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WO1994026765A1 - Utilisation des genes de m. tuberculosis, m. bovis et m. smegmatis conferant une resistance a l'isoniazide - Google Patents

Utilisation des genes de m. tuberculosis, m. bovis et m. smegmatis conferant une resistance a l'isoniazide Download PDF

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Publication number
WO1994026765A1
WO1994026765A1 PCT/US1994/005398 US9405398W WO9426765A1 WO 1994026765 A1 WO1994026765 A1 WO 1994026765A1 US 9405398 W US9405398 W US 9405398W WO 9426765 A1 WO9426765 A1 WO 9426765A1
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tuberculosis
nucleic acid
ala
isoniazid
enzyme
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PCT/US1994/005398
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English (en)
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William R. Jacobs
Asesh Banerjee
Desmond Collins
Theresa Mary Wilson
Geoffrey William De Lisle
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Albert Einstein College Of Medicine Of Yeshiva University, A Division Of Yeshiva University
Agresearch
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Priority to AU69496/94A priority Critical patent/AU6949694A/en
Publication of WO1994026765A1 publication Critical patent/WO1994026765A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)

Definitions

  • polynucleotide includes genes, expressed mRNA, proteins, promoters, enhancers and enzymes.
  • This invention relates to the identification, cloning sequencing and characterization of M. tuberculosis. M. bovis and M. sme ⁇ matis polynucleotides which determine whether or not tuberculosis mycobacteria (i.e. M. tuberculosis. M. bovis or M. africanum) will be resistant to the anti-tuberculosis drugs isoniazid and ethianomide.
  • polynucleotides which include genes, and the enzymes they encode may be used to produce probes capable of identifying the nucleic acids in mycobacteria which encode isoniazid resistance, which probes can be used in the treatment and prevention of tuberculosis, to assess the susceptibility of various mycobacterial strains to isoniazid, and to determine whether various antibiotic drugs are effective against the M. tuberculosis complex.
  • This invention further relates to diagnostic kits containing said probes.
  • bovis pS5 mRNA can be produced, which compounds may be used to eliminate isoniazid resistant or isoniazid sensitive mycobacteria. Further, the polynucleotides of this invention may be used to generate vaccines for tuberculosis or novel recombinant BCG vaccines.
  • Tuberculosis (which includes infection caused by M. tuberculosis, M. bovis and . africanum) remains the largest cause of human death in the world from a single infectious disease, and is responsible for one in four avoidable adult deaths in developing countries. In addition, in 1990, there was a 10% increase in the incidence of tuberculosis in the United States. Further, . bovis causes tuberculosis in a wide range of animals, and is a major cause of animal suffering and economic loss in animal industries. isoniazid-resistant isolates appear to be catalase negative. Previous studies have shown that low-level isoniazid resistance correlates better with the co-acquisition of ethionamide resistance than with loss of catalase activity.
  • Drug resistance can often be mediated by an accumulation of mutations in the polynucleotide (gene) which encodes the target. These mutations result in amino acid substitutions which result in reduced binding of drugs to their target enzymes.
  • rifampicin resistance is often mediated by mutations in the gene encoding the ⁇ ' subunit of RNA polymerase, or trimethoprim resistance can be mediated by mutations in dihydrofolate reductase.
  • trimethoprim resistance can be mediated by mutations in dihydrofolate reductase.
  • isoniazid blocks mycolic acid biosynthesis in the M. tuberculosis complex.
  • the inhibition of mycolic acid biosynthesis in cell-free extracts of mycobacteria has confirmed that isoniazid specifically inhibits mycolic acid biosynthesis.
  • the target enzymes had not yet been identified prior hereto.
  • tuberculosis cells but appears n °t to be the target of action of isoniazid.
  • isoniazid-resistance can be accounted for by the loss of catalase activity, in some studies only 10% of the enzymes themselves, could then be used to eliminate isoniazid resistance of various strains of the M. tuberculosis complex.
