increase in MIC, which is relevant. That all three proteins have a role in mefloquine export is a plausible possibility. The reason that they were not overexpressed in the DNA array and GFP library screening is unknown, but there is a similar study for Candida albicans in which export pump genes were not identified in DNA array upon exposure to itraconazole, although they represent the main mechanism of resistance (6).
Rv3661 was the protein that, when overexpressed, resulted in the greater increase in the MIC. Rv3661 was also expressed (5.8-fold) in the GFP promoter screening, suggesting that targets might undergo compensatory overexpression when the pathogen is exposed to subinhibitory concentrations of antimicrobials.
In Streptomyces azureus, the gene is associated with mycelium formation, and clones that do not contain a functional gene are aerial mycelium negative. There is some suggestion that the Rv3661 gene encoding a hypothetical protein has homology to the MinD protein gene of Bacillus subtilis, the gene for a septum site-determining protein (13). How the inhibition of a gene associated with cell differentiation can be lethal is unknown. The fact that the function of the protein encoded by Rv3661 is not known in mycobacteria, except for some suggestive similarity, might be the reason. The identification of a novel drug target in mycobacteria may have an important impact on the treatment of mycobacterial infections.
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ACKNOWLEDGMENTS |
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This research was supported by a grant from the National Institute of Allergy and Infectious Diseases, R03AI48363.
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FOOTNOTES |
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REFERENCES |
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