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WO1996019578A9 - PROCEDES ET COMPOSITIONS DE DETECTION ET DE TRAITEMENT D'INFECTIONS MYCOBACTERIENNES A L'AIDE D'UN OPERON ahpCF - Google Patents

PROCEDES ET COMPOSITIONS DE DETECTION ET DE TRAITEMENT D'INFECTIONS MYCOBACTERIENNES A L'AIDE D'UN OPERON ahpCF

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Publication number
WO1996019578A9
WO1996019578A9 PCT/NZ1995/000133 NZ9500133W WO9619578A9 WO 1996019578 A9 WO1996019578 A9 WO 1996019578A9 NZ 9500133 W NZ9500133 W NZ 9500133W WO 9619578 A9 WO9619578 A9 WO 9619578A9
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WIPO (PCT)
Prior art keywords
inh
ahpc
operon
mycobacteria
ahpf
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PCT/NZ1995/000133
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English (en)
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WO1996019578A2 (fr
WO1996019578A3 (fr
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Priority to EP95941938A priority Critical patent/EP0805863A2/fr
Priority to AU43185/96A priority patent/AU708456B2/en
Publication of WO1996019578A2 publication Critical patent/WO1996019578A2/fr
Publication of WO1996019578A3 publication Critical patent/WO1996019578A3/fr
Publication of WO1996019578A9 publication Critical patent/WO1996019578A9/fr

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  • the invention relates to mate ⁇ als and methods used in the diagnosis and treatment of mycobacterial diseases, and more specifically to DNA sequence(s) associated with resistance to isoniazid and its analogs in mycobacteria, methods for isolating such sequence(s), and the use of such sequence(s) in human and animal medical practice.
  • M. bovis. and M. a ricanum 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, M. bovis causes tuberculosis in a wide range of animals, and is a major cause of animal suffering and economic loss in animal industries.
  • INH isoniazid (isonicotinic acid hydrazide, INH), rifampicin, and pyrazinamide.
  • INH was first reported to be active against M. tuberculosis in 1952, and particularly active against M. tuberculosis and M . bovis. However, mutants resistant to INH have emerged since then, and today such mutants account for as many as 26% of the clinical M. tuberculosis isolates in certain U.S. cities.
  • INH-resistant strains are associated with a loss of catalase activity, and deletions of the catalase-peroxidase gene ( atGJ correlate with INH resistance in certain M. tuberculosis isolates. Furthermore, transfer of the wild-type M. tuberculosis katG gene to INH-resistant M. smegmatis: M. tuberculosis and M. bovis confers INH sensitivity, suggesting that catalase-peroxidase activity is required for INH-sensitivity. However, in some studies only 10 to 25% of the INH-resistant isolates appear to be catalase negative, indicating that INH resistance can be due to other tactors.
  • INH resistance inh ⁇
  • inh ⁇ another gene involved in INH resistance, inh ⁇ , was identified and alterations in this gene, particularly in its promoter region, have been found to occur in some but not all INH- resistant isolates that were catalase positive.
  • INH-resistant M. tuberculosis strains whose resistance cannot be explained by loss or mutation of either the ka Q or inhA genes.
  • Drug resistance can be caused by many mechanisms, including mutations in the drug target that reduce the binding of the drug or mutations that lead to increased production of the target.
  • the mechanism by which INH inhibits mycobacteria and its precise target of action are unknown.
  • Biochemical evidence has suggested that both INH and ethionamide (ETH, a structural analog of INH) block mycolic acid biosynthesis in mycobacteria. It has also been suggested that the cellular response to oxidative stress, particularly levels of hydrogen peroxide, is involved in INH-susceptibility.
  • INH has been found to inhibit mycolic acid biosynthesis in cell-free extracts of mycobacteria, and the target protein has been identified as InhA, an enzyme which has significant homology to the lipid biosynthetic enzyme, enoyl ACP reductase. INH does not appear to act directly on this enzyme so an intermediate or intermediates appear to be involved. In addition, in certain cases, low-level INH resistance correlates not with the loss of catalase activity but with the coacquisition of ETH resistance, suggesting that the two drugs may share a common target. Furthermore, it has been proposed that the reactive oxygen intermediates formed during interactions between INH and peroxidases may suggest a mode of action for INH.
  • This invention is based upon the discovery of an operon in mycobacteria, designated as ahpCF. It contains two large open reading frames (ORFs) that may encode the two protein components of the enzyme, alkyl hydroperoxide reductase (Ahp reductase). Mutations within this operon, particularly within the promoter region preceding the first open reading frame, upregulate the expression of the operon and confer INH resistance.
  • ORFs alkyl hydroperoxide reductase
  • the present invention provides isolated and recombinant polynucleotide sequences and polypeptides encoded therein that are associated with resistance to INH and its structural analogs in members of the genus mycobacteria, particularly those of the M. tuberculosis complex, including M. tuberculosis.
  • the polynucleotides of the invention have many uses. For example, they are useful in assessing the susceptibility of various strains of the M. tuberculosis complex to INH type antibiotics, as decoys and antisense oligonucleotides to prevent the expression of polypeptides associated with INH resistance, and for the expression of the polypeptides encoded therein.
  • polypeptides encoded in the polynucleotides and/or antibodies directed to them may also have use in immunoassays for the detection of INH-resistant strains, in the determination of whether an INH-type antibiotic may be effective against tuberculosis, and in the treatment of individuals for infection with these strains.
  • embodiments of the invention include the following: An isolated polynucleotide comprised of a nucleotide sequence of at least 15 nucleotides ofan ahpCF operon of mycobacteria.
  • nucleotide sequence includes a sequence from a promoter region of the operon.
  • polynucleotide as described above wherein the polynucleotide is a recombinant expression vector comprised of control sequences operably linked to a segment encoding the AhpC and/or AhpF polypeptide or fragment or variant thereof.
  • a host cell comprised of a polynucleotide selected from the group of polynucleotides described above.
  • a method of treating an individual for infection caused by a member of the mycobacterial complex comprising: (a) providing a composition comprised of a polynucleotide capable of inhibiting mRNA activity from an ahpCF operon of the infecting species and a suitable excipient; and
  • a method of assessing susceptibility of a strain of mycobacteria in a biological sample to INH comprising:
  • step (b) comprises a promoter region of the operon.
  • PCR polymerase chain reaction
  • the method may be further comprised of providing a comparable portion of wild- type INH-sensitive ahpCF operon from the mycobacteria, and the determination of whether a mutation exists in the biological sample is by comparison with the wild-type ahpCF operon.