  • inhA which encodes the enzyme (InhA) comprising the target of action of M. tuberculosis, M. avium and M. smegmatis for isoniazid
  • pS5 polynucleotide
  • the DNA sequences of these polynucleotides can be used to prepare anti-DNA or anti-mRNA oligonucleotides which are administered and which inhibit the mRNA activity of the polynucleotides, thereby preventing expression of the enzymes and eliminating isoniazid resistance so as to treat and prevent tuberculosis infection.
  • These DNA sequences can be used to produce oligonucleotide probes capable of identifying the nucleic acids in tuberculosis mycobacteria which encode isoniazid resistance, which probes may be used in diagnostic kits.
  • the polynucleotides of this invention can be used to assess the susceptibility of various strains of the M. tuberculosis complex in clinical samples to the antibiotic isoniazid.
  • the chromosomal DNA of the inhA polynucleotide (gene) of M. tuberculosis or the DNA of the pS5 polynucleotide (gene) of M. bovis from a clinical sample is isolated.
  • Oligonucleotides are then prepared using the DNA sequences.
  • the region of the gene from the clinical sample which is identified by the oligonucleotides is amplified, for example using PCR, and then single strand conformation polymorphism analysis is performed to determine whether a mutation gene exists in the tuberculosis from the clinical sample, the presence of a mutation indicating that the tuberculosis in the clinical sample is resistant to isoniazid.
  • the polynucleotides of this invention can also be used to determine whether various antibiotic drugs are effective against tuberculosis.
  • the polynucleotides which encode the enzymes are overexpressed so as to obtain said enzymes, which enzymes are then combined with a purified reagent to obtain a mycolic acid.
  • the mycolic acid is assayed to measure its level of biosynthesis, and then a drug is combined with the mycolic acid synthesis system to determine whether the drug blocks the biosynthetic activity of the system. If the biosynthesis of mycolic acid is blocked, then the drug is effective against tuberculosis.
  • Figure 1 represents minimal inhibitory concentrations of K. smeqmatis transformed with various cosmid clones from M. tuberculosis, M. smeqmatis, M. bovis and M. avium;
  • Figure 2 represents the DNA sequences of isoniazid resistant polynucleotides from M. tuberculosis, M. bovis and M. smeqmatis.
  • M. smeqmatis sequence the single point mutation that is different (in comparison to the isoniazid sensitive strain) is the key feature which determines that this polynucleotide is isoniazid resistant
  • Figure 3 represents the subcloning strategy used to demonstrate that the isoniazid resistance phenotype is conferred by the inhA open reading frame;
  • Figure 4 represents an analysis comparing the inhA open reading frame to the envM open reading frames of E. Coli and S. typhimurium. The close homology therebetween suggests that inhA is involved in lipid biosynthesis;
  • Figure 5 represents the percent inhibition of cell-free extracts derived from M. smeqmatis transformants containing various recombinant inhA plasmids in the presence of isoniazid;
  • Figure 6 represents an allelic exchange experiment
  • Figure 7 is comprised of Figure 7A, Figure 7B and Figure 7C, and represents the nucleic acid sequence of the
  • Figure 8 is comprised of Figure 8A, Figure 8B and
  • Figure 9 is comprised of Figure 9A, Figure 9B,
  • Figure 9C and Figure 9D represents the amino acid sequence of the pS5 M. bovis operon
  • Figure 10 represents the amino acid sequence of a fragment of the pS5 operon encoded by nucleic acid residues 1256-2062;
  • Figure 11 represents the amino acid sequence of a fragment of the pS5 operon encoded by nucleic acid residues 494-1234;
  • Figure 12 represents the nucleic acid sequence of the pS5 M. bovis operon
  • Figure 13 represents the vector map of pYUB18.
  • This invention is directed to polynucleotides which encode the enzymes of M. tuberculosis, M. bovis, M. avium and M. smegmatis which are the targets of action of isoniazid. Mutations of these polynucleotides confer isoniazid resistance onto mycobacteria.
