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ABSTRACT |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The increase in multidrug-resistant tuberculosis and high mortality among those co-infected with HIV-1 necessitates new therapeutic approaches directed at Mycobacterium tuberculosis. We hypothesized that a dominant-negative mutation in the DNA-dependent RNA polymerase gene would inhibit transcription of all genes by blocking access of the wild-type enzyme to promoters. An evolutionarily invariant lysine was substituted with arginine by site-directed mutagenesis in the rpoB gene. The dominant-negative rpoB gene product inhibited a transposon-derived kanamycin-resistance gene in both M. smegmatis and M. tuberculosis H37Rv, leading to growth inhibition of the mycobacteria on solid media containing kanamycin. The dominant-negative mutant rpoB gene is a potential suicide gene especially for the treatment of multidrug-resistant tuberculosis once a delivery strategy is also developed.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Tuberculosis has had a resurgence predominantly due to the epidemic of AIDS and the immunosuppressive effects of the HIV-1 virus (1, 2). Complicating these epidemics has been the increase in multidrug-resistant tuberculosis (MDR-TB), with few therapeutic options generally requiring the use of second-line drugs that have greater toxicity and less efficacy. A novel approach to this problem would be to develop a therapy based on a suicide gene.
One of the most potent drugs used in the treatment of tuberculosis is rifampicin, whose molecular basis of action has been well characterized in Escherichia coli (3, 4). Rifampicin binds
to the 
subunit of RNA polymerase and leads to abortive initiation of transcription (5). The DNA-dependent RNA polymerase of E. coli is a multifunctional enzyme consisting of
four different subunits: 
, ', and 
. The core enzyme (2')
can perform RNA polymerization, while site-specific transcription initiation requires the holoenzyme (2'). The genes encoding subunits , , ', and are rpoA, rpoB, rpoC, and rpoD,
respectively. The mechanism of action of rifampicin on RNA
polymerase has also been shown to be similar in Mycobacterium smegmatis (6). Molecular genetic analysis of rifampicin-resistant mutants of E. coli revealed that drug resistance is the
result of mutations in the subunit (7). Analogous to E. coli,
the subunit of Mycobacterium tuberculosis (8) and Mycobacterium leprae (9) has also been shown to be responsible for
rifampicin resistance. Telenti and colleagues (8) mapped 64 of
66 rifampicin-resistant M. tuberculosis clinical isolates to mutations in the rpoB gene corresponding to mutated sites previously identified in E. coli.
Since inhibition of transcription and cell growth is responsible for the potency of rifampicin, we postulated that a mutation engineered in the M. tuberculosis rpoB gene which encodes a
mutant subunit would resemble the behavior of normal polymerase inactivated by rifampicin (10). The sites for mutagenesis
were chosen based on affinity cross-linking mapping of amino acids in the vicinity of the RNA polymerase active center (11).
In E. coli, when the evolutionarily invariant lysine1065 was mutated to arginine, the resultant mutant subunit acted in a
transdominant fashion to block the initiation-to-elongation transition by forming stable promoter complexes (10). This
dominant-negative mutant protein blocked the access of wild-type enzyme to promoters and thus acted as a nonspecific repressor of transcription, resulting in inhibition of cell growth.
We reasoned that since the dominant-negative mutation localized to a region that is highly conserved among living organisms, engineering an analogous mutation in the rpoB gene of
M. tuberculosis may have similar effects and result in growth
inhibition of M. tuberculosis. Inhibition of gene expression and
growth would make this mutant rpoB gene a potential candidate as a suicide gene for M. tuberculosis.
In this report, we demonstrate that the mutant rpoB gene product of M. tuberculosis indeed acts as a repressor of gene expression in both M. smegmatis and M. tuberculosis. By analogy to E. coli, expression of the mutant rpoB gene product is expected to be dominantly lethal to M. tuberculosis. Therefore, the dominant-negative mutant rpoB gene could be delivered as a suicide gene, perhaps as a form of gene therapy, especially for the treatment of MDR-TB.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Strains and Plasmids
Plasmids pLN2 and pLUC10 were obtained from Dr. Thomas M. Shinnick (Centers for Disease Control and Prevention, Atlanta, GA). pLN2 (12) contained the wild-type rpoB gene of M. tuberculosis H37Rv in pMV261 (13), which contained the origin of replication of mycobacteria and E. coli and the Tn903-derived aph gene conferring kanamycin resistance. pLUC10 (14) contained the firefly luciferase gene under the control of the mycobacterial heat shock protein hsp60 promoter in the pMV261 vector. M. smegmatis mc2155 was obtained from Dr. William R. Jacobs (Albert Einstein Medical School, Bronx, NY) (15).