  • a method of assessing susceptibility of a strain of mycobacteria m a biological sample to INH comprising:
  • step (c) is performed by comparing the level of messenger RNA transcribed from the ahpC gene in the biological sample with the level of messenger RNA transcribed by the ahpC gene in the control sample, or alternately, wherein step (c) is performed by comparing the level of AhpC polypeptide in the biological sample with the level of AhpC polypeptide in the control sample.
  • a method for producing a compound that inhibits alkyl hydroperoxide reductase activity comprising:
  • a recombinant mycobacterial vaccine comprised of attenuated mutants selected from the group consisting of BCG, M. tuberculosis, and M. bovis. wherein the mutants are host cells containing a mutated ahpCF operon.
  • Figure 1 presents a DNA sequence from M. bovis ATCC 35729
  • Figure 2 shows the amino acid sequences for two large open reading frames (ORFs) encompassed within the DNA sequence shown in Figure 1.
  • Figure 3 shows the alignment of the amino acid sequences of the polypeptides from ORF1 of ahpCF from M. bovis ATCC 35729 with some other sequences of related amino acid homology.
  • Figure 4 shows a comparison of the amino acid translation of ORF2 ofahpCF from M. bovis with AhpF from E. coli.
  • Figure 5 is a schematic diagram ofpYUB18. showing significant features of the genome and restriction nuclease cleavage sites.
  • Figure 6 presents a DNA sequence from the parental M. bovis
  • Figure 7 is a half-tone reproduction of a Southern blot, in which INH sensitive and resistant strains were probed for the catalase gene katG.
  • Figure 8 is a drawing of subfragments of the ahpCF operon, showing which elements of the operon are capable of conferring INH resistance.
  • Figure 9 is a half-tone reproduction of a primer extension analysis performed on RNA isolated from M. smegmatis using the primer 5'- AGCGGTGAGCTGGTAGGCGGGGAATTGATC (SEQ ID NO: 15).
  • the invention stems from the discovery of ahpCF. an operon comprising two ORFs that encodes polypeptides that are a target for INH in a strain of the M. tuberculosis complex. It is probable that mutations of this operon render mycobacteria INH-resistant. Methods of screening for INH-resistance are routine, and are known within the art.
  • the operon was identified using a genetic strategy.
  • a genomic library was constructed in a shuttle cosmid vector from an INH-resistant mutant of M. bovis. Transferral of the library into a wild type (i.e., INH-sensitive) M. smegmatis strain
  • R£CTIFIED 6 SHEET (RULE 91) allowed the identification of clones that consistently conferred INH-resistance.
  • I he DNA fragments that conferred INH-sensitivity to M. smegmatis were subjected to DNA sequencing. The derived DNA sequence is shown in Figure 1. Sequence analysis revealed two ORFs separated by 26 base pairs indicating the two ORFs are part of the same operon.
  • the first ORF (ORFl) does not have a ribosome binding site immediately upstream of the first potential start codon, TTG at positions 712-714.
  • the next six potential start codons also lack ribosome binding sites.
  • the first potential start codon with a likely upstream ribosome binding site is ATG at positions 880-882. This has a ribosome binding site GAGGAG at positions 870-875.
  • ORFl is therefore shown as beginning at positions 880-882.
  • ORFl (positions 880-1467) encodes a protein of 25 kDa which has significant homology to the polypeptide encoded in the coding region of ORFl of the ahpCF operon from other bacteria ( Figure 3 and Table 1). Because of this high homology, ORFl is denoted as ahpC.
  • ORF2 The second large ORF (ORF2) begins 26 bp downstream from ahpC at positions 1493-1495. It has a potential ribosome binding site GGGA at positions 1485-1488.
  • ORF2 (positions 1493-2152) encodes a protein of 22 kDa which is 19% identical to ahpF. of E. coli. Comparisons at the DNA level revealed a small amount of terminal sequence that was homologous between ORF2 of the M. bovis ahpCF operon, and an ORF downstream of the putative ahpC gene of M avium. There are also DNA sequences downstream of the putative ahpC gene in M. leprae that have high homology to ORF2, but these are not associated with a large ORF.
  • ORF2 of M. bovis has been denoted ahpF.
  • a polynucleotide from an INH resistant strain of M. bovis that encodes the ahpCF operon has been identified, isolated, cloned, sequenced and characterized.
  • the nucleic acid sequence of the coding strand for this polynucleotide is shown in Figure 1.
  • Figure 2 shows the putative amino acid sequences of the polypeptides encoded in the polynucleotide.
  • the corresponding polynucleotide of the INH sensitive parent has also been obtained and sequenced, revealing that INH resistance is conveyed by a point mutation in the transcriptional start site of the ahpCF promoter region. Examples 9 and 10 (infra) suggest that the mutation leads to increased production of the ahpC gene product, which in turn is responsible for conveying INH resistance.
  • ahpCF promoter Disclosed herein are two point mutations in the ahpCF promoter currently known to be associated with INH resistance.
  • This invention also contemplates alternative alterations to the ahpCF operon which would affect the expression and/or function of the gene products thereof, thereby imparting various degrees of sensitivity or resistance to INH.
  • Any of the uses of the invention disclosed herein should be considered generic, and can be adapted, if necessary, for application to any of the contemplated ahpCF alterations.
  • any mutation in the promoter region which improves the binding of an intracellular component involved in transcription or a positive controlling elements of transcription would increase the transcription rate.
  • Any mutation in the promoter region which impairs the binding of a negative controlling element like a repressor protein would have the same effect.
  • the ahpCF operon could be duplicated in the strain, also resulting in increased expression of AhpC, and resistance to INH.
  • mutations in the coding region of ahpC could be associated with INH resistance; in particular, those that altered the active site of AhpC so as to increase its catalytic activity. It is desirable to be able to test and treat any alteration which imparts INH resistance.
  • This invention also contemplates a converse set of alterations to the ahp h operon which would result in decreased expression and/or function of the gene products, which may increase sensitivity to INH, compared with the wild type.
  • the ahpCF operon associated with INH resistance in strains of the M. tuberculosis complex provides the practitioner of ordinary skill with compositions and methods useful in the diagnosis and treatment of pathogenic states resulting from infection with mycobacteria, particularly INH-resistant strains.
  • target of action for INH refers to one or both of the polypeptides AhpC and AhpF, encoded in an ahpCF operon of mycobacteria, and preferably in members of the M. tuberculosis complex.
  • ahpCF operon refers to a polynucleotide that encodes polypeptides that are present in mycobacteria, wherein the polypeptides have substantial amino acid homology and equivalent function to the AhpC and AhpF proteins of M. bovis.
  • substantial amino acid homology means at least about 60% homology, generally at least about 70% homology, even more generally at least about 80% homology, and at times at least about 90% homology to any of the indicated polypeptides.