  • polynucleotides In order to enable the elimination of isoniazid resistance conferred by these polynucleotides (inhA for M. tuberculosis and pS5 for M. bovis) , the polynucleotides have been identified, isolated, cloned, sequenced and characterized. The DNA sequences of the polynucleotides are used to develop treatments for tuberculosis and other mycobacterial infections, and can be used to determine whether various strains of the M. tuberculosis complex in clinical samples are resistant to isoniazid. In addition, these polynucleotides are used to assess whether antibiotic drugs are effective against the M. tuberculosis complex. The inventors have employed a genetic strategy to identify the targets of action of the M.
  • FIG. 1 represents the minimal inhibitory concentrations of M. smegmatis formed with the various cosmid clones.
  • a 3 kb BamHI DNA fragment from the M. smegmatis cosmid that retained its ability to confer isoniazid-resistance was used as a probe for Southern analysis and found to strongly hybridize to all of 11 different mycobacterial species tested, demonstrating that the inhA gene is highly conserved among mycobacteria.
  • the DNA sequences for the DNA fragments that conferred isoniazid-resistance were determined for DNAs from the isoniazid-sensitive strains of M. smegmatis and M. tuberculosis, and isoniazid-resistant strains of M. smegmatis and M. bovis.
  • the inhA genes are preceded by another open reading frame that shares 40% identity with acetyl CoA reductases.
  • the inhA genes are positioned within operons with the ORF1
  • the inhA gene has its own promoter.
  • ORF1 has been designated mabA, a gene which is involved in mycolic acid biosynthesis.
  • Figure 6 represents the allelic exchange experiment
  • Polynucleotides from M. smegmatis and M. tuberculosis (inhA) which encode the target of action for isoniazid and ethionamide have been identified, isolated, cloned, seguenced and characterized.
  • the nucleic acid sequences for these polynucleotides are shown below as well as in Figures 7 and 8, respectively.
  • GCGCGATCGA CTTCATCCCG GACAAGCGGG TCGGCACGGT CGAGGAGGTC GCGGGCGCGG 780 TCAGCTTCCT GGCCTCGGAG GACGCCTCCT ACATCGCGGG CGCGGTCATC CCCGTCGACG 840
  • CTGCACTTCG GCGGGGTGTC ACCGATCAAC GGCATCAACC GGGACCTGAT CGTCGCGATC 1920 GAGGCCGAAC TCGCCCGACG CGGCCGCAAC CTTCCGGTCT ACTTCGGCAA CCGCAACTGG 1980
  • GCAGCAGTGT CAACGGCGCA CCGTGCACGC CGGCCTGCTC GGCGTGACCC GCCCCGGGCG 2760
  • GCCATACGCG CCGAGCGCAC CACCCGCGTG AGGGGGCGCA GCGCCGAGTC GGCGATCTGA 2880
  • ACCTCCGACG AACTCTGCAG ACCGCTCGGG ATCAGACCCG CACTCACCGC GATGATGGCG 2940 TCGACATGGG CGGCGTTCTC CAGCACCCGC ACAGCCCGGG TCGGCGCGTG GTCGGGGACG 3000
  • GAAGGTCATC AACGCCAACC TCACCGGGGC GTTCCGGGTG GCTCAACGGG CATCGCGCAG 600
  • M. bovis for isoniazid has been identified, isolated, cloned, seguenced and characterized.
  • the amino acid sequence for this gene is shown below, as well as in Figure 9.
  • CGCTCTTCCC AGACTTGCAG CCCCGGGGCA CGGCGGCGGT TGGTGTCGAT GATCGCGGCG 120 GGAAGATCCG CGTCGATCCA CTTGGCGCCA TGGAAGGCAG AAGCCGAGTA GCCGGCCAGC 180
  • GGCGTCAATA CACCCGCAGC CAGGGCCTCG CTGCCCAGAA AGGGATCCGT CATGGTCGAA 360
  • GTGTGCTGAG TCACACCGAC AAACGTCACG AGCGTAACCC CAGTGCGAAA GTTCCCGCCG 420 GAAATCGCAG CCACGTTACG CTCGTGGACA TACCGATTTC GGCCCGGCCG CGGCGAGACG 480
  • CAG CAT GCA GCG CAA CAA ATT CGG TCG AAT GAT ATT CAT AGG TTC GGT 915 Ser Met Gin Arg Asn Lys Phe Gly Arg Met He Phe He Gly Ser Val 130 135 140
  • fragments of the pS5 operon may be responsible for conferring isoniazid resistance:
  • the pS5 operon of M. bovis has the following nucleic acid seguence:
  • SEQ ID NO: 6 GTTCGCTCCG GCGCGGTCAC GCGCATGCCC TCGATGACGC AGATCTCGTC GGGCTCGATG 60
  • the polynucleotides of the invention may be obtained by expressing a DNA sequence coding said polynucleotides in a host cell or organism.
