EP0907751A2

EP0907751A2 - Dna, rna and a protein useful for detection of a mycobacterial infection

Info

Publication number: EP0907751A2
Application number: EP97921666A
Authority: EP
Inventors: Mahavir Dr. Singh; Christiane Dipl.-Chem. Honisch; Clara Dr. Espitia; Carlos Dr. Moreno
Original assignee: Helmholtz Zentrum fuer Infektionsforschung HZI GmbH
Current assignee: Helmholtz Zentrum fuer Infektionsforschung HZI GmbH
Priority date: 1996-04-29
Filing date: 1997-04-18
Publication date: 1999-04-14
Also published as: WO1997041252A3; JP2000509981A; CA2252542A1; WO1997041252A2

Abstract

The invention concerns a DNA, RNA and a protein useful for identifying and combating mycobacterial infections.

Description

DNA, RNA AND A PROTEIN USEFUL FOR DETECTION OF A MYCOBACTERIAL INFECTION

Description Technical field The invention is in the field of clinical medicine, molecular biology and genetic engineering. More particularly, it relates to the molecular methods of tuberculosis diagnosis using newly identified DNA sequences which can be used as probes for DNA hybridization and or for DNA amplification leading to the identification of pathogenic mycobacteria causing disease in humans and animals.

Background

Tuberculosis, an infectious disease mainly caused by respiratory infection with Mycobacterium tuberculosis, represents an important subject of multidisciplinary investigation owing to the urgent need for rapid and reliable diagnostic tests and effective vaccines for disease control.

An estimated 8 million persons are developing tuberculosis each year and this number will be rising for the foreseeable future. Especially immuno- comprimised people, e.g. Human Immunodeficiency Virus-infected individuals (Selwyn et al., 1989; Barnes et al., 1991) and the population of countries with insufficient public health systems (Grzybowski, 1991 ; Kochi, 1991) are the most endangered groups of this "global disease" (WHO, 1992). Emergence of multiple drug resistant strains is posing major threat to human health not only in developing countries, but also in developed countries. A rapid and specific diagnosis of tuberculosis is still a problem.

One approach to address this problem is to use the specific humoral or cellular response of the host to infer the presence of disease. Mycobacteria are rich in antigens that stimulate the production of antibodies and serology is simple and readily applicable as a rapid diagnostic test (Wilkins, 1994). Unfortunately the usefulness of serological tests are often limited by their lack of specificity and by their inability to destinguish between active disease, prior sensitization by contact with M. tuberculosis or cross-sensitization to other mycobacteria. Another means of achieving the correct diagnosis are to develop increasingly sensitve methods to detect the causative bacilli or their products. Such techniques include amplification of a defined region of bacterial DNA via polymersase chain reaction (PCR) (Shankar et al., 1991), immunoassays for detecting antigen, gas liquid chromatography and mass spectrometry for detecting specific mycobacterial lipids. Of these, PCR is being evaluated most intensely and appears to hold greatest promise.

Attempts have been made to develop methods for the detection of chromosomal DNA of the M. tuberculosis complex in patient's sputum (Glennon, 1994). While the possibility of developing a DNA probe to distinguish between the M. tuberculosis complex and other mycobacterial strains has been reported, strain differentiation within the individual members of the complex is still a problem.

In this study we report the isolation of novel genomic clones containing as yet unreported genes and DNA, and the identification of novel M. tuberculosis chromosomal DNA regions specific for species of the M. tuberculosis complex. In addition, amplification of (i) a 377 bp fragment specific for the M. tuberculosis complex and (ii) of a 380-bp fragment showing sequence similarities with the genome of Mycobacterium asiaticum, Mycobacterium gastri, Mycobacterium gordonae and Mycobacterium kansasii are described. The utility of the 377-bp and the 380-bp fragment for the differentation of species and strains of mycobacteria is reported. In addition to other ORF identified in this study, a novel ca. 15kDa recombinant protein showing high homology to a family of transposase was overproduced in Eschericha coli as a thioredoxin fusion and purified. The ca. 15kDa and ca. 3 lkDa proteins described in this study are different from the 35 kDa ORF belonging to an insertion element identified by Mariani et al. (1993). Disclosure of invention

The present invention is based on novel DNA sequences cloned from the genome of Mycobacterium tuberculosis, which can be used for strain differentiation and for the diagnosis of tuberculosis.

Accordingly, the DNA sequences of the cloned fragments is an aspect of the invention.

The cloned DNA fragments are found to code for at least 7 proteins of about 9kDa, 15kDa, 17kDa,31kDa, 55kDa, 74kDa and 77kDa, the sequences of which are another aspect of the invention.