  • the operon includes regions that are responsible for the control of transcription or expression of the encoded polypeptide, including promoter regions, enhancer regions, ribosomal binding regions, and sites capable of binding any other component involved in the regulation of transcription.
  • AhpC polypeptide and “AhpF polypeptide” refer to polypeptides encoded in individual open reading frames within the ahpCF operon in mycobacteria, in either INH-resistant or INH-sensitive variants.
  • AhpCF polypeptide refers to any polypeptide encoded within the ahpCF operon.
  • polynucleotide refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double- and single-stranded DNA and RNA.
  • modifications for example, labels which are known in the art (e.g., Sambrook, et al.), methylation, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates), those containing pendant moieties, such as, for example, proteins (including for e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine), those with intercalators (e.g., acridine, psoralen), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids), as well as unmodified forms of the polynucleotide.
  • labels which are known in
  • the invention includes as an embodiment isolated polynucleotides comprised of sequences encoding polypeptides associated with isoniazid (INH) resistance in mycobacteria or active fragment thereof. These isolated polynucleotides contain less than about 50%, preferably less than about 70%, and more preferably less than about 90% of the chromosomal genetic material with which the sequence encoding the polypeptide is usually associated in nature.
  • An isolated polynucleotide "consisting essentially ol" a sequence encoding an INH resistance associated polypeptide lacks other sequences encoding other polypeptides derived from the mycobacterial chromosome.
  • INH isoniazid
  • the invention also includes as embodiments recombinant polynucleotides containing a region encoding ahpCF operon gene products for mycobacteria.
  • polynucleotide intends a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation: (1) is not associated with all or a portion of a polynucleotide with which it is associated in nature; or (2) is linked to a polynucleotide other than that to which it is linked in nature; or (3) does not occur in nature.
  • a purified or recombinant polynucleotide comprised of a sequence encoding AhpC or AhpF of mycobacteria or variant or active fragment thereof may be prepared by any technique known to those of skill in the art using the polynucleotide sequences provided herein. For example, they can be prepared by isolating the polynucleotides from a natural source, or by chemical synthesis, or by synthesis using recombinant DNA techniques.
  • polypeptide refers to a polymer of amino acids and does not refer to a specific length of the product; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also does not refer to or exclude post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids
  • polypeptides with substituted linkages as well as the modifications known in the art, both naturally occurring and non-naturally occurring.
  • cloning vectors and expression vectors comprised of a sequence encoding AhpC and/or AhpF or variant or fragment thereof. 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.
  • cloning vector selected may vary according to the host cell intended to be used, useful clomng vectors will generally have the ability to self replicate, may possess a single target for a restriction endonuclease, and may carry genes for a readily selectable marker (e.g., antibiotic resistance or sensitivity markers). Suitable examples include plasmids and bacterial viruses, e.g., pUC18, mpl8, mpl9, pBR322, pMB9, ColEl, pCRl, RP4, phage DNAs, and shuttle vectors (e.g., pSA3 and pAT28. Preferred vectors include pBLUSCRIPT Ilks (TM) (Stratagene), and pYUB18.
  • TM pBLUSCRIPT Ilks
  • Expression vectors generally are replicable polynucleotide constructs that encode a polypeptide operably linked to suitable transcriptional and translational regulatory elements.
  • suitable transcriptional and translational regulatory elements examples include promoters, enhancers, ribosomal binding sites, and transcription and translation initiation and termination sequences.
  • the regulatory elements employed in the expression vectors containing a polynucleotide encoding AhpC, AhpF or an active fragment thereof would be functional in the host cell used for expression. It is also contemplated that the regulatory sequences may be derived from the ahpCF operon; thus, a promoter or terminator sequence may be homologous (i.e., from mycobacteria) to the coding sequence.
  • the invention also includes recombinant host cells comprised of any of the above described polynucleotides that contain a sequence encoding an AhpC and/or AhpF polypeptide of mycobacteria.
  • the polynucleotides may be inserted into the host cell by any means known in the art.
  • "recombinant host cells”, “host cells”, “cells”, “cell lines”, “cell cultures”, and other such terms denoting microorganisms or higher eukaryotic cell lines cultured as unicellular entities refer to cells which can be, or have been, used as recipients for recombinant vector or other transfer DNA, and include the progeny of the original cell which has been transformed.
  • progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation.
  • Hosts which may be used include prokaryotic cells (e.g., bacterial cells such as E. coli. mycobacteria, and the like) and eukaryotic cells (e.g., fungal cells, insect cells, animal cells, and plant cells, and the like).
  • prokaryotic cells are generally preferred, and E. coli and M. smegmatis are particularly suitable.
  • Transformation refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion, for example, direct uptake, transduction, f-mating or electroporation.
  • the exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host cell genome.
  • polynucleotides comprised of sequences encoding AhpC and/or AhpF maybe of use in the detection of mycobacteria in biological samples, and mutant forms of these polypeptides associated with INH resistance may be of use in the detection of INH- resistant forms.
  • a “biological sample” refers to a sample of tissue or fluid isolated from an individual, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs, and also samples of in vitro cell culture constituents (including but not limited to conditioned medium resulting from the growth of cells in cell culture medium, putatively infected cells, recombinant cells, and cell components).
  • Particularly useful samples in the diagnosis of human pulmonary tuberculosis are sputum and lung aspirates, and cultures thereof.
  • the term “clinical sample” is synonymous with "biological sample”.
  • the term "individual” as used herein refers to vertebrates, particularly members of the mammalian or avian species, and includes but is not limited to domestic animals, sports animals, and primates, including humans.
  • oligomers of approximately 8 nucleotides or more can be prepared, either by excision from recombinant polynucleotides or synthetically, which hybridize with the mycobacterial sequences in the plasmids and are useful in identification of the INH-resistant and INH-sensitive mycobacteria.
  • the probes are a length which allows the detection of the AhpC and/or AhpF encoding sequences by hybridization.
  • nucleotides While 6-8 nucleotides may be a workable length, sequences of about 10-12 nucleotides are preferred, about 15 nucleotides are more preferred, and about 20 nucleotides appears optimal.
  • probes can be prepared using routine methods, including automated oligonucleotide synthetic methods. For use as probes, complete complementarity is desirable, though it may be unnecessary as the length of the fragment is increased.
  • a polynucleotide comprising all or part of the nucleic acid sequences of an ahpjCE operon, and particularly polynucleotides with sequences from the promoter region, may be used as probes for INH-resistance.
  • the probes may be labelled, for example with radioactive isotopes.
  • Usual isotopes include, for example P and P.
  • the probes are capable of hybridizing to the genetic elements associated with INH-resistance.