  • a DNA molecule such as the nucleic acid sequence from residues 1256-2062, or 494-1234 of Figure 12, can be expressed in a host cell or organism.
  • promoters which comprise DNA molecules which consist of part or all of the nucleic acid sequence from residues 1-493 of Figure 12, can be used in the cloning and/or expression of a nucleic acid sequence.
  • DNA expression vectors containing polynucleotides of the invention can be prepared by transforming host cells capable of expressing the polynucleotides of the invention or fragments thereof with a DNA sequence encoding the polynucleotides of the invention. The transformed host cells are then cultured, and the expressed polynucleotide is recovered.
  • polynucleotides and fragments of polynucleotides of the invention can be prepared several ways. For example, they can prepared by isolating the polynucleotides from a natural source, or by synthesis using recombinant DNA techniques. In addition, variants of the polynucleotides of the invention can be prepared by utilizing site-specific mutagenesis of the DNA encoding the native amino acid sequences (see Adelman et al., DNA, Vol. 2, p. 183 (1983)). If recombinant DNA techniques are used, DNA encoding the polynucleotide must be obtained. This DNA can be isolated from mycobacteria. Alternatively, the DNA may be produced as intron free cDNA using conventional techniques. In addition, the DNA can be produced in the form of synthetic oligonucleotides.
  • the DNA is obtained, it is treated so that it is suitable for insertion into appropriate cloning and/or expression vectors.
  • the DNA is cleaved utilizing restriction enzymes.
  • the nucleic acid is then recovered.
  • the DNA is then tailored using conventional techniques, such as treatment with polymerase 1, phenol and chloroform.
  • the DNA is then extracted and precipitated by ethanol.
  • religation is performed by providing equimolar amounts of the desired components, appropriately tailored for correct matching and treatment with an appropriate ligase, such as T. DNA ligase.
  • Suitable cloning vectors may be constructed according to standard techniques, or may be selected from the large number of cloning vectors available in the art. While the cloning vector selected may vary according to the host cell intended to be used for expressing the polynucleotide-encoding DNA, useful cloning vectors will generally have the ability to self replicate, will possess a single target for any particular restriction endonuclease and will carry genes for a readily selectable marker such as isoniazid resistance. Two major types of cloning vectors which may be used are plasmids and bacterial viruses. Examples of such vectors include pUC18, pl8, Mpl9, pBR322, pf*!39, ColEl, and pCRl from E. coli.
  • a wide range of host plasmids include RP4, phage DNA's such as lambda and M13, and shuttle vectors such as pSA3 and pAT28.
  • phage DNA's such as lambda and M13
  • shuttle vectors such as pSA3 and pAT28.
  • the most suitable cloning vectors are pBluesript Ilks (Stratagene), and pYUB18, as shown in Figure 13.
  • the cloning vector must incorporate an expression control sequence.
  • a typical expression control sequence can be described as having a promoter region, a 5' untranslated region, a polypeptide coding sequence, a 3' untranslated region and a transcription termination region.
  • a promoter which may be used comprises all or part of the nucleic acid sequence from residues 1-493 shown in Figure 12.
  • Hosts which may be used in this invention include bacteria, yeasts, fungi, insects and animal and plant cells.
  • Procaryatic hosts are generally preferred.
  • Mycobacterial and bacterial hosts including E. coli and M. smec ⁇ tatis, are particularly suitable. Of the mycobacterial hosts,
  • M. smeornatis mc 155 is preferred.