The use of the DNA sequence for detecting specific fragments by hybridization or by DNA amplification is another aspect of the invention.

The use of the cloned DNA or of the proteins coded by the cloned DNA for the purpose of serology, skin testing, vaccine development or drug design is another aspect of the invention.

The object underlaying the invention is solved by the following three main embodiments with their preferred embodiments.

According to a first embodiment the invention concerns a DNA

(a) having sequence (I) according to figure 9, wherein optionally one or more condons can be replaced by condons coding for the same amino acid(s) ,

(b) having a sequence complementary to said of (a) ,

(c) being single-stranded, wherein its strand is hybridizable with that of the DNA according to (a) or (b) ,

(d) being double-stranded, the sequences of its single strands being defined as in (a) and (b) , respectively,

(e) being double-stranded, its single strands being hybridizable with those of the DNA according to (d) , or

(f) being a subsequence of the sequences according to (a) to (e) .

Further the invention concerns a DNA according to (c) or (e) , its single strands being hybridizable with those of the DNA according to (a) , (b) , (d) and (f) , respectively, at a temperature of at least 25 °C and at a concentration of NaCl of 1 M.

Further the invention concerns a RNA being a transcript of a DNA according to the invention.

Further the invention concerns a protein being encoded by a DNA according to the invention.

Further the invention concerns a protein having the amino a. d sequence (II) according to figure 13.

The protein according to the invention can be an about 74 kDa protein. Further the invention concerns a protein having the amino acid sequence (III) according to figure 14.

The protein according to the invention can be an about 77 kDa protein.

Further the invention concerns a protein having the amino acid sequence (IV) according to figure 15.

The protein according to the invention can be an about 9 kDa protein.

Further the invention concerns a protein having the amino acid sequence (V) according to figure 16.

The protein according to the invention can be an about 55 kDa protein.

The protein according to the invention can be a recombinant protein, especially a protein produced by means of a bacterial strain, a yeast strain, a fungal strain or a cell line of a higher eucaryote.

The protein according to the invention can be encoded by a DNA sequence according to the first embodiment of the invention and can be recovered by a method comprising the following steps: (i) subjecting proteins encoded by said DNA sequence to a usual test for diagnosis of tuberculosis,

(ii) selecting a protein showing an inhibitory effect and (iii) isolating and recovering said protein.

Further the invention concerns a DNA, RNA or protein according to the invention which can be used for

(i) diagnosis of tuberculosis in humans and animals and/or (ii) diagnosis of other mycobacterial infections in humans or animals, each especially by means of samples taken from humans or animals .

Further the invention concerns a use of a DNA according to the invention for the identification of mycobacteria in media samples.

The foregoing use can comprise the steps of (i) isolating the mycobacterium, (ii) preparing crude or purified genomic DNA,

(iii) hybridizing it to a DNA according to the first embodiment of the invention and

(iv) detecting the fragment pattern using conventional methods such as a radioactivity assay, chemiluminiscence or fluorescence.

Further the invention concerns a use, wherein as samples clinical samples are used.

Further the invention concerns a use of a DNA according to the invention or of a protein according to the invention for

(i) epidemeological purposes and/or

(ii) vaccination follow-up for humans or animals suffering from mycobacterial infections, especially tuberculosis.

Further the invention concerns a use of a DNA according to the invention or of a protein according to the invention for the development of drugs useful for combating mycobacterial infections of humans or animals, especially tuberculosis, especially for testing and recovering of substances inhibiting mycobacterial infections in humans and animals, especially tuberculosis. According to a second embodiment the invention concerns a DNA

(a) having sequence (VI) according to figure 2, wherein optionally one or more condons can be replaced by condons coding for the same amino acid(s) ,

(b) having a sequence complementary to said of (a) ,

(f) being a subsequence of the sequences according to (a) to (e) .

Further the invention concerns a DNA according to (c) or (e) , its single strands being hybridizable with those of the DNA according to (a) , (b) , (d) and (f) , respectively, at a temperature of at least 25 °C and at a concentration of NaCl of

1 M.

Further the invention concerns a protein having the amino acid sequence (VII) according to figure 5.

The protein according to the invention can be an about 15 kDa protein.

Further the invention concerns a protein having the amino acid sequence (VIII) according to figure 6. The protein according to the invention can be an about 31 kDa protein.

The protein according to the invention can be a recombinant protein, especially a protein produced by means of a bacterial strain, a yeast strain, a fungal strain or a cell line of a higher eucaryote .

The protein according to the invention can be a encoded by a DNA sequence according to the second embodiment of the invention and can be recovered by a method comprising the following steps: (i) subjecting proteins encoded by said DNA sequence to a usual test for diagnosis of tuberculosis,

(i) diagnosis of tuberculosis in humans and animals and/or

(ii) diagnosis of other mycobacterial infections in humans or animals, each especially by means of samples taken from humans or animals .