  • the probe may be the entire nucleotide sequence depicted in Figure 1. However, shorter probes are preferred.
  • a preferred embodiment of this invention is a probe useful for detecting either of the point mutations in the promoter region of the ahpCF operon that convey INH resistance. Particularly preferred are probes capable of hybridizing to the promoter region of either the INH resistant or INH sensitive strains, but not both.
  • Promoter region in this context means any region or subregion of the ahpCF operon upstream from the coding region of the ahp_C gene, and is at or near a site involved in promoting gene transcription.
  • probes which encompass the actual codon which is mutated in INH resistant strains are those encompassing the 5-mer sequences GGTAC, GGCAC, CGTAA, and CGCAA, optionally extended in the 5' and/or the 3' direction according to the sequence in Figure 1 to include other residues; probes encompassing the 7-mer sequences CGGTACG, CGGCACG, TCGTAAC, TCGCAAC, optionally extended in the same fashion; probes encompassing the 10-mer sequences TCACGGYACG (SEQ ID NO: 16), CACGGYACGA (SEQ ID NO: 17), ACGGYACGAT (SEQ ID NO: 18), CGGYACGATG (SEQ ID NO: 19), ATGTCGYAAC (SEQ ID NO:20), TGTCGYAACC (SEQ ID NO:21), GTCGYAACCA (SEQ ID NO:22), TCGYAACCAA (SEQ ID NO:23), optionally extended in the same fashion, where Y is T or C. It is
  • the biological sample to be analyzed such as blood or serum
  • the biological sample to be analyzed may be treated, if desired, to extract the nucleic acids contained therein.
  • the resulting nucleic acid from the sample may be subjected to gel electrophoresis or other size separation techniques; alternatively, the nucleic acid sample may be dot blotted without size separation.
  • the probes are usually labeled. Suitable labels, and methods for labeling probes are known in the art, and include, for example, radioactive labels incorporated by nick translation or kinasing, biotin, fluorescent probes,
  • the nucleic acids extracted from the sample are then treated with the labeled probe under hybridization conditions of suitable stringencies.
  • the probes can be made completely complementary to the allelic form of polynucleotide that has been targeted. With this goal, high stringency conditions usually are desirable in order to prevent false positives.
  • the stringency of hybridization is determined by a number of factors during hybridization and during the washing procedure, including temperature, ionic strength, length of time, and concentration of formamide. These factors are outlined in, for example, Maniatis, T. (1982).
  • amplification techniques in hybridization assays.
  • Such techniques include, for example, the polymerase chain reaction (PCR) technique described which is by Saiki et al. (1986), by Mullis, U.S. Patent No. 4,683,195, and by Mullis et al. U.S. Patent No. 4,683,202.
  • PCR polymerase chain reaction
  • This technique may be used in conjunction with other techniques, for example, in single-strand conformation polymorphism analysis (see infra., in the Examples).
  • Suitable reagents to perform a diagnostic procedure can be provided in the form of a diagnostic kit.
  • the reagent supplied will depend on the nature of the assay, and may be a polynucleotide probe, a polypeptide, an antibody, or an enzyme substrate.
  • the reagent may be labeled; alternatively, the reagent may be unlabeled and the ingredients for labeling may be included in the kit in separate containers.
  • the kit may also contain other suitably packaged reagents and materials needed for the particular hybridization protocol, for example, standards, as well as instructions for conducting the test. If the kit is to be used for an assay system which includes PCR technology it may also include primers for the PCR reaction.
  • the ahpCF operon sequences and polypeptides encoded therein may also be used for screening for drugs against mycobacteria, particularly members of the mycobacterial complex, and more particularly M. tuberculosis and M. bovis.
  • it can be used to express the INH-resistant and INH-sensitive polypeptides, or fragments thereof, encoded in allelic forms of ahpCF. Utilizing these polypeptides in in vitro assays, one could monitor the effect of candidate drugs on alkyl hydro peroxidase activity. Drugs that inhibit this enzyme are candidates for therapy of mycobacterial diseases.
  • Drugs that may be tested for effectiveness in this type of system include INH, ETH, rifampicin, streptomycin, ethambutol, ciprofloxacin, novobiocin and cyanide.
  • the ahpCF operon sequences may also be used to design polynucleotides that can be used for treatment of mycobacterial infections, including those caused by * tuberculosis. M. avium. and M. bovis.
  • One method of treating a mycobacterial infection utilizing the ahpCF operon is by providing antisense polynucleotides or triplex forming polynucleotides which can be used to inhibit the transcription or translation of mRNA from the ahpCF operon, for example antisense polynucleotides, triplex forming polynucleotides, decoys, and ribozymes.
  • antisense polynucleotides triplex forming polynucleotides, decoys, and ribozymes.
  • these types of polynucleotides are also included within the invention.
  • These polynucleotides may be prepared by a variety of techniques known in the art, including chemical synthesis and recombinant technology. After preparation they can then be administered, either alone or in combination with other compositions to treat mycobacterial infections, including tuberculosis.
  • the compositions containing these polynucleotides may also include suitable excipients.
  • the sequence ofahpCF can also be used to assess the susceptibility of various strains of mycobacteria, and particularly of M. tuberculosis or M. bovis. in a clinical sample to INH. This susceptibility comparison is based upon the detection of a mutant allele as compared to the wild-type ahpCF allele that is INH-sensitive. Procedures to perform this type of assessment will be readily evident to those of skill in the art. For example, one procedure to perform this assessment is described in the Examples, and is based upon isolation of the chromosomal DNA of the bacterium, and amplification of the ahpCF region by PCR using primers specific for the region, based upon the ahpCF sequences provided herein.
  • a mutation associated with INH resistance can then be detected by single-strand conformation polymorphism analysis, or by direct sequencing of the amplified region.
  • a probe encompassing the sequence associated with INH resistance can be used in a direct hybridization assay, such as a blotting assay. INH resistance would be indicated by a positive result with a probe specific for a sequence associated with resistance, or by a negative result with a probe specific for a sequence associated with INH sensitivity.
  • the activity of the ahpC gene can be used to determine the INH susceptibility of a mycobacteria strain in a biological sample.
  • Activity in this context means the level of transcription of the gene into messenger RNA, which may be followed by translation of the RNA into a peptide, which in turn may be followed by other events such as a display of enzymatic activity by the transcribed protein.
  • suitable assays for measuring the activity ot ahpC 1. hybridization blot assays and single strand protection assays using a polynucleotide specific for ahpC messenger RNA; 2.
  • the assay is usually conducted both on the sample to be tested, and on a control sample.
  • the control sample should contain a strain of mycobacteria with a known susceptibility to INH. It can be either INH susceptible, or INH resistant.