  • the exogenous protein is then isolated using routine methods such as freeze-thaw extraction. Purification is then performed utilizing conventional techniques.
  • a DNA molecule comprising all or part of the nucleic acid sequences of the polynucleotides of the invention may be used as probes for identifying nucleic acids which code for polynucleotides associated with isoniazid resistance.
  • a probe may be labelled, for example, with radioactive isotopes, including 32P and 33P labels.
  • the probes of the invention are capable of hybridizing to the genetic elements associated with isoniazid resistance.
  • the probes of the invention are specific for sense-strand and anti-sense strand of the DNA which encodes for the isoniazid resistance gene.
  • the probe may be the entire nucleotide sequence depicted in Figure 12. More useful probes may be from 100-1000 base pairs. However, shorter probes are preferred.
  • diagnostic kits for use in detecting the presence of polynucleotides which confer isoniazid resistance in a clinical sample.
  • the diagnostic kits of the invention include nucleic acid probes, as discussed hereinabove, which may be labelled.
  • the diagnostic kits of the invention may also include a lysing agent, a denaturing solution, a neutralizing solution, an alkaline fixation solution and a saline citrate.
  • Clinical samples may be tested to determine whether the samples contain polynucleotides which confer isoniazid resistance.
  • Clinical samples are tested with labelled probes under conditions such that the probes will hybridize to the isoniazid resistance-associated genes.
  • hybrid DNA is detected in the sample. These procedures are performed using standard techniques.
  • polynucleotides which are associated with isoniazid resistance are amplified in a clinical sample. After amplification, the detection of the presence or absence of the amplified DNA in the sample indicates whether the sample contains polynucleotides which confer isoniazid resistance.
  • antibodies can be raised which are immunoreactive with epitopes on the polynucleotides of the invention which confer isoniazid resistance. These antibodies can be used in diagnostic immunoassays, and can be passively administered to prevent and treat tuberculosis and other mycobacterial infection and disease.
  • M. bovis isoniazid resistant strains In order to select M. bovis isoniazid resistant strains, a virulent wild-type New Zealand strain of M. bovis was cloned by four serial passages using a combination of liquid tween albumin broth (TAB) and 7H10 pyruvate agar media. A single colony of M. bovis was inoculated into TAB and incubated until visible growth was apparent. An appropriate dilution of this TAB was then inoculated onto solid media to obtain discrete colonies. After incubation, a single colony was inoculated into TAB and the process was repeated. The G4 strain was obtained after four cloning cycles.
  • TAB liquid tween albumin broth
  • the isoniazid resistant strains were obtained by inoculating G4 into a series of TABs containing different concentrations of isoniazid and then incubated. Of the TABs with luxuriant growth, the one with the highest concentration of isoniazid, was inoculated onto solid media containing isoniazid. A colony from this inoculation was used to inoculate a TAB containing isoniazid and incubated. When visible growth was apparent, this was used to inoculate a series of TABs containing varying concentrations of isoniazid. This selection procedure was repeated to obtain a series of clones of M. bovis with increasing resistance to isoniazid. The last strain selected, G4/100, is resistant to 100 ⁇ g/ml of isoniazid.
  • a cosmid library of G4/100 was made in the shuttle vector pYUB18 (as shown in Figure 13). This vector replicates independently in E. coli and various mycobacterial species. It contains a selectable kanamycin gene and a cos site.
  • the cosmid library was made by performing the following steps: (a) Partial Sau3AI digestion of purified chromosomal
  • M. smegmatis organisms were selected by growth on media containing kanamycin. Approximately 1200 kanamycin resistant clones were patched onto media containing isoniazid. Four isoniazid resistant clones were identified.
  • M. smeqmatis cultures of M. smeqmatis (5 ml) were incubated with cycloserine and ampicillin for 3 hours before harvest. The cells were pelleted and resuspended in 0.25 ml of 40 mM Tris-acetate, 2 mM EDTA, pH 7.9. To this, 0.5 ml of lysing solution was added (50 MM Tris, 3% SDS) and the solution was mixed for 30 minutes. The sample was heated to 60°C for 20 minutes, cooled for 10 minutes and the DNA was extracted by adding 0.8 ml of phenol (containing 50 mM NaCI). This was centrifuged and the upper layer containing the DNA was removed.