Construction of Mutations in the rpoB Gene of M. tuberculosis
The dominant-negative mutation was engineered into the wild-type rpoB gene derived from pLN2 using the Chameleon Double-stranded mutagenesis kit (Stratagene, La Jolla, CA). Site-directed mutagenesis was performed using the mutagenic primer TCTCCGACGGTGACcgGCTGGCCGGCCGGC, resulting in the substitution of lysine with arginine (underlined codon). The resulting dominant-negative mutant rpoB gene contained in the pLN2dn plasmid was screened by the gain of a new BstEII restriction site (Figure 1A) and confirmed by DNA sequencing. To demonstrate that functional expression of the mutant rpoB gene encoding the arginine substitution was responsible for the inhibitory phenotype, a frame-shift mutation was introduced upstream of the arginine by digesting the four-base protruding 3' termini created by the BstXI restriction enzyme. Hence, the resulting pLN2dn-BstXI plasmid would encode a mutant protein that no longer contained the arginine residue.
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Transformation of M. smegmatis and M. tuberculosis and Selection for Kanamycine Resistance
Plasmid DNA used for transformation was prepared by Wizard Maxipreps (Promega, Madison, WI). Competent bacteria were prepared according to Jacobs and colleagues (16). M. smegmatis strain mc2155 and M. tuberculosis H37Rv were cultivated in 100 ml of Middlebrook 7H9 with 10% ADC enrichment and 0.05% Tween 80 at 37° C with shaking until absorbance at 600 nm reached 0.8 and were then harvested by centrifugation for 15 min at 3,500 rpm, 4° C. Cells were washed twice and resuspended in cold 10% glycerol. The number of competent M. smegmatis for each electroporation was estimated to be 1 × 109 by the formula of Wayne (17). Electroporation was preformed using the Bio-Rad Gene Pulser in a disposable cuvette with a 0.2-cm gap after mixing 200 µl of competent M. smegmatis with 10 µg of total DNA on ice for 5 min. After electroporation, cells were cultured in Middlebrook broth for 2 h before plating in agar containing 50 µg/ml of kanamycin. Kanamycin-resistant transformants were scored as colonies after 3 d of incubation at 37° C.
Southern Analysis
Individual colonies of M. smegmatis were isolated and cultured in 500 ml of Middlebrook 7H9 medium with 10% ADC enrichment and 50 µg/ml of kanamycin for 2 d. Cells were harvested by centrifugation and incubated in GTE (25 mM Tris-HCl [pH 8.0], 10 mM EDTA, 50 mM glucose, and 10 mg/ml lysozyme) overnight at 37° C. Cells were lysed by gentle inversion in 0.2 M NaOH/1% SDS at 4° C for 10 min, and 1.5 volumes of 5 M potassium acetate were added at 4° C for 30 min. After centrifugation at 10,000 rpm at 4° C for 30 min, RNase A (100 µg/ml) was added to the supernatant and incubated at 37° C for 30 min. The supernatant was extracted with phenol:chloroform:isoamyl alcohol (25: 24:1) and precipitated with 2.5 volumes of ethanol. DNA was either analyzed directly by Southern analysis or transformed and propagated in E. coli XL-1-Blue by culturing overnight in LB containing 50 µg/ml of kanamycin. Kanamycin-resistant cells were grown in large scale (500 ml) from which DNA was extracted using the Wizard Maxipreps.
DNA (5 to 10 µg/ml) was digested with the appropriate restriction
enzymes overnight and separated on a 0.8% agarose gel and transferred to Hybond-N nylon membrane (Amersham) in 20× SSC after denaturation in 0.4 N NaOH/1.5 NaCl. The DNA was fixed on the filter by ultraviolet cross-linking. The filter was prehybridized and hybridized in Church buffer (7% SDS, 1% bovine serum, albumin, 1 mM EDTA, [pH 8.0], 0.25 M Na2HPO4 [pH 7.2]) containing DNA
probe labeled by random priming at 64° C overnight. Filters were
washed with 2× SSC/0.1% SDS at room temperature for 10 min, 1×
SSC/0.1% SDS at 65° C for 10 min, and 0.1× SSC/0.1% SDS at 65° C
for 10 min. Filters were exposed to X-OMAT AR film (Kodak) at
80° C for several hours.