  • strain in the control sample is INH sensitive, then similar activity of ahpC in the test sample indicates INH sensitivity; a higher activity indicates INH resistance. If the strain in the control sample is INH resistant, then similar activity of ahpC in the test sample indicates INH resistance, while lower activity indicates a degree of INH sensitivity. Depending on the nature of the assay, it may or may not be necessary that the testing of the biological sample and the control sample be contemporaneous.
  • the ahpCF promoter region may be used to promote the transcription and expression of heterologous genes.
  • An example of heterologous expression of the lacZ gene is described infra.
  • the ahpCF promoter is isolated, or prepared by polynucleotide synthesis from the sequence data. A fusion polynucleotide is formed, in which the promoter is operably linked to a gene that is not ahpC. The construct is then transfected into the desired species of mycobacteria using a suitable vector plasmid.
  • compounds which block the activity of AhpC or AhpF polypeptides are also used to promote the transcription and expression of heterologous genes.
  • ahpCF (which may be enzymes) can be prepared utilizing the sequence information ofahpCF. This is performed by overexpressing AhpC and/or AhpF, purifying the polypeptide(s), and then performing X-ray crystallography on the purified polypeptide(s) to obtain its(their) molecular structure. Next, compounds are created which have similar molecular structures to all or portions of the polypeptide(s). The compounds are then combined with the polypeptide(s) and attached thereto so as to block the biochemical activity of the polypeptide(s).
  • the ahpCF polynucleotide may also be used to produce a vaccine to confer immunity against the product of the gene.
  • the ahpCF polynucleotide may also be used to prepare attenuated strains of mycobacteria or BCG, and recombinants thereof.
  • a mutated ahpCF operon is selected or engineered which has a lower level of expression than wild type mycobacteria, which may render it even more susceptible to INH than the wild type.
  • the level of expression would be chosen to maximize INH sensitivity, while leaving the strain with the minimum amount ofahpCF activity that is consistent with viability and the desired growth rate.
  • this would be combined with other attenuation strategies, such as alteration of the mycobacteria virulence gene.
  • the resulting strain could be used in a vaccine to stimulate immunity against mycobacteria. Genes expressing proteins from other pathogens could be transfected into the attenuated strain, thereby providing protection against other diseases.
  • the list of pathogens which would be suitable for this strategy is extensive, and includes leprosy, polio, malaria, AIDS, hepatitis B, hepatitis C, and tetanus.
  • polypeptides of the invention include those encoded in allelic variants of ahpCE, and are in purified or recombinant form. These polypeptides include fragments of the entire polypeptides encoded in the ORFs.
  • polypeptides of the invention include variants of AhpC and/or AhpF which differ from the native amino acid sequences by the insertion, substitution, or deletion of one or more amino acids. These variants may be prepared chemically, or by alteration of the polynucleotide sequences encoding AhpC or AhpF, using techniques known in the art, for example, by site-specific primer directed mutagenesis.
  • polypeptides can be purified by any means known in the art, including, for example freeze-thaw extraction, salt fractionation, column chromatography, affinity chromatography and the like.
  • the polypeptides of the invention may find use as therapeutic agents lor treatment of mycobacterial infection. "Treatment” as used herein refers to prophylaxis and/or therapy.
  • the AhpC and/or AhpF polypeptides can be prepared as discrete entities or incorporated into a larger polypeptide, and may find use as described herein.
  • the immunogenicity of the epitopes of AhpC and/or AhpF may also be enhanced by preparing them in mammalian or yeast systems fused with or assembled with particle-forming proteins such as, for example, that associated with hepatitis B surface antigen. See, e.g., U.S. Pat. No. 4,722,840.
  • Vaccines may be prepared from one or more immunogenic polypeptides derived from AhpC and/or AhpF.
  • the polypeptides of this invention can also be used in a polypeptide vaccine.
  • AhpC and/or AhpF are provided in a suitable form for administration to human or other mammalian subjects.
  • Vaccines can be prepared for injection, or for oral or intranasal administration.
  • the polypeptide(s) may be prepared to render them more immunogenic by a technique or combination of techniques known in the art, such as aggregation with a cross-linking agent like glutaraldehyde, fragmentation, linking to a carrier like keyhole limpet hemocyanin (KLH), and cyclization.
  • KLH keyhole limpet hemocyanin
  • such vaccines are prepared as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared.
  • the preparation may also be emulsified, or the protein encapsulated in liposomes.
  • the active immunogenic ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine.
  • adjuvants which may be effective include but are not limited to: aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-( 1 '-2'-dipalmitoyl-sn-glycero-3 -h ydroxyphos ⁇ horyloxy)-ethylamine (CGP 19835 A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 e
  • an adjuvant may be determined by measuring the amount of antibodies directed against an immunogenic polypeptide containing an AhpC or AhpF antigenic sequence resulting from administration of this polypeptide in vaccines which are also comprised of the various adjuvants.
  • the vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are • suitable for other modes of administration include suppositories and, in some cases, oral formulations or formulations suitable for distribution as aerosols.
  • binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably l%-2%.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%-95% of active ingredient, preferably 25%-70%.
  • the proteins may be formulated into the vaccine as neutral or salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric, maleic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • the vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and/or therapeutically effec ⁇ tive.
  • the quantity to be administered which is generally in the range of 5 micrograms to 250 micrograms of antigen per dose, depends on the subject to be treated, capacity of the subject's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be administered may depend on the judgment of the practitioner and may be peculiar to each subject.
  • the vaccine may be given in a single dose schedule, or preferably in a multiple dose schedule.
  • a multiple dose schedule is one in which a primary course of vaccination may be with 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reenforce the immune response, for example, at 1-4 months for a second dose, and if needed, a subsequent dose(s) after several months.
  • the dosage regimen will also, at least in part, be determined by the need of the individual and be dependent upon the judgment of the practitioner.
  • the vaccine containing the immunogenic AhpC and/or AhpF antigen(s) may be administered in conjunction with other immunoregulatory agents, for example, immune globulins, as well as antibiotics.
  • the AhpCF antigens may be used for the preparation of antibodies.
  • the immunogenic polypeptides prepared as described above are used to produce antibodies, including polyclonal and monoclonal. If polyclonal antibodies are desired, a selected mammal (e.g., mouse, rabbit, goat, horse) is immunized with an immunogenic polypeptide bearing AhpC or AhpF epitope(s). Serum from the immunized animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to an AhpCF epitope contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art, see for example, Mayer and Walker (1987).
  • Monoclonal antibodies directed against AhpCF epitopes can also be readily produced by one skilled in the art.
  • the general methodology for making monoclonal antibodies by hybridomas is well known.
  • Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., M. Schreier et al. (1980); Hammerling.et al. (1981); Kennett et al. (1980); see also, U.S. Patent Nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,466,917; 4,472,500;
  • Panels of monoclonal antibodies produced against AhpC or AhpF epitopes can be screened for various properties; i.e., for isotype, epitope affinity, etc.
  • Antibodies both monoclonal and polyclonal, which are directed against AhpCF epitopes are particularly useful in diagnosis, and those which are neutralizing may be useful in passive immunotherapy.
  • Monoclonal antibodies in particular, may be used to raise anti-idiotype antibodies.
  • Anti-idiotype antibodies are immunoglobulins which carry an "internal image" of the antigen of the infectious agent against which protection is desired. See, for example, Nisonoff, A., et al. (1981) and Dreesman et al. (1985). Techniques for raising anti-idiotype antibodies are known in the art. See, for example, Grzych (1985), MacNamara et al. (1984), and Uytdehaag et al. (1985). These anti-idiotype antibodies may also be useful for treatment, vaccination and/or diagnosis of mycobacterial infections, as well as for an elucidation of the immunogenic regions of AhpCF antigens.
  • AhpCF polypeptides are useful in immunoassays to detect the presence of anti ⁇ bodies to mycobacteria.
  • Anti-AhpCF antibodies are useful in immunoassays to detect the presence of AhpCF antigens.
  • relatively high levels of AhpC would indicate that a strain of mycobacteria in a biological sample has a highly active ahpCF operon, and is probably INH resistant.
  • Protocols may be based, for example, upon competition, direct reaction, or sandwich type assays. Protocols may also, for example, use solid supports, or involve immunoprecipitation.
  • An immunoassay will involve the interaction between at least one epitope derived from either AhpC or AhpF, and an antibody specific for the epitope. A first component of this interaction (the epitope or the antibody) will be supplied as a reagent, the other component (the antibody or the epitope, respectively) will be supplied either as a control sample, or as a biological sample in which the level of the component is to be determined.
  • the formation of the complex between antibody and epitope is detected by many techniques known in the art, including precipitation and nephelometry. Often, the complex is detected by supplying the reagent with a label, either before or after the complexing reaction.
  • the labels may be, for example, enzymatic, fluorescent, chemiluminescent, radioactive, or dye molecules. As- says which amplify the signals from the probe are also known; examples of which are assays which utilize biotin and avidin, and enzyme-labeled and mediated immunoassays, such as ELISA assays.
  • the label may be attached directly to the reagent, or supplied through a reaction with a second reagent, such as anti-immunoglobulin or protein A.
  • an immunoassay for an anti-AhpCF antibody(s) will involve selecting and preparing the test sample suspected of containing the antibodies, such as a biological sample, then incubating it with an antigenic (i.e., epitope-containing) AhpCF polypeptide(s) under conditions that allow antigen-antibody complexes to form, and then detecting the formation of such complexes.
  • the polypeptide component may comprise a single epitope or a combination of epitopes derived from AhpC and or AhpF.
  • the epitopes may be natural isolates, or provided in separate recombinant polypeptides, or together in the same recombinant polypeptides. Suitable incubation conditions are well known in the art.
  • the immunoassay may be, without limitations, in a heterogenous or in a homogeneous format, and of a standard or competitive type.
  • the polypeptide is typically bound to a solid support to facilitate separation of the sample from the polypeptide after incubation.
  • solid supports that can be used are nitrocellulose (e.g., in membrane or microtiter well form), polyvinyl chloride (e.g., in sheets or microtiter wells), polystyrene latex (e.g., in beads or microtiter plates, polyvinylidine fluoride (known as Immulon), diazotized paper, nylon membranes, activated beads, and Protein A beads.
  • Dynatech IMMULON 1 or IMMULON 2 (TM) microtiter plates, or 0.25 inch polystyrene beads (Precision Plastic Ball) can be used in the heterogeneous format.
  • the solid support containing the antigenic polypeptide is typically washed after separating it from the test sample, and prior to detection of bound antibodies. Both standard and competitive formats are known in the art.
  • Complexes formed comprising anti-AhpCF antibody are detected by any of a number of known techniques, depending on the format.
  • unlabeled anti-AhpCF antibodies in the complex may be detected using a conjugate of antixenogeneic Ig complexed with a label, (e.g., an enzyme label).
  • the test sample typically a biological sample
  • anti-AhpCF antibodies under conditions that allow the formation of antigen-antibody complexes.
  • the antibody component may be, for example, a monoclonal antibody directed towards an AhpCF epitope(s), a combination of monoclonal antibodies directed towards epitopes of one mycobacterial antigen, monoclonal antibodies directed towards epitopes of different mycobacterial antigens, polyclonal antibodies directed towards the same antigen, or polyclonal antibodies directed towards different antigens. It may be desirable to treat the biological sample to release putative bacterial components prior to testing.
  • a "sandwich assay" may be employed, where antibody bound to a solid support is incubated with the test sample; washed; incubated with a second, labeled antibody to the analyte, and the support is washed again. Analyte is detected by determining if the second antibody is bound to the support.
  • a competitive format which can be either heterogeneous or homogeneous, a test sample is usually incubated with antibody and a labeled, competing antigen is also incubated, either sequentially or simultaneously.
  • M. bovis ATCC 35729 and M. bovis ATCC 35728 were selected for study. Both strains were shown to be catalase negative and not to have the inhA mutation that characterized another INH resistant M. bovis strain (Banerjee et al. Science 263:227, 1994).
  • Plasmid pYUB 18 is a multicopy E. coli-mycobacteria shuttle cosmid that contains a selectable kanamycin gene and a cos site (J.T. Beslile et al., J. Bacteriol. 173:6991 (1991); S.B. Snapper et al., Mol. Microbiol. 4:1911 (1990); W.R. Jacobs et al., Methods Enzymol. 204:537 (1991)).
  • a restriction enzyme map of pYUB18 showing some significant features of the genome is shown in Figure 5.
  • the cosmid library was prepared as follows using standard techniques. Chromosomal DNA was purified from ATCC 35729, and subjected to partial digestion with Sau3Al: fragments between about 30-50 kb were purified by sucrose gradient purification and ligated to linearized pYUB18. Resulting cosmids were packaged into S- phage using a commercial kit (GIGAPACK GOLD (TM), Stratagene) according to the manufacturer's directions, and transfected into E. coli: approximately 10,000 colonies were obtained. The colonies were pooled and the plasmids amplified, using standard plasmid preparation techniques.