  • lysing solution 50 MM Tris, 3% SDS
  • pS5 The smallest plasmid obtained which conferred an isoniazid resistance phenotype on M. sme ⁇ matis was 2.3 Kb in size and was designated pS5.
  • pS5 was cloned into the vector pBluesript II KS+ (Stratagene, CA) .
  • This vector contains the T3 and T7 promoters which were used for the sequencing. Sequencing was carried out using the dsDNA cycle sequencing system from GIBCO BRL, Life Technologies, according to the manufacturer's instructions.
  • radioactively labelled nucleotide was either [ ⁇ - P] ATP or [ ⁇ - 33P] ATP (Amersham) .
  • the sequencing program used was
  • GCG Sequence Analysis Software Package
  • the enzyme was first isolated by pelleting a culture of M. bovis and resuspending in 50 mM potassium phosphate buffer, pH 7. This was added to a tube containing 0.5 g zirconium beads (Biospecs Products) and vortexed for 5 minutes. The sample was centrifuged briefly and the supernatant was diluted to 4 ml with 50 mM potassium phosphate buffer and then filter sterilized through 0.22 ⁇ m filters.
  • the catalase activity was assayed by incubating an aliquot of the above sample with 3 ⁇ M H,0_ in a total volume of 3 ml for 5 minutes. The reaction was stopped by adding 1.5 ml of titanium tetrachloride reagent (1.5 mg/ml
  • the G4 strain and the other virulent M. bovis strains contained similar levels of catalase activity. No catalase activity was detected in the G4/100 strain.
  • G4(S5) was electroporated into G4 to produce G4(S5).
  • G4(S5) grew on media containing a level of isoniazid that prevented growth of G4.
  • G4(S5) was also shown to have catalase activity similar to that of G4.
  • these polynucleotides can be used in the treatment of M. tuberculosis, M. avium, M. smegmatis, M. bovis and other mycobacterial infection.
  • One method of treating mycobacterial infection such as tuberculosis utilizing these polynucleotides is preparing anti-DNA or anti-RNA oligonucleotides which can be used to inhibit mRNA activity of the inhA operon of M. tuberculosis or M. bovis.
  • These oligonucleotides can be prepared utilizing the wild-type DNA sequence of the inhA operon of M. tuberculosis or the pS5 operon of M.
  • oligonucleotides can then be administered, either alone or in combination with other compositions, to treat mycobacterial infection, including tuberculosis.
  • These oligonucleotides can be administered orally, enterally, subcutaneously, intraperitoneally or intravenously.
  • the DNA sequences of these polynucleotides can also be used to assess the susceptibility of various strains of the M. tuberculosis complex in a clinical sample to isoniazid. In order to perform this, first the chromosomal DNA of the M. tuberculosis complex from a clinical sample must be isolated.
  • Oligonucleotides are prepared, for example using an oligonucleotide synthesizer, utilizing the wild-type inhA polynucleotide DNA sequence of M. tuberculosis depicted in Figure 8 or the DNA sequence of M. bovis depicted in Figure 12. Regions of the inhA polynucleo ide of M. tuberculosis or the PS5 polynucleotide of M. bovis from the clinical sample which are identified by the oligonucleotides are then amplified (for example by using polymerase chain reaction (PCR)) to obtain double stranded DNA. Next, single strand conformation polymorphism analysis is performed in order to determine whether a mutated gene exists in the M. tuberculosis complex organisms from the clinical sample. If a mutation exists, this indicates that the M. tuberculosis complex organisms from the clinical sample are resistant to isoniazid.
  • PCR polymerase chain reaction
  • PCR or other type of amplification is performed after substitution of half of the dCTP by 0.5 ⁇ l of
  • Electrophoresis is then performed at room temperature and constant power (6W for a 50 x 32 x 0.4 cm gel) overnight. Gels are then dried and exposed for autoradiography overnight.