Luciferase Assays
Lysates of M. smegmatis electroporated with the pLUC10 plasmid were tested for luciferase activity according to the method of Cooksey and colleagues (12). After electroporation, cells were incubated at 37° C with shaking for 3 d in 10 ml of complete Middlebrook 7H9 broth supplemented with 0.05% Tween 80 and 50 µg/ml of kanamycin. Cells were harvested by centrifugation at 3,500 rpm, 10 min, 4° C, and washed twice in cold phosphate-buffered saline, pH 7.2. The pellets were resuspended in 500 ml of a detergent lysis buffer (1% Triton X-100, 25 mM Tris [pH 7.8 with H3PO4], 10 mM trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid [CDTA], 10 mM dithiothreitol, 50% glycerol) for 5 min; cell suspensions were sonicated in ethanol-soaked ice for 5 min with an Ultrasonic Homogenizer 4710 series (Cole Parmer, Chicago, IL) equipped with a 2-mm microtip and then centrifuged for 10 min at 12,000 rpm, 4° C. Cell supernatants were collected and concentration of proteins were measured by Bio-Rad Protein Assay. Cell extracts were tested for luciferase activity by adding 50 µl of the cell extract into 300 µl of luciferase reaction buffer (15 mM MgSO4, 0.1 mg/ml bovine serum albumin, 1 mM dithiothreitol, 25 mM glycylglycine [pH 7.8], 1 mM ATP [pH 7.0], pH 7.3 to 7.8) using a Monolight 2010 luminometer (Lumat LB-9501; Berthold). For each sample, 100 µl of 1 mM D-luciferin (Analytical Luminescence Laboratory, San Diego, CA) was injected into the tube by the automatic injector with a measuring time of 10 s. The mean value of 15 cycles in relative light units (RLU) was recorded.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Mutagenesis of M. tuberculosis rpoB Gene
There was significant homology in the amino acid sequence of
the rpoB subunit product of M. tuberculosis H37Rv when
compared with that of E. coli in the vicinity of the dominant-negative mutation (lysine [K] 1065
arginine [R]) (14) (Figure
1A). The corresponding lysine residue in the rpoB gene of
H37Rv in the plasmid pLN2 was therefore substituted with
arginine by site-directed mutagenesis (described in METHODS).
The resulting of pLN2dn plasmid containing the dominant-negative (dn) mutant rpoB gene with the lysine-to-arginine
substitution was screened by the gain of a new BstEII restriction site, and the mutation was confirmed by DNA sequencing
(data not shown). To demonstrate that the inhibitory effect of
the dominant-negative mutant rpoB gene product is due to
its functional expression, a frame-shift mutation was constructed upstream of the substituted arginine by deletion of
four base pairs at the BstXI restriction site. The resulting
pLN2dn-BstXI plasmid contained the frame-shifted, dominant-negative rpoB gene which would no longer produce a
protein containing the arginine residue that conferred the inhibitory phenotype. A schematic representation of these plasmids is shown in Figure 1B.
Inhibition of Kanamycin-resistant Colony Formation by the Dominant-negative Mutant rpoB Gene Product
To investigate the effects of expression of the dominant-negative mutant rpoB gene produce on gene expression, pLN2 and pLN2dn plasmids were introduced by the electroporation into mycobacteria and selected for expression of the Tn903-derived aph gene conferring resistance to kanamycin. Electroporation of the pLN2 plasmid which contained the wild-type rpoB gene of M. tuberculosis H37Rv into M. smegmatis (Figure 2A) resulted in growth of thousands of kanamycin-resistant colonies. In striking contrast, electroporation of the pLN2dn plasmid which contained the dominant-negative mutant rpoB gene into M. smegmatis resulted in only two kanamycin-resistant colonies. However, when cultured in liquid medium containing kanamycin, these two colonies failed to grow and hence were not kanamycin resistant. Similar results were obtained when the pLN2 and pLN2dn plasmids were electroporated into M. tuberculosis H37Rv (Figure 2B).