Further the invention concerns a use of a DNA according to the invention for the identification of mycobacteria in media samples .

The foregoing use can comprise the steps of

(i) isolating the mycobacterium,

(ii) preparing crude or purified genomic DNA,

(iii) hybridizing it to a DNA according to the second embodiment of the invention and (iv) detecting the fragment pattern using conventional methods such as a radioactivity assay, chemiluminiscence or fluorescence.

(i) epidemeological purposes and/or

Further the invention concerns a use of a DNA according to the invention or of a protein according to the invention for the development of drugs useful for combating mycobacterial infections of humans or animals, especially tuberculosis, especially for testing and recovering of substances inhibiting mycobacterial infections in humans and animals, especially tuberculosis.

According to a third embodiment the invention concerns a DNA

(a) having sequence (IX) according to figure 3, wherein optionally one or more condons can be replaced by condons coding for the same amino acid(s) ,

(b) having a sequence complementary to said of (a) ,

(f) being a subsequence of the sequences according to (a) to (e) .

Further the invention concerns a protein having the amino acid sequence (X) according to figure 7.

The protein according to the invention can be an about 17 kDa protein.

The protein according to the invention can be a recombinant protein, especially a protein produced by means of a bacterial strain, a yeast strain, a fungicidal strain or a cell line of a higher eucaryote.

The protein according to the invention can be encoded by a DNA sequence according to the third embodiment of the invention and can be recovered by a method comprising the following steps: (i) subjecting proteins encoded by said DNA sequence to a usual test for diagnosis of tuberculosis,

(i) diagnosis of tuberculosis in humans and animals and/or

(ii) diagnosis of other mycobacterial infections in humans or animals, each especially by means of samples taken from humans or animals.

The foregoing use can comprise the steps of

(i) isolating the mycobacterium,

(ii) preparing crude or purified genomic DNA,

(iii) hybridizing it to a DNA according to the third embodiment of the invention and

(iv) detecting the fragment pattern using conventional methods such as a radioactivity assay, chemiluminiscence or fluorescence .

(i) epidemeological purposes and/or

Further the invention concerns a use of a DNA according to the invention or of a protein according to the invention for the development of drugs useful for combating mycobacterial infections of humans or animals, especially tuberculosis, especially for testing and recovering of substances inhibiting mycobacterial infections in humans and animals, especially tuberculosis. The invention is explained in detail by the following figures and experimental data.

Fig. 1 shows a restriction endonuclease map of the 7.2 kb M. tuberculosis chromosomal region;

Fig. 2. shows a 2253 bp M. tuberculosis chromosomal region including BamHI, EcoRI and Kpnl restriction sites and oligonucleotides for screening the lambda gt 11 M. tuberculosis library (Primer 1 and Primer 2 underlined) and for amplification of the 377 bp region (377 bp region in bold, Primer 3 and Primer 4 underlined) ; amino acid sequences of the about 15 kDa and the about 31 kDa proteins are shown above the DNA sequences and are marked with arrows (small arrow about 15 kDa ORF 1, strong arrow about 31 kDa ORF 2) ;

Fig. 3. shows a DNA sequence of the 440 bp M. tuberculosis chromosomal region including the 380 bp region (in bold) used in PCR experiments and the amino acid sequence of the ORF 3 shown below the complementary DNA strand (< ORF 3) ;

Fig. 4 is an overview of the isolated lambda gtll-clone C9-2; 7.2 kb insert fragment, sequenced chromosomal regions and ORF 1, ORF 2 and ORF 3 marked with arrows;

Fig. 5 shows the amino acid sequence of the about 15 kDa protein (ORF 1) ;

Fig. 6 shows the amino acid sequence of the about 31 kDa protein (ORF 2) ;

Fig. 7 shows the amino acid sequence of the about 17 kDa protein; Fig. 8 A shows SDS-PAGE of the insoluble pellet fraction (lane 1) and the purified about 15 kDa recombinant antigen (lane 2) ; lane A3 shows protein molecular weight standards (2.850 to 43.000 molecular weight range);

Fig 8 B shows SDS-PAGE of the purified about 15 kDa thioredoxin fusion protein (lane 1) and the two protein bands obtained after enterokinase cleavage (lane 1) ;

Fig. 9 shows a DNA sequence of M. tuberculosis;

Fig. 10 is a schematic drawing of the clone Mtub-Clara-Klon; the open reading frames of about 9 kDa (bp 3536 to bp 3829) , 55 kDa (bp 2111 to bp 3829) , 74 kDa (bp 1538 to bp 3829) and 77 kDa (bp 2698 to bp 2 on the complimentary strand) proteins are shown by arrows and the corresponding coding regions are numbered;

Fig. 11 A shows are southern hybridization with genomic DNA from different mycobacteria digested with PvuII (1: M. tuberculosis H37Rv; 2: M. avium; 3: M. kanssasi ; 4: M. necroti ; 5: M. fortui tum; 6: M. phlei ; 1 : M. smegma tis ; 8: M. vaccae) ;

Fig. 11 B shows a finger-print obtained using the DNA (BamHI digest) of (1) M. tuberculsosis H37 RV, (2) M. tuberculosis H37 Ra, (3) M. bovis BCG, and (4) M. tuberculosis H37Rv digested with Sail;

Fig. 12 shows a finger-print with DNA from different M. tuberculosis clinical isolates (numbered 1 to 12) digested with PvuII restriction enzyme; the 4 kb Sal I fragment (Mtub-Klar- Klon) was used as probe;

Fig. 13 shows an amino acid sequence of the protein of about 74 kDa (molecular weight 74999, length 764)

Fig. 14 shows a glycine rich protein of about 77 kDa (molecular weight 77056, length 899); Fig. 15 shows the amino acid sequence of the about 9 kDa proline rich protein (molecular weight 9356, length 98) ; and

Fig. 16 shows the proline rich protein of about 55 kDa (molecular weight 55982, length 573) .

Modes for Carrying out the invention

We were interested in identifying and cloning novel DNA sequences from the genome of Mycobacterium tuberculosis for use in rapid and specific diagnosis of tuberculosis. Our strategy was to go for new repeated elements and insertion elements which are present only in M.tuberculosis or in the strains of M. tuberculosis complex.

Examples The following examples further describe the isolation and sequencing of M. tuberculosis-ONA containing putative IS-element (Insertion Element) and repeat sequences, e.g., PGRS-elements (Polymorphic GC-Rich-Sequences) and the use of the as yet unreported DNA sequences for strain identification and diagnosis of tuberculosis.

Escherichia coli strains, phages and plasmids: The Escherichia coli K12 strain

Y1090r - (Huynh et al., 1985) was used to propagate the λgtl 1 library and the E. coli K12 strain GI724 (Invitrogen, Leek, The Netherlands) was the host for the production of the ca. 15kDa protein fused to thioredoxin.

The recombinant DNA library of M. tuberculosis genomic DNA in the λgtl 1 ex¬ pression vector was constructed by Young et al. (1985).

The plasmid vector pTrxFus (Invitrogen, Leek, The Netherlands) was used to make an in-frame fusion with thioredoxin as an amino-terminal fusion partner.

Mycobacterial strains and preparation of cell extracts: The mycobacterial strains used in this study are shown in Table 1 (Results and Discussion). All organisms

were grown on Loewenstein medium. For preparing cell extracts a loop of bacteria was suspended in 0.5 ml of 10 mM Tris/base, 1 mM EDTA (pH 7.4) followed by addition of 0.5 ml glass beads (150-212 microns, Sigma, Deisenhofen, Germany). The suspension was incubated at 80°C for 10 min followed by a 1 min treatment in a Mini-Bead Beater (Biospec Products).

DNA sequence analysis: Similarity comparisons were done using the BLAST program (Pearson and Lipman, 1988; NCBI computing facility).

All DNA manupulations were done according to standard procedures (see Maniatis et al. 1982).

DNA sequencing: DNA sequencing analysis was performed by the dideoxynucleotide- chain termination method using a PCR sequencing kit (ABI PRISMTM £>ye Terminator Cycle Sequencing Ready Reaction Kit, Perkin Elmer, Warrington, Great Britain) on a 373A DNA Sequencer (Applied Biosystems, Warrington. Great Britain). DNA sequences were determined for both strands by primer walking.

1. Clone containing putative IS-Element 1 .1 Isolation of the clone C9-2 containing a putative IS element: In our attempt to isolate new mycobacterial insertion elements, a λgtl 1 M. tuberculosis library was screened with oligodeoxyribonucleotide primers based on conserved regions of different insertion elements. The library was screened as described by Young and Davis (1985). Briefly, phage-infected cells of the strain E.coli Y1090r " were plated in top agar on Luria-Bertani plates

(7.0 x 10⁶ PFU per 85 mm plate) and incubated for 6-8 h at 42°C. Nylon membranes (Biodyne B Transfer Membrane, 0.45 μm, Pall, Portsmouth, England) were overlaid on plates. The filters were treated with 0.5 N NaOH, 1.5 M NaCl and the DNA was fixed via UV-crosslinking. Screening was performed using 3'-end labeled oligonucleotides of the sequence 5'- TGACGCGAGTGGGTGTGATTTCG-3' and 5'-GTGGTCGAGCCGTTGATGCCG-3' (Fig.2, PRIMER 1 and PRIMER 2) . Digoxigenin-labeling of the oligodeoxyribonucleotide primers was carried out using a DIG Oligonucleotide 3'-End Labeling Kit (Boehringer Mannheim, Germany). Hybridzation was done at 45°C in hybridization buffer (Boehringer Mannheim, Germany) overnight. Then the membranes were washed under stringent conditions for 5 min twice in 2 x SSC, 0.1% SDS and for 15 min twice at 37°C in 0.1 x SSC, 0.1% SDS. Chemi¬ luminescent detection was carried out with the help of a DIG Luminescent Detection Kit (Boehringer Mannheim, Germany). Plaques were purified by three rounds of plating to obtain single plaques. Phage DNA was isolated using a Nucleobond AX L50 Kit (Machery-Nagel, Dϋren, Germany) and restriction mapping of the selected clone was performed by standard procedures (Maniatis et al., 1982).

Several positive clones were obtained. Detailed analysis of one of the clones (C9-2) is presented here. The recombinant phage was mapped with the restriction endonucleases BamHI, EcoRI and Kpnl (Fig. 1). EcoRI digestion revealed a 7.2 kb DNA insert fragment.

1.2 DNA sequencing of the cloned fragment:

Two M. tuberculosis chromosomal regions of 2253-bp and 440-bp of this fragment were sequenced (Fig.2 and Fig.3). DNA sequencing of the 2253-bp region revealed the presence of a putative insertion element between bp 401 and bp 1378 containing inverted repeats flanked by duplications of 4 base pairs. The cloned fragment reported here is novel and is located at a different position than the 2.1 kb Pstl/EcoRI fragment reported by Mariani et al. (1993), because the DNA sequence of the adjoining regions on the left and the right ends of the putative IS-element were completely different in our clone C9-2 as compared to that reported by Mariani et al. (1993). Fig.4 gives an overview of the 7.2 kb insert fragment and the sequenced chromosomal regions.

1.3 Novel Proteins coded by the cloned DNA:

During the molecular characterization of the clone, novel ORFs were identified. The complete ORF of the ca. 15kDa protein is located on the 2253-bp fragment coded by a 408-bp fragment, corresponding to a coding capacity of 136 amino acids. The ca. 15kDa protein (Fig.5) is a novel product showing limited homology in the N-terminus of a 34kDa ORF reported by Mariani et al. (1993). We also identified an ORF of about 31 kDa (Fig. 2 and Fig. 6) coded by the cloned DNA (bp 515 till bp 1378). This 31kDa ORF did not show any homology in the N-terminus to any known sequence in the database. The C-terminus of the ca. 3 lkDa protein showed homology to a 34kDa ORF (Mariani et al., 1993). We have not used the DNA sequence showing homology to the sequence reported by Mariani et al. (1993) as far as the claims of this patent application are concerned. An ORF (ORF 3, Fig. 3 and Fig.7) on the complementary strand to the 3'-end of the insert fragment of the recombinant λ-clone C9-2 was identified, which had not been reported earlier. This sequence showed homology to a family of transcription regulators in microorganism. In addition, some homology was observed with a putative two-component system mtrA-mtrB isolated from M.tuberculosis H37Rv (Via et al., 1996) and to PhoP of Bacillus subtilis (Lee and Hulett, 1992). Based on this data, the DNA sequence (440-bp fragment, Fig. 3) and the derived polypeptide might play a role in regulation of virulence in mycobacteria.

1.4 Cloning, expression and purification of the ca. 15kDa protein fused to thioredoxin

The λgtl 1 clone C9-2 (Fig. 4) was used as template to amplify a PCR fragment of 951- bp (Fig. 2, sequence position 451-1378) including the ORF for the ca. 15kDa protein (Fig. 5) and cleavage sites for the restriction endonucleases Smal and Sail at the 5¹- and 3'-ends. Amplification of the Smal-Sall mycobacterial DNA fragment for insertion into pTrxFus (Invitrogen. Leek, The Netherlands) was done using the oligonucleotide primers with the sequence 5'-TCTAGACATATGACGCGAGTGGGTGTGATTTCG-3' (PRIMER 7, forward) and 5'-CATATGGTCGACCTAGGGCGTGTCTCCCAA-3' (PRIMER 8, reverse) corresponding to sequence positions 451-474 and 1378-1361 (Fig. 2). Composition of the reaction mix was the same as described above with 400 ng phage DNA as template. The probe was amplified in 30 cycles consisting of the same conditions as described. Cleavage sites were introduced by appropriate primers. After digestion with both restriction endonucleases the product was inserted in pTrxFus (Invitrogen, Leek, The Netherlands) to form the plasmid pCH3-8.

The E. coli strain GI724 was electroporated with the plasmid pCH3-8. Bacterial cultures (200 ml of Induction Medium (Invitrogen, Leek, The Netherlands) supplemented with 100 μg/ml ampicillin) grown at 30°C were induced to synthesize the fusion protein by tryptophan addition (lOOμg/ml) and temperature shift to 37°C. Cells were collected after 4 hours (10 000 x g, 5 min. 4°C), resuspended in 4 ml Osmotic Shock Solution (Invitrogen, Leek, The Netherlands), broken by three rounds of alternate sonication on ice (10 sec.) and shock freezing in liquid nitrogen, and pelleted (10 000 x g, 15 min, 4°C). Most of the fusion protein accumulated in the form of inclusion bodies and only a small fraction was present as soluble protein inside the cells.The pellet containing the inclusion bodies was resuspended (denaturation) in 10 ml 6 M guanidine/HCl (pH 8.5), incubated for 2 hours at room temperature and pelleted again (10 000 x g, 30 min, 4°C). The recombinant fusion protein was refolded by dialysing against 50 mM Tris/HCl (pH 8.0). Anion exchange chromatography was done with the help of a BioCAD perfusion system (Perseptive Biosystems) on a Poros column HQ/M (Perseptive Biosystems).

SUBSTITUTE SHEET (RULE 261 Elution was performed using a linear NaCl gradient (0-1M). The fusion protein concentration was deteπnined with the BioRad Protein Assay Kit (BioRad, Munich, Germany). Purity was assessed by densitometry (Molecular Dynamics, Software Image Quant) and analytical SDS- PAGE and coomassie staining.

The ca. 15kDa protein fused to thioredoxin was refolded as described above. Further purification of the ca. 15kDa protein fused to thioredoxin was carried out by anion exchange chromatography (Fig. 8, A lane 3 and B lanel). After enterokinase cleavage of the purified ca. 15kDa protein fused to thioredoxin two protein bands were detectable on SDS-PAGE (Fig. 8, lane 2). By western blotting with a thioredoxin monoclonal antibody the lower 1 IkDa band was identified to be thioredoxin. The upper band corresponds to the ca. 15kDa recombinant protein of M. tuberculosis. This is the first report of expression and purification of the ca. 15kDa protein of M. tuberculosis in E. coli.

1.4. Species specific diagnosis of mycobacteria : Deprotected and desalted Oligonucleotide primers were obtained from Gibco BRL (Eggenstein, Germany) or Eurogentec (Seraing, Belgium).

The oligodeoxyribonucleotide primers with the sequence 5'-GTCCATGTGCCGCCG CTG-3' (PRIMER 3, forward) and 5'-CTGCGCGGCTCCCGGCA-3' (PRIMER 4, reverse), specific for the DNA regions of the 2253-bp M. tuberculosis chromosomal region shown in Fig. 2 were used in PCR experiments to amplify a 377-bp fragment.

For amplification of a 380-bp fragment from the 440-bp chromosomal fragment, the oligodeoxyribonucleotide primers with the sequences 5'-CGAGGCTGAACGGCT TTG-3' (PRIMER 5, forward) and 5'-TCAACGTCCGCGGCAAGC-3' (PRIMER 6, reverse) corresponding to the DNA region shown in Fig. 3 were used. Amplifications were performed in 0.2 ml Micro Amp Reaction Tubes (Perkin Elmer, Norwalk, Connecticut, USA) in a final volume of 100 μl using a GeneAmp® PCR Kit (Perkin Elmer, Branchburg, New Jersey, USA). Reaction mixtures contained 10 mM Tris/HCl (pH 8.3), 50 mM KCl, 3 mM MgCl2, 200 μM dNTP, 0.1 μM Primer, 30-100 ng chromosomal DNA from mycobacterial cell extracts (Table

1) and 2.5 U AmpliTaq® DNA polymerase. All components of a PCR reaction except for the template are included in the Kit. The reactions were performed using the automated Thermal Cycler Gene Amp PCR System 9600 (Perkin Elmer, Norwalk, Connecticut, USA). The samples were amplified by 40 cycles consisting of denaturation at 96°C for 2 min, annealing of the primers at 25°C for 1 min and primer extension at 72°C for 3 min.

After amplification, 10 μl of each product was electrophoresed in a horizontal 1.5% agarose gel. Gels were precasted using a 1 :10 000 dilution of SYBR Green I stock reagent (Eugene,

Leiden, The Netherlands) in 10 mM Tris/HCl, 1 mM EDTA (pH 8.0).

For DNA sequencing the appropriate 377-bp and 380-bp PCR products from the mycobacterial cell extract samples (Table 1) were purified from an 1.5% agarose gel using a Gel Extraction

Kit (QIAGEN, Hilden, Germany).

1.4.1. The 377-bp region:

The 377-bp region (Fig.2) of the isolated and sequenced 2253-bp M. tuberculosis chromosomal fragment and the 380-bp region (Fig.3) of the identified 440-bp chromosomal fragment were examined for their suitability for strain differentiation (Table 1 ). A PCR-product of the predicted size and a 100% DNA sequence homology in the 377-bp region was detected only in the members of the M. tuberculosis complex. No amplification product was obtained from other mycobacteria (Table 1). Therefore, the PCR primers of the 377-bp region are useful for the rapid discrimination of M. tuberculosis complex (M. tuberculosis, Mycobacterium bovis, Mycobacterium bovis BCG, Mycobacterium africanum and Mycobacterium microti) from other mycobacteria.

1.3.2. The 380-bp region:

A predominant amplification product of correct size of the 380-bp region was obtained from the chromosomal DNA samples of the M. tuberculosis complex including the vaccine strain M. tuberculosis BCG, the tuberculosis isolate Tubl 18 and the mycobacterial species M. asiaticum. M. gastri, M. gordonae and M. kansasii. Thus, this fragment can be used for the identification of above mycobacterial species, since no amplification product was obtained from other mycobacterial species (Table 1).

2. Clone containing PGRS Element 2.1. Cloning of DNA fragment containing PGRS elements:

We screened Lawrist cosmid library of M. tuberculosis DNA using a degenerate oligonucleotide of the sequence 5'-

C/GGCC/GGCC/GGGC/GACC/GGGC/GGGC/GGCCGGCTCC/GGG- 3' which was designed in such a way that it contained GC rich regions as well as it coded for a putative proline rich polypeptide. Colony hybridization using labelled oligonucleotide was performed using standard procedures (Maniatis et al.1982). Filters were prehybridized and probed at 42°C overnight in a solution containing 6xSSC, 1 mM Sodium phosphate, ImM EDTA, 0.05% skimmed milk, 0.5%SDS. Filters were washed twice in 2xSSC;0,3%SDS for 15 min at 65°C. First screeinig yielded six positive clones which were recheked by hybridization with the oligonucleotide. Three clones gave strong signal and restriction mapping of the clones showed identical restriction pattern. Further restriction mapping and Southern hybridization of one of the clones called identified an about 4kb Sail fragment that hybridized strongly to the oligonucleotide.

2.2. DNA sequencing of the cloned fragment: The ca. 4kb Sail fragment was subcloned in pUC19 and the clone was named Mtub-Clara-Klon. Entire insert was sequenced by primer walking method. The DNA sequence is presented in figure 9. There were unusual difficulties in obtaining the sequence of the recobminant clone because of the high GC rich content and due to the presence of unusual repeats.

2.3. Proteins coded by the cloned DNA:

We identified at least 4 ORF (open reading- frames) belonging to a ca. 9kDa. 55kDa, 74kDa and a 77kDa protein (Fig. 10). Interestingly, the amino acid sequence of the 9kDa, 55kDa ,74kDa and the 77kDa proteins didnot show strong homology to any sequences reported so far for Mycobacteria (Genbank and Swissprot Databases) . In addition, the 9kDa, 55kDa and the 74kDa proteins have an unusually high content proline, nevertheless, no strong homology with the known proline rich antigens ( Laqueyrerie et al. 1995; Infect.Immun.63.4003) of mycobacteria was observed. Unexpectedly, the amino acid sequence showed restricted homology to Mucein like proteins from eucaryotes. The 77kDa protein is highly rich in amino acid glycine and may be a cell wall protein of Mycobacterium tuberculosis. Such proteins have not been reported from M. tuberculosis.

2.3. DNA finger-printing: The ca. 4kb Sail fragment was used to probe (Southern hybridization) genomic DNA of different mycobacteria digested by PvuII (Fig. 1 1). The results show that each strain showed a characteristic pattern making the differentiation of M. tuberculosis-Rv, M. tuberculosis-Ra, M. bovis and the M. tuberculosis Erdman strain. The ca. 4kb Sail fragment is also suitable for finger printing of clinical isolates, since hybridization of the probe to the genomic DNA of clinical isolates from tuberculosis patients also yielded strain specific finger print (Fig. 12). No hybridization to the genomic DNA of M. smegmatis, M. vaccae, M. avium. M. chelonie, M. fortituim, M. phlei was observed.

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Table 1 Distribution of the 377-bp sequence and the 380-bp sequence in different mycobacteria

Species Presence of the 377-bp Presence of the 380-bp fragment fragment

M. tuberculosis M. tuberculosis H37Rv + + complex M. tuberculosis H37Ra + +

M. bovis

M. africanum + + M. microti + +

vaccine strain M. bovis BCG

Species Presence of the 377-bp Presence of the 380-bp fragment fragment

clinical tuberculosis Tub 118 isolate

M. asiaticum ro cn

M. avium

M. chelonae

M. flavescens

M. fortuitum

M. parafortuitum

M. gastri +

M. gordonae +

M. intracellularβ

M. kansasii

M. lufu

M. marinum

M. nonchromogenium

Species Presence of the 377-bp Presence of the 380-bp v fragment fragment

M. pergrinum

M. phlei

M. scrofulaceum

M. simiae

M. smegmatis

M.terrae

M. ulcerus

M. vaccae ro cn

M. xenopi

M. thermoresistibile

M. triviale

none Nocardia asteroides tuberculosis Rodococcus equi strains

•

Claims

1 . ( I ) DNA

(b) having a sequence complementary to said of (a) ,

(f) being a subsequence of the sequences according to (a) to (e) ; or

(II) DNA

(b) having a sequence complementary to said of (a) ,

(f) being a subsequence of the sequences according to (a) to (e) ; or

(III) DNA

(a) having sequence (IX) according to figure 3, wherein optionally one or more condons can be replaced by condons coding for the same amino acid(s) , (b) having a sequence complementary to said of (a) ,

(f) being a subsequence of the sequences according to (a) to (e) .

2. A DNA according to claim 1 (I) (c) , (I) (e) , (II) (c) , (II) (e) , (III) (c) or (III) (e) , its single strands being hybridizable at a temperature of at least 25 °C and at a concentration of NaCl of

1 M.

3. RNA being a transcript of a DNA according to claim 1 or 2.

4. Protein being encoded by a DNA according to claim 1 or 2.

5. Protein having the amino acid sequence (II) according to figure 13.

6. An about 74 kDa protein according to claim 4 or 5.

7. Protein having the amino acid sequence (III) according to figure 14.

8. An about 77 kDa protein according to claim 4 or 7.

9. Protein having the amino acid sequence (IV) according to figure 15.

10. An about 9 kDa protein according to claim 4 or 9. W

- 29 -

11. Protein having the amino acid sequence (V) according to figure 16.

12. An about 55 kDa protein according to claim 4 or 11.

13. Protein having the amino acid sequence (VII) according to figure 5.

14. An about 15 kDa protein according to claim 4 or 13.

15. Protein having the amino acid sequence (VIII) according to figure 6.

16. An about 31 kDa protein according to claim 4 or 15.

17. Protein having the amino acid sequence (X) according to figure 7.

18. An about 17 kDa protein according to claim 4 or 17.

19. A protein according to any of claims 4 to 18, wherein the protein is a recombinant protein, especially a protein produced by means of a bacterial strain, a yeast strain, a fungal strain or a cell line of a higher eucaryote.

20. A protein being encoded by a DNA sequence according to claim 1 or 2 and which can be recovered by a method comprising the following steps:

(i) subjecting proteins encoded by said DNA sequence to a usual test for diagnosis of tuberculosis,

21. DNA according to claim 1 or 2, RNA according to claim 3 or protein according to any of claims 4 to 20 which can be used for (i) diagnosis of tuberculosis in humans and animals and/or (ii) diagnosis of other mycobacterial infections in humans or animals, each especially by means of samples taken from humans or animals .

22. Use of a DNA according to any of claims 1, 2 or 21 for the identification of mycobacteria in media samples.

23. Use according to claim 22, comprising the steps of (i) isolating the mycobacterium,

(ii) preparing crude or purified genomic DNA, (iii) hybridizing it to a DNA according to claim 1 or 2 and (iv) detecting the fragment pattern using conventional methods such as a radioactivity assay, chemiluminiscence or fluorescence .

24. Use according to any of claims 22 to 23, wherein as samples clinical samples are used.

25. Use of a DNA according to any of claims 1 to 2 or of a protein according to any of claims 4 to 20 for

(i) epidemeological purposes and/or

26. Use of a DNA according to any of claims 1 to 2 or of a protein according to any of claims 4 to 20 for the development of drugs useful for combating mycobacterial infections of humans or animals, especially tuberculosis, especially for testing and recovering of substances inhibiting mycobacterial infections in humans and animals, especially tuberculosis.