  • An example of using single strand conformation polymorphism is described by Telenti et al . in "Detection Of Rifampicin-Resistance Mutations In Mycobacterium Tuberculosis", Vol. 341, pages 647-650 (March, 1993), which is incorporated herein by referenced.
  • the polynucleotide sequences can also be used to determine whether a drug is effective against tuberculosis. This is performed by overexpre ⁇ sing the M. tuberculosis inhA polynucleotide or the M. bovis pS5 polynucleotide (i.e., a gene which encodes the target of action enzyme for isoniazid) so as to obtain the enzyme. The enzyme is then combined with a purified reagent, such as a fatty acid or NADP, to obtain a mycolic acid. The mycolic acid is then assayed to measure its level of biosynthesis, such as by thin layer chromatography or spectrophotometry.
  • a purified reagent such as a fatty acid or NADP
  • a drug is combined with the system used to produce mycolic acid to determine whether the drug blocks mycolic acid biosynthesis. If there is a blockage, this indicates that the drug is effective against tuberculosis.
  • Drugs which may be tested for their effectiveness against tuberculosis by this method include isoniazid, ethionamide, rifampicin, streptomycin, ethambutol, ciprofloxacin, novobiocin and cyanide. Further, the polynucleotide which encodes an enzyme involved in mycolic acid biosynthesis can be used for treating tuberculosis.
  • the gene mabA which is depicted in Figure 7 from nucleic acid residue 96 to nucleic acid residue 863, and in Figure 8, from nucleic acid residue 224 to nucleic acid residue 967, has been isolated by the inventors and identified as a gene which encodes an enzyme involved in mycolic acid biosynthesis of tuberculosis.
  • a compound which blocks the biochemical activity of the enzyme encoded by the mabA gene is administered. The compound blocks the enzyme activity, thereby preventing mycolic acid biosynthesis. This causes the tuberculosis to organisms die.
  • compounds which block the activity of enzymes encoded by the polynucleotides can be prepared. This is performed by overexpressing the enzyme and purifying the enzyme, and then performing X-ray crystallography on the purified enzyme to obtain the molecular structure of the enzyme. Next, compounds are created which have a similar molecular structure to the enzyme. The compounds are then combined with the enzyme and attached thereto so as to block the biochemical activity of the enzyme. Since the enzyme is blocked, it is unable to confer isoniazid resistance on tuberculosis organisms. Further, tuberculosis-specific purified mycolic acid compounds can be produced by adding the enzyme encoded by the polynucleotide to the chemical reaction which produces mycolic acids.
  • polynucleotide DNA sequences can be used to produce or improve tuberculosis vaccines.
  • M. tuberculosis complex strains that have become isoniazid resistant often have reduced virulence and can be administered as vaccines.
  • mutated genes of M. tuberculosis and M. bovis can be added to BCG or tuberculosis vaccines to provide attenuated mutant tuberculosis vaccines.
  • These vaccines can be used to treat and prevent a wide variety of diseases, including tuberculosis, AIDS, leprosy, polio, malaria and tetanus.
  • GCGCGATCGA CTTCATCCCG GACAAGCGGG TCGGCACGGT CGAGGAGGTC GCGGGCGCGG 780 TCAGCTTCCT GGCCTCGGAG GACGCCTCCT ACATCGCGGG CGCGGTCATC CCCGTCGACG 840
  • CTGCACTTCG GCGGGGTGTC ACCGATCAAC GGCATCAACC GGGACCTGAT CGTCGCGATC 1920
  • GCAGCAGTGT CAACGGCGCA CCGTGCACGC CGGCCTGCTC GGCGTGACCC GCCCCGGGCG 2760
  • GCCATACGCG CCGAGCGCAC CACCCGCGTG AGGGGGCGCA GCGCCGAGTC GGCGATCTGA 2880
  • ACCTCCGACG AACTCTGCAG ACCGCTCGGG ATCAGACCCG CACTCACCGC GATGATGGCG 2940 TCGACATGGG CGGCGTTCTC CAGCACCCGC ACAGCCCGGG TCGGCGCGTG GTCGGGGACG 3000
  • GAAGGTCATC AACGCCAACC TCACCGGGGC GTTCCGGGTG GCTCAACGGG CATCGCGCAG 600
  • GGCGTCAATA CACCCGCAGC CAGGGCCTCG CTGCCCAGAA AGGGATCCGT CATGGTCGAA 360 GTGTGCTGAG TCACACCGAC AAACGTCACG AGCGTAACCC CAGTGCGAAA GTTCCCGCCG 420
  • CAG CAT GCA GCG CAA CAA ATT CGG TCG AAT GAT ATT CAT AGG TTC GGT 915 Ser Met Gin Arg Asn Lys Phe Gly Arg Met He Phe He Gly Ser Val 130 135 140 CTC CGG CAG CTG GGG CAT CGG CAA CCA GGC CAA CTA CGC AGC CTC CAA 963 Ser Gly Ser Trp Gly He Gly Asn Gin Ala Asn Tyr Ala Ala Ser Lys 145 150 155

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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne l'identification, le clonage, le séquençage et la caractérisation de polynucléotides permettant de déterminer la résistance mycobactérienne à l'isoniazide antibiotique. Ces polynucléotides codent l'expression d'une enzyme qui est la cible de M. tuberculosis, M. smegmatis, M. Bovis et M. avium pour l'isoniazide. Les polynucléotides ont éte soumis au séquençage de l'ADN et peuvent être utilisés dans le traitement et la prévention d'injections mycobactériennes dont la tuberculose. On peut également utiliser lesdits polynucléotides pour déterminer la sensibilité ou la résistance de diverses souches de M. tuberculosis à divers médicaments antibiotiques.
PCT/US1994/005398 1993-05-13 1994-05-13 Utilisation des genes de m. tuberculosis, m. bovis et m. smegmatis conferant une resistance a l'isoniazide WO1994026765A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU69496/94A AU6949694A (en) 1993-05-13 1994-05-13 Use of genes of m. tuberculosis, m. bovis and m. smegmatis which confer isoniazid resistance

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
NZ24762093 1993-05-13
NZ247620 1993-05-13
US6240993A 1993-05-14 1993-05-14
US08/062,409 1993-05-14
US22174294A 1994-03-31 1994-03-31
US08/221,742 1994-03-31

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WO1994026765A1 true WO1994026765A1 (fr) 1994-11-24

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AU (1) AU6949694A (fr)
WO (1) WO1994026765A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO1999061625A1 (fr) * 1998-05-27 1999-12-02 Isis Innovation Limited N-acetyltransferases mycobacteriennes
US6335436B1 (en) 1995-02-10 2002-01-01 Hybridon, Inc. Oligonucleotides active against drug-resistant bacteria
FR2837836A1 (fr) * 2002-03-29 2003-10-03 Centre Nat Rech Scient UTILISATION DE LA PROTEINE Maba (FABG1) DE MYCOBACTERIUM TUBERCULOSIS POUR LA CONCEPTION ET LE CRIBLAGE D'ANTIBIOTIQUES

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6335436B1 (en) 1995-02-10 2002-01-01 Hybridon, Inc. Oligonucleotides active against drug-resistant bacteria
WO1999061625A1 (fr) * 1998-05-27 1999-12-02 Isis Innovation Limited N-acetyltransferases mycobacteriennes
FR2837836A1 (fr) * 2002-03-29 2003-10-03 Centre Nat Rech Scient UTILISATION DE LA PROTEINE Maba (FABG1) DE MYCOBACTERIUM TUBERCULOSIS POUR LA CONCEPTION ET LE CRIBLAGE D'ANTIBIOTIQUES
WO2003082911A2 (fr) * 2002-03-29 2003-10-09 Centre National De La Recherche Scientifique (Cnrs) Utilisation de la proteine maba (fabg1) de mycobacterium tuberculosis pour la conception et le criblage d'antibiotiques
WO2003082911A3 (fr) * 2002-03-29 2004-04-01 Centre Nat Rech Scient Utilisation de la proteine maba (fabg1) de mycobacterium tuberculosis pour la conception et le criblage d'antibiotiques

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