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To further demonstrate that expression of the dominant-negative rpoB gene product resulted in the inhibition of expression of the kanamycin-resistant gene, the pLN2dn-BstXi plasmid containing a frame-shift mutation upstream of the dominant-negative mutation was tested. In contrast to electroporation of the pLN2dn plasmid which yielded no kanamycin-resistant colonies, electroporation of the pLN2dn-BstXI plasmid into M. smegmatis yielded many kanamycin-resistant colonies (Figure 2C). Electroporation of the pMV261 empty vector control or the pLN2 plasmid also yielded many kanamycin- resistant colonies. Thus, the functional expression of the dominant-negative rpoB gene with the lysine-to-arginine substitution but not the frame-shifted, dominant-negative rpoB gene inhibited expression of the kanamycin-resistance gene in mycobacteria.
To prove that the kanamycin-resistant colonies selected contained the corresponding plasmid, DNA was isolated from individual kanamycin-resistant colonies and Southern analysis was performed. Southern analysis of DNA from electroporation of the pLN2dn plasmid could not be performed because no kanamycin-resistant colonies were obtained (Figure 2C). Hence, DNAs from two kanamycin-resistant colonies selected after electroporation of M. smegmatis with pMV261, pLN2, and pLN2dn-BstXi were analyzed. The DNAs of all the kanamycin-resistant colonies hybridized with the aph probe containing the kanamycin-resistance gene (Figure 3), demonstrating the presence of the plasmid in these colonies.
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To ascertain that functional expression of the dominant-negative rpoB gene also inhibited expression of the kanamycin-resistant gene in M. tuberculosis, M. tuberculosis H37Rv was electroporated with the plasmic pLN2, pLN2dn, or pLN2dn-BstXI, expressing the wild-type, dominant-negative mutant or frame-shifted, dominant-negative mutant rpoB gene product, respectively. Expression of the wild-type or frame-shifted, dominant-negative rpoB gene product in H37Rv yielded similar number of kanamycin-resistant colonies, whereas expression of the dominant-negative ropB gene product yielded no colonies (Table 1). Hence, the dominant-negative rpoB gene product inhibited the growth of kanamycin-resistant colonies in both M. smegmatis and M. tuberculosis H37Rv.
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Inhibition of Luciferase Gene Expression by the Dominant-negative Mutant rpoB Gene Product
The effects of expression of the dominant-negative rpoB gene product on the expression of a reporter gene, the firefly luciferase gene, under the control of the mycobacterial heat shock promoter was investigated. High levels of luciferase activity were obtained when the pLUC10 plasmid was electroporated with the vector control pMV261 plasmid or pLN2 plasmid containing the wild-type rpoB gene (Figure 4). In contrast, when the pLN2dn plasmid encoding the dominant-negative rpoB mutant protein was electroporated with pLUC10, a dramatic reduction in luciferase activity was observed. There was no inhibitory effect when the frame-shift mutant pLN2dn-BstXI plasmid was electroporated with pLUC10, as demonstrated by the high luciferase activity comparable to that electroporated with vector control.
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Southern Analysis of Kanamycin-resistant Colonies
To demonstrate the uptake and replication of both plasmids in the luciferase assay, kanamycin-resistant colonies were isolated after electorporation of M. smegmatis with the pLUC10 plasmid alone or with the pMV261 vector, pLN2 plasmid, or pLN2dn-BstXI plasmid. DNAs were isolated and Southern analysis was performed and probed with the luciferase gene or rpoB gene. As shown in Figure 5A, Southern analysis using the luciferase probe demonstrated hybridization to DNAs from all kanamycin-resistant colonies resulting from electroporation of the pLUC10 plasmid. When probed with the rpoB gene, hybridization was observed only from DNAs resulting from electroporation with the pLN2 or pLN2dn-BstXI plasmid (Figure 5B). Because DNAs isolated from kanamycin-resistant colonies selected after electroporation of pLUC10 plasmid with the pLN2 or pLN2dn-BstXI plasmid hybridized to both the luciferase and rpoB probes, we have demonstrated that both plasmids are present in a single colony. Kanamycin-resistant colonies resulting from electroporation of the pMV261 vector alone or together with the pLUC10 plasmic or the pLN2 plasmid alone also contained the corresponding plasmid when probed with the aph gene (data not shown) or the rpoB gene (Figure 5B).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Based on the mutation engineered in the rpoB gene of E. coli, we introduced an analogous mutation in the rpoB gene of M. tuberculosis H37Rv and showed that expression of the latter in mycobacteria also resulted in the inhibition of gene expression. By analogy to E. coli, expression of the dominant-negative mutant rpoB gene of M. tuberculosis was dominantly lethal and therefore could be used as a suicide gene in the form of gene therapy for the treatment of tuberculosis.