  • TM GAPACK GOLD
  • the cosmid library was then transformed into M. smegmatis strain mc 155 by electroporation. Transformants were selected by growth on medium containing kanamycin. Approximately 2000 kanamycin resistant clones were patched onto media containing 25 Tg/mL INH. Twelve INH resistant clones were identified. The first ORF is preceded by putative -10 and -35 promoter regions, indicated in
  • FIG. 2 by single underlining.
  • the potential ribosome binding site is indicated in Figure 2 by double underlining.
  • This DNA sequence of the ahpCF operon of ATCC 35729 has been submitted to GenBank, and is assigned the accession number U24084.
  • cosmids were extracted from the transformants.
  • Cultures of M. smegmatis (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% sodium dodecylsulfate (SDS)) and the solution was mixed for 30 minutes.
  • SDS sodium dodecylsulfate
  • the sample was heated to 600C for 20 minutes, cooled for 10 minutes and the DNA was extracted by adding 0.8 ml phenol (containing 50 mM NaCl). This was centrifuged and the upper layer containing the DNA was removed. To precipitate the DNA, a half volume of 7.5 M ammonium acetate was added, incubated on ice for 30 minutes and then centrifuged for 30 minutes. The DNA was resuspended in 10 mM Tris, 1 mM EDTA. Southern blot hybridization with a probe of the inhA gene of restriction digests of the cosmids revealed that only one of the cosmids contained the inhA gene.
  • a library of 2-4 kb partial Sau3AI fragments of one (pUHA210) of the remaining 11 cosmids that did not contain inhA was subcloned into pYUB 18.
  • a plasmid, pUHA211 , conferring INH resistance on M. smegmatis was selected and isolated in the same way as its parent cosmid pUHA210.
  • the sequence of the insert in pUHA211 was obtained as follows.
  • the insert in pUHA211 was cloned into the vector pBLUESCRIPT II KS+ (TM) (Stratagene, California) to form pUHA212.
  • This vector contains the T3 and T7 promoters which were used for the sequencing.
  • Sequencing of pUHA212 was carried out using the dsDN A • cycle sequencing system from GIBCO BRL, Life Technologies, according to the manufacturer's directions.
  • the radioactive labelled nucleotide was [K- 3 P] ATP, available from Amersham.
  • the sequencing program used was, GCG Sequence Analysis Software Package.
  • the nucleic acid sequence for the insert in pUHA212 and the amino acid sequence from two large open reading frames encompassed within it are shown in Figure 2.
  • the AhpC polypeptide predicted from the M. bovis ahpC gene sequence shows homology with a number of thiol-specific antioxidant enzymes from bacteria, yeast, and human. AhpC and AhpF in E. coli and S. typhimurium have been proposed to be involved in the cellular response to oxidative stress. In M. smegmatis. the effect of hydrogen peroxide on INH-susceptibility suggests these genes may be involved in INH resistance.
  • Figure 3 provides a comparison of the AhpC from M. bovis ATCC 35729 with related sequences obtained from GenBank, and aligned using the computer algorithm PILEUP.
  • the thiol-specific antioxidant enzymes are sometimes referred to as "protector" proteins.
  • M. bovis AhpC has 33% amino acid sequence identity to the AhpC component of alkyl hydroperoxide reductase, an E. coli enzyme which is a heterodimer of AhpC and AhpF chains.
  • M. bovis AhpC also has 65% identity to the gene product ofdirp22 (dirA).
  • DirA iron-repressible genes from Corynebacterium diptheriae
  • Other related proteins are AhpC from S. typhimurium.
  • AhpC from C. pasteurianum.
  • TSA from cerevisiae. and PAG from H. sapiens.
  • the thiol-specific antioxidant of S. cerevisiae exists as a homodimer of 25 kDa subunits, each containing two cysteine residues: Cys-47 and Cys-170.
  • M. bovis AhpC has two cysteine residues in equivalent positions.
  • INH and peroxidases form intermediates like carbonyl, peroxy and isonicotinyl radicals (Shoeb et al., Antimicrob. Ag. Chemother 27:404, 1985) and this oxidation process has been proposed as a mechanism for the mode of action of INH (Johnsson and Schulz, 1994).
  • AhpC may act as a protector protein to confer INH resistance.
  • the predicted M. bovis AhpF has only a low level of homology with AhpF from other organisms.
  • Figure 4 shows a best-fit comparision of the protein encoded in ORF2 (top) with the AhpF from E. coli (below). Vertical lines indicated identity; dots between the sequences indicate similarity; asterisks indicate residues that are 10 amino acids apart.
  • the M. bovis AhpF has a Cys-X-X-Cys (SEQ ID NO:24) motif (underlined), which is characteristic of a family of disulphide oxidoreductases such as thioredoxin, disulphide isomerase, and DsbA, a periplasmic protein required for disulfide bond formation in E. coli.
  • DNA was harvested from M. bovis ATCC 35723 which is the INH-sensitive parent strain from which M. bovis ATCC 35729 was derived. This DNA was digested with the two restriction enzymes NotI and Bglll and the resulting fragments were cloned into pBluescript II KS+. A clone containing a plasmid which incorporated the ahpCF operon was identified by colony hybridization with a probe of part of the insert of pUHA212. The wild type ahpCF operon incorporated in this plasmid, denoted pUHA313, was sequenced in a similar manner to the pUHA212 insert. The DNA sequence of the ahpCF operon of ATCC 35723 (the INH sensitive parent) is shown in Figure 6 (SEQ ID NO: 14). The sequence has been submitted to GenBank, and is assigned the accession number U24083.
  • the panel comprised 5 strains of M. bovis: ATCC 35729, the INH resistant strain characterized in Example 3; ATCC 35723, the INH sensitive strain from which it was derived; ATCC 35728, another INH resistant strain derived from the same parent; WAg201 , a different INH sensitive parent; and WAg405, an INH resistant strain derived therefrom.
  • the panel also comprised 2 strains of M. tuberculosis: ATCC 27294, an INH sensitive parent strain; and ATCC 35822, an INH resistant descendant strain of ATCC 27294.
  • Catalase activity of an INH-sensitive strain of M. bovis was determined.
  • the enzyme was first isolated from the strain by pelleting a culture of M. bovis. resuspending it in 50 mM potassium phosphate buffer, pH 7, and adding it to a tube containing 0.5 g zirconium beads (Biospecs products), and vortexing for 5 min. The sample was centrifuged briefly, the supernatant collected and diluted to 4 ml with 50 mM potassium phosphate buffer, and filter sterilized through 0.22 Tm filters. Catalase catalyzes the conversion ofH 2 O 2 to H 2 O and O 2 .
  • Catalase activity was assayed by incubating an aliquot of supernatant, prepared as above, with 3 Tm H 2 O 2 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 TiCl 4 in 4.5 M H 2 SO 4 ). The absorbance was read at 410 nm and the catalase activity was calculated using a standard curve of the amount of hydrogen peroxide versus wavelength at 410 nm; the activity was expressed as Tmol/min/mg protein.
  • Catalase activity was detected in all strains that were sensitive to INH, but none of the strains that were resistant to INH. There is an approximate correlation between catalase activity and the virulence of the strain. However, certain strains (notably ATCC 35728) can be virulent even in the absence of detectable catalase activity.
  • DNA preparations from different M. bovis strains were digested with restriction nucleases and subjected to Southern analysis by standard techniques, using a 4.5 kb polynucleotide probe for the katG gene.
  • DNA preparations from different M. bovis strains were digested with restriction nucleases and subjected to Southern analysis by standard techniques, using a 4.5 kb polynucleotide probe for the katG gene.
  • a katG gene encoding functional catalase activity was integrated back into the genomes of ATCC 25728 and ATCC 35729, using similar techniques to those outlined elsewhere in this disclosure. As a result, INH resistance was reduced from 64 Tg/ml to 1 Tg/ml and 0.5 Tg/ml, respectively.
  • mutation of the inhA gene can also confer INH resistance, it was important to establish the status of inhA in the strains analyzed herein. Mutations that confer INH resistance have been mapped to two well-separated nucleotides in the coding region of inhA (Banerjee et al., Science 263:227, 1994), and also to the promoter region (Kapur et al., Arch. Pathol. Lab. Med. 119:131, 1995). Therefore, the relevant regions were retrieved from each of the mycobacteria strains of the panel, and the nucleotide sequence was determined.
  • the promoter region of inhA was amplified using primer 5'- CAGCGCTTTTGCACGCA (SEQ ID NO:25) and 5'-CCAGGACTGAACGGGAT (SEQ ID NO:26) to produce a 350 bp fragment and the PCR product cloned using TA cloning kit (Invitrogen). Both strands were sequenced by cycle sequencing with the amplification primers.
  • Results are shown in Table 2.
  • the sequence of the inhA promoter was compared to that of WAg201, and if identical is denoted in the Table as wild type (W.T.).
  • the coding region of inhA was sequenced near the previously reported mutations at positions 16 and 94 of the protein.
  • the presence of any mutation affecting position 16 was determined using primers 5'-GCGAGCTATATCTCCGG (SEQ ID NO:27) and 5'- CATGATCGGCAGCAGCG (SEQ ID NO:28).
  • the presence of any mutation affecting position 94 was determined using primer 5'-TCAGCGCATCACCGACC (SEQ ID NO:29) and 5'-CATGATCGGCAGCAGCG (SEQ ID NO:28).
  • the PCR products were cloned and sequences as described above. Sequences identical to WAg201 are denoted as W.T.
  • primers were designed to amplify a 200 bp region of the promoter upstream from the start codon.
  • the promoter region was amplified using primers 5'-GCAACGTCGACTGGCTC (SEQ ID NO:30) and 5'-CGGTCCTCGAACTCGTC (SEQ ID NO:31).
  • the PCR product was cloned into pKSl l+ using the TA CLONING KIT (TM) (Invitrogen), and both strands of the promoter region were sequenced using the amplification primers and an additional primer 5'-GTGGCATGACTCTCCTC (SEQ ID NO:32). Nucleotide sequences near the previously determined mutation site of ATCC
  • Primer extension analysis revealed that the mutation in position 72 occurs at the transcriptional start site.
  • Total RNS was isolated from a pelleted culture of M. smegmatis which had been transformed with the integrating vector pYUB178::aphC using TRIzolTM (Gibco/BRL) and the manufacturer's protocol.
  • Primer extension analysis was performed as described by Levin and Hatfull (1993) except that the annealing conditions used were 30°C for 16 hours and the reverse transcriptase temperature used was 50°C for 2 hours. Results are shown in Figure 9.
  • H37Rv (ATCC 27294) GGCACGATGG AATGTCGCAA (SEQ ID NO:33)
  • the 2.1 kb insert of ATCC 35729 was treated with restriction nucleases NotI (N), EcoRI (E), P_sjl (P), and figJII (B), producing the fragments shown in Figure 8.
  • N restriction nucleases NotI
  • E EcoRI
  • P P_sjl
  • B figJII
  • the object of the next experiment was to determine the effect of the mutation in the ahpCF operon of mycobacteria strains on the function of the promoter region.
  • the strains tested were ATCC 35723 (wild type, INH sensitive), ATCC 35729 (one mutation, INH resistant), and ATCC 35728 (two mutations, INH resistant).
  • Primers 5'-CTCGGATCCACTGCTGAACCACTGCTT-3' (SEQ ID NO:36) and 5'-CTCGGATCCGACTCTCCTCATCATCAA-3' (SEQ ID NO:37) were used to amplify the promoter region of the ahpC gene from different strains.
  • the amplified product was digested with BamHI and ligated into the BamHI cloning site ofpYUB76. This is a mycobacterial shuttle vector with a promoterless reporter gene, lacZ. immediately downstream of the cloning site.
  • the vector constructs were electroporated into E. coli and selected on media containing 25 Tg/ml kanamycin and 40 Tg/ml X-Gal.
  • Colonies containing the wild-type promoter were colorless, while the colonies containing a mutated promoter were pale blue. All three constructs were sequenced to verify that no errors had been introduced by the amplification procedure. The constructs
  • Results are shown in Table 4. The values shown are the mean ⁇ standard deviation for three independent experiments.
  • the ahpC promoter with a single mutation (ATCC 35729) showed a six-fold higher level of activity than the wild type.
  • the ahp£ • promoter with two mutations (ATCC 35822) showed a 10-fold higher level of activity.

Abstract

Les modes de réalisation de l'invention sont basés sur l'identification et la caractérisation d'un opéron déterminant la résistance mycobactérienne à l'antibiotique isoniaside (INH) et ses analogues. Cet opéron, appelé ahpCF, code des polypeptides, AhpC et AhpF, lesquels sont susceptibles de se combiner pour former une réductase d'hydropéroxyde d'alkyle active pouvant être soit une cible directe pour l'INH soit agir pour conférer une résistance à l'INH. L'invention concerne également la séquence de l'opéron ahpCF mutant illustrant que la résistance à l'INH peut être conférée par une mutation dans la région promotrice. L'invention concerne également des polynucléotides ainsi que des polypeptides utiles dans le diagnostic et le traitement.
PCT/NZ1995/000133 1994-12-20 1995-12-20 PROCEDES ET COMPOSITIONS DE DETECTION ET DE TRAITEMENT D'INFECTIONS MYCOBACTERIENNES A L'AIDE D'UN OPERON ahpCF WO1996019578A2 (fr)

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