WO1998032862A9 - L-alanine dehydrogenase of mycobacterium marinum - Google Patents

Abstract

Tuberculosis is an infectious disease which kills more than 3 million people every year. Although both a vaccine and various methods of diagnosis and treatment are available the efficacy of these measures is in urgent need of improvement, given that the number of new cases is again on the rise. Research focuses, among other things, on the characterization of antigens secreted in the early stages of the infection as they constitute the first point of contact of the immune system with the pathogen. The 40 KD-antigen discussed in this article is present $I(in vivo) as a hexamer and, despite its high molecular weight and lack of a signal sequence, is present extracellularly after only a few days of growth. Functionally it is an L-alanine dehydrogenase and reacts with the monoclonal antibody HBT-10 directed against this protein. HBT-10 was the first known antibody specific to a protein of M.tuberculosis which did not cross-react with the vaccine strain M.bovis BCG.

Description

 L-ALANINE DEHYDROGENASE OF MYCOBACTERIUM MARINUM

1.5 Task

The 40 kD antigen treated in this paper is in many ways an interesting object for in-depth studies.

The antigen had already been cloned into an expression vector for Escheήchia coli (Konrad & Singh, unpublished). Therefore, the expression and purification of the recombinant protein should be optimized. With a homogeneous protein fraction then the crucial biochemical parameters of the enzyme should be determined. From such data, experience has shown that conclusions can be drawn about the physiological function of an enzyme. The question was whether the hypothetical role of the enzyme in cell wall biosynthesis can be underlined or disproved. In case of refutation, other possible functions should be identified.

In addition, biochemical starting points for a targeted influencing of the enzyme in vivo can result. In this context, again the physiological function is the key point of all efforts. If the antigen plays an essential role in the bacterium, targeted efforts to eliminate the gene or protein could provide opportunities to prevent the tuberculosis pathogen from growing at a defined point. The protein would then be an ideal drug target. If, as postulated (Delforge et al., 1993), the 40 kD antigen also represents a virulence factor, then such activities could influence the natural virulence of the bacterium. Therefore, this point should be checked by different approaches. The ability to discriminate against the strains of M. tuberculosis and M.bovis BCG by means of mAb HBT-10 makes it possible to develop methods that can distinguish an infection from a vaccine. This is not possible with the conventional screening methods, the PPD or the Mantoux test (Bass Jr. et al., 1990, Huebner et al., 1993). By analyzing the distribution of the gene or gene product, the basis should be laid for enabling rational process development for such a test. In addition, it should be investigated whether the presence of a functional enzyme correlates with any other parameters. Particular emphasis was placed on taxonomic and virulent relationships. Certain natural lifestyles or the entry into certain growth phases could also be related to alanine dehydrogenase. These questions should be investigated.

2. Materials and Methods

2y living material

2ΛΛ bacteria

2.1.1.1 E. coli strains

The strain Escherichia coli was used to optimize the expression of the recombinant 40 kD antigen (Table 2.1). In addition, it has already overproduced cloned mycobacterial antigens (Table 2.2).

Tab. 2.1: Used expression strains and their relevant properties

Strain genotype and relevant phenotype origin / reference

E. coli CAG 629 / ac (am) pfto (am) f (am) supC ^ls rpsL ma / (am) Ion C.Gross ΛfpR165-Tn10 (TetR)

E. coli DH5α sυpEAA Δ / acU169 (φ80 lacΣ ΔM15) hsdRM recA Hanahan (1983) endM gyrA96 thiA relM

E. coli TG2 sυpE hsdAδ thiA {lac-proAB) Δ (srf-recA) 306 :: Tn10 (Tet ^R ) Sambrook et al. (1989) F ^' {traD35 proA * lad * / acZM15)

E. coli SURE hsdR mcrA mcrB mvr endA supE44 / Λ / -1 λ-gy / Α96 Stratagene re / A1 lac recB recJ sbcC umuC uvrC (F'proAB / acl ^q Z ΔM15 Tn10 (TetR))

E.CO// BL 321 mc105 nadB ⁺ puή ⁺ Studier (1975)

E.coli N 4830 suo his ilv ga / KΔ8 AchlO pgl (λ ΔBam N ⁺ cl _lsδ57 ΔHI) Gottesman et al. (1980)

E. coli 538 genotype unknown Bayer AG ^~ off. 2.2 (1/2): Producers of mycobacterial antigens and their characteristics It is stated which antigen is produced by the respective strains. The last two columns show the growing conditions (see also 2.5.2).

Strain Origin / Reference (s) Product Antibiotics Induction

E. coli BLZ, (pKAM1301) J. van Embden GST-36 kD antigen, Ap IPTG M.leprae

E. coli BL21 / plys5 J. van Embden 70 kD antigen, Ap + Cm IPTG

(pKAM3601) M.leprae

Eco // CAG629 (pMS9-2) Singh et a /. (1992) 38 kD antigen, Ap heat M. tuberculosis

E. coli CAG629 (p S14-1) Cherayil & Young (1988) 28 kD antigen, Ap heat

Daie & Patki (1990) M.leprae

Singh et al. (Unpublished).

E. coli 15 (pHISK16 Verbon et al., (1992) 16 kD antigen, Ap IPTG

+ pREP4) Vordermeier et al. (1993) M. tuberculosis

E.CO// M1697 V. Mehra His-30 kD antigen, Ap + Km IPTG M. tuberculosis

E.CO./ 1698 V. Mehra His-30 kD Antigen, Ap + Km IPTG M.leprae

E. coli POP (pKA 2101) J. van Embden 70 kD antigen, Ap M. tuberculosis heat

E. coli POP (pR! B1300) Thole ef a /. (1987) 65 kD antigen, Ap heat

<- van Eden et al. (1988) M.bovis BCG

E. coli POP (pZW1003) Mehra et al. (1986) 65 kD antigen, Ap Heat van der Zee et al. (not published) M.leprae

E.CO./TB1 (pKAM1101) di Guan ef a /. (1987) MBP-36 kD antigen, Ap heat Maina ef a /. (1988) M.leprae Thole ef a /. (1990) Tab. 2.2 (2/2): Producers of mycobacterial antigens and their characteristics

It is indicated which antigen is produced by the respective strains. The last two columns show the growing conditions (see also 2.5.2).

Strain Origin / Reference (s) Product Antibiotics Induction

E.CO./TB1 (pKAM4101) J. van Embden MBP-2nd 65kD-Ap Heat Antigen, M.leprae

E.CO./TB21-8/2 Khanolar-Young et al. (1992) MBP-10 kD antigen, Ap IPTG Mehra ef a /. (1992) M. tuberculosis

E. coli TG2 - 50/55 Sal C.Espitia; M. Singh 50/55 kD, large frag., Ap IPTG large M. tuberculosis

2.1.1.2 Mycobacterial strains

Tab. 2.3 (1/3): Mycobacteria used and their origin

Trunk abbreviation Exact name, origin

M.africanum 1 Afr1 M.africanum no.5544, RIV

M.asiaticum 1 Asi1 M.asiaticum 3250, portals

M.avium 1 Avi1 M. avium Myc 3875, serotype 2, RIV

M.bovis 3 Bov3 M.6ows nr.8316, RIV

M.bovis BCG 2 BCG2 M.bovis Copenhagen, Seruminstitut Copenhagen

M.fao ^'s BCG 4 BCG4 M.bovis BCG P ₃ , RIV

M.chelonae 7 Che7 M.chelonei 1490, P.Dirven

M. Flavescens 1 Fla1 M. Flavescens ATCC 14474, RIV

M.fortuitum 11 For11 M.fortuitum ATCC 6841, RIV

M.gastή Gas1 M.gastri ATCC 25220, RIV

M.gordonae 3 Gor3 M.gordonae 8960, Portaals Table 2.3 (2/3): Mycobacteria used and their origin

Trunk abbreviation Exact name, origin

M. Intracellulare 1 Int 1 M. intracellulare 6997, ATCC 15985, Portaals

M. Intracellulare 5 Intra M. intracellularly IWG MT3, RIV

M. kansasii 1 Kan1 M. kansasii Myc 1012, RIV

M.lufu 1 Luf1 M.lufu 2 ^, RIV

M.marinum 3 Mar3 M.marinum L66, Portals

M.microti 1 id M.microti nrA 27 Q, Portals

M.nonchromogenium 1 Non1 M. nonchromogenium ATCC 25145, RIV

M. parafortuitum 1 Paf1 M. parafortuitum nr.6999, Portals

M.pereg num 1 Perl M.peregήnum, Patient Bakker, TB6849, Antonie Ziekenhuis

M.phlei 1 Phl1 M.phlei 258 (Ph), Portals

M.phleiA Phl4 M.phlei Weybήάge R82, Tony Eger

M.crofulaceum 1 Scr1 M.crofulaceum Myc 3442, RIV

M.scrofulaceum 8 Scrδ M.crofulaceum Myc 6572, RIV

M.simiae 1 Sim1 M.simiae 784, Tony Eger

M.smegmatis 1 Sme1 M.smegmatis ATCC 14460, RIV

M.smegmatis 3 Sme3 M.smegmatis 8070, Poriaals

M. Terrae Ter2 M. Terrae, RIV

M.thermoresistibile 1 The1 M.thermoresistibile nr.7001, Poriaals

M. trivial 1 Tri1 M. trivial 8067, Poriaals

M. tuberculosis H37R _V H37R _V M. tuberculosis H37R _V , RiV

M. tuberculosis H37R _a H37R _a M. tuberculosis H37R _a , no.19529, RIV

M. tuberculosis 1Tub1 M.f (7% uterus / s 4514, RIV

M. tuberculosis M. tuberculosis 49 Tub49 C _3, Sang-Hae Cho, Korea

M. tuberculosis 60 Tub60 M. tuberculosis S ₂ , Sang-Hae Cho, South Korea Tab. 2.3 (3/3): Mycobacteria used and their origin

Trunk abbreviation Exact name, origin

M. tuberculosis 118 Tub118 M. tuberculosis Myc 16293, Hannoufi

M. tuberculosis 130 Tub130 M. tuberculosis, psύenl yy, bar code 3J265, Dr. Bijlmer. The Hague

M. tuberculosis 132 Tub132 M. tuberculosis Myc 16770, RIV

M. tuberculosis 145 Tub145 M. tuberculosis 416138N, patient N.Wielaart, Reg.No. 7,796,267, WKZ, Utrecht

M. tuberculosis 146 Tub146 M. tuberculosis, Abdi Hussein

M. tuberculosis 163 Tub163 M. tuberculosis 925, patient isolate No. 32, INH> 1, StrR, RifS, EthS

M. ulcerus 1 Uld M. ulcerus 932, portals

M.vaccae 3 Vac3 M.vaccae ATCC 25950, RIV

M. xenopi 7 Xen7 M. xenopi code 132, patient Alois Necas, H.Kristanpul, Prague

2.1.1.3 Other bacterial strains

Tab. 2.4: Further used bacterial strains

Tribe origin

Listeria monocyiogenes ETUC Andreas Lignau

Listeria innocua Andreas Lignau

Nocardia asteroides 702774 Juul Bruins

Rodococcus equi no. 10P388 VMDC, Utrecht 2.1.2 cell culture

The mice were used macrophages cell line J774. This cell line was originally established from a tumor of a female BALB / c mouse (Ralph & Nakoinz, 1975). J774 is used for phagocytosis assays, IL-1 production, and for a variety of biochemical studies. It has receptors for immunoglobulins and complement. Furthermore, J774 produces lysozyme in large quantities and constitutively secretes IL-1 (Ralph & Nakoinz, 1976; Snyderman et al., 1977). Bacteria are absorbed by phagocytosis. Direct cytolysis of foreign organisms is relatively rare.

2.2 Nucleic acids

2.2.1 Plasmids

pJLA604Not

Fig. 2.1: The plasmid pJLA604Not and its relevant functional sections

This 4.9 kb plasmid, a derivative of pJLA 604 (Schauder et al., 1987), was used as an expression vector (Figure 2.1). The plasmid pJLA604Not (Konrad & Singh, unpublished) differs from pJLA604 in that the Nde \ - -5 -

Removed interface, and instead a Λ / ofl interface was installed. The translational reading frame starts with the ATG codon of the SpΛI site. Transcription starts at lambda promoters P _R and P, but is effectively repressed at temperatures of 28-30 ° C by the cl _ts857 gene product. Induction is achieved by raising the temperature to 42 ° C. At this temperature, the temperature-sensitive lambda repressor becomes inactive and can no longer repress transcription. Transcription ends at the fd terminator. In addition, the vector has the atpE translation initiation region (TIR) of E. coli. This section is very useful for the initiation of translation because it has little disruptive secondary structure and thereby ensures a high expression rate (McCarthy et al., 1986). As a selection marker, the plasmid has the β-lactamase gene coding for ampicillin resistance.

As a negative control plasmid pJLA603 was also used, which is identical to pJLA604 except for a few bases in the cloning site.

pMSK12

Fig. 2.2: The plasmid pMSK12 and its relevant functional sections This is a derivative of the plasmid pJLA604Not, in which between the Sph - and the

The 40kD antigen of Mycobacterium tuberculosis was cloned (Figure 2.2, Konrad & Singh, unpublished).

2.2.2 Oligonucleotides

All oligonucleotides (Table 2.5) were prepared by Ms. Astrid Hans (GBF, Braunschweig) on a 394 DNA / RNA synthesizer (Applied Biosystems). The oligonucleotides were purified with an oligonucleotide purification cartridge (Applied Biosystems).

Tab. 2.5: Oligonucleotides used

Name Sequence Orientation

AlaDH-F1 5'-ATGCGCGTCGGTATTCCG-3 'for ard

AlaDH-F1 + 5'-GCGCGTCGGTATTCCGACCG-3 "forward

AlaDH-F2 5 ^• -GAGACCAAAAACAACGAA-3 ^, forward

AlaDH F4 S forward ^{'-GAATTCCCATCAGCAATCTTGCAGA-S'}

AlaDH-F5 5'-GCCCCGATGAGCGAAGTC-3 'forward

AlaDH-F6 5'-GGGGCCGTCCTGGTGCC-3 'forward

AlaDH-F7 5'-GACGTCGACCTACGCGCTGAC-3 'forward

AlaDH-RI δ'-CTCGGTGAACGGCACCCC-S 'reverse

AlaDH 2 5'-GGCCAGCACGCTGGCGGG-3 'reverse

AlaDH-R3 5'-CACCCGTTCGGACAGTAA-3 'reverse

AlaDH-R4 5'-CGCGGCCGACATCATCGC-3 'reverse

AlaDH-R5 5'-GGCCGACATCATCGCTTCCC-3 'reverse

AlaDH-R6 5'-CGAGACTAATTTGGGTGCCTTGGC-3 'reverse

AlaDH-R7 S'-ATTTGGGTGCCTTGGC-S 'reverse

AlaDH-RM 5'-GGCGGCGAGTCGACCGGC-3 'reverse - 71 -

The localization of the oligos on the AlaDH gene is schematized in Fig. 2.3.

tcgagagggg taatcatgcg cgtcggta "ccgaccgaga ccaaaaacaa cgalttccaa 45 ttccgggtgg ccatcacccc ggccggcg" gcggaactaa cccgtcgtgg ccatgaggtg 105 ctcatccagg caggtgccgg aςagggctcg gctatcaccg acgcgςattt caaggcggca 165 ggcgcgcaac tςgtcggcac cgccgaccag gtςtgggccg acgctgattt attgctcaag 225 ςtcaaagaac cgatagc gc ggaatacggc cgcctgcgac acgggcagat cttgttcacg 285 ttcttgcatt tggccgcgtc acgtgcttgc accgatgcgt tgttggattc cggcaccacg 345 ro OB üüfÜT ^{£ CGAG £ CCGT CC5ga CC} ^ ^C ^ acggcgcac tacccctgc tgecccgatg 405 agcgaag ^ cg cccgtcςact cgccgcccag gttggcgctt accacctgat gcgaacccaa 465 ggggccgcg gtgtgctgat g gcgggςtg cccggcgtcg aaccggccga cgtcgtggtg 525 atcggcgccg gcaccgccgg ctacaacgca gcccgcatcg ccaacggcat gggcgcgacc 585 gttacggttc tagacatcaa catcgacaaa cttcggcaac tcgacgccga gttctgcggc 645 cggatceaea ctcgctac c atccgcctac eagctcgagg g gccgtcaa acgtcccgac 705 c ggtgattg gggccgtcct ggtgccaggc gccaaggcac ccaaattagt ctcgaa tca 765 cttgtcgcgc atatgaaacc aggtgcggta ctggtcgata tagccatcga ccε ggcggc 825 tgtttcga ag gctacacgacc gaccacctac caccacccga cgttcgccgt gcacgcsacg 865 ctgttttact gcgtggcssaa catgcccgc tcggtgccga agacgtcgac ctcscccgctg 945

tcggccgctc gttacgccga gcacacgtcg ggagtaaggg aag'cga gat gtcggccgcg

Fig. 2.3: The ve ⁱ v ^/ ended oligos and their location on the AlaDH gene

2.3 recipes

All of the solutions described under this point were made according to Sambrook et al. (1989).

2.3.1 culture media

LB

10 g Bacto tryptone (Difco), 5 g Bacto yeast extract (Difco), 10 g NaCl to 1000 ml H ₂ 0, pH 7.0, autoclave - 1Ä -

eat

12 g bacto tryptone (Difco), 24 g bacto-yeast extract (Difco), 4 ml glycerol (87%), 2.31 g

KH ₂ P0 ₄ , 12.54 g K ₂ HPO ₄ ad 1000 ml H ₂ 0, the phosphate solutions are separated from the others

Components autoclaved and mixed afterwards

soc

2% Bacto-tryptone (Difco), 0.5% Bacto-yeast extract (Difco), 10mM NaCl, 2.5mM KCl, 10mM MgC- _2, 10mM MgSO ₄₎