Substitution of an invariant amino acid (lysine1065 arginine) in the rpoB gene product of E. coli resulted in the production of a mutant subunit of RNA polymerase that inhibited transcription by assembling into the holoenzyme and
forming stable promoter complexes that only synthesized promoter-specific dinucleotides without chain elongation (10). By
forming stable complexes, the mutant protein acted in a transdominant fashion to block access of the wild-type enzyme
complex to promoters, inhibiting global gene expression and
leading to cell growth inhibition. Because of the latter, expression of the dominant-negative mutant rpoB gene was dominantly lethal. Hence, this mutant gene is a suicide gene.
Because the pLN2dn plasmid encoding the dominant-negative mutant protein can be propagated in E. coli as kanamycin-resistant cells but cannot be propagated in M. tuberculosis H37Rv and M. smegmatis, the dominant-negative rpoB gene product of M. tuberculosis specifically represses gene expression only in mycobacteria. The inability to form kanamycin-resistant colonies after electroporation with the pLN2dn plasmid was due to the functional expression of the dominant-negative rpoB mutant protein because introduction of a frame-shift mutation upstream of the dominant-negative mutation in the pLN2dn-BstXI abrogated the inhibitory effect of pLN2dn. Southern analysis of DNA isolated from kanamycin-resistant colonies selected after electroporation with the pMV261 vector containing the rpoB gene (pLN2 and pLN2dn-BstXI) and the luciferase gene (pLUC10) demonstrated the presence of the corresponding plasmids in M. smegmatis. Hence, both plasmids can be propagated and co-selected in the cells of the kanamycin-resistant colonies.
Since the mutant polymerase inhibits in a dominant fashion not only the transcription machinery of M. tuberculosis but
also of M. smegmatis, the mutant subunit of M. tuberculosis
must participate in the RNA polymerase enzyme complex of
both mycobacterial species. As a potent inhibitor of gene expression and growth of mycobacteria, the dominant-negative
rpoB gene can potentially be used as a suicide gene for the killing of not only M. tuberculosis but also other clinically significant mycobacterial species such as M. leprae and M. avium.
Recombinant mycobacteriophages provide efficient delivery systems for introducing genes into a wide range of mycobacterial species (18). Temperate shuttle plasmids which were vectors constructed by inserting E. coli cosmids into nonessential regions of the mycobacteriophage TM4 genome have been used to introduce and express the kanamycin-resistant gene in M. smegmatis (19) and the firefly luciferase gene in M. tuberculosis for rapid assessment of drug susceptibilities (20). Importantly, efficient delivery of suicide genes to mycobacteria will require the development of vectors that can deliver genes to mycobacteria, e.g., through a bronchoscopic or aerosol delivery. In the current epidemic of MDR-TB that defies conventional chemotherapeutic treatments, gene therapy offers an alternative approach for the treatment of tuberculosis.
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Footnotes |
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Supported by Grants MO1 RR00096, HL51494, and CDC CCU210075, and by the Stony Wold-Herbert Foundation.
Correspondence and requests for reprints should be addressed to Dr. Kam-Meng Tchou-Wong, Division of Pulmonary and Critical Care Medicine, New York University Medical Center, 550 First Ave., MSB 147, New York, NY 10016.
(Received in original form November 21, 1996 and in revised form June 12, 1997).
Acknowledgments: The writers thank Natalie Little for editorial assistance. They would like to thank Dr. T. M. Shinnick for the gift of pLN2 and pLUC10 and Dr. W. R. Jacobs for M. smegmatis mc2155.
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References |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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1.
Bloom, B. R., and
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Snapper, S. B.,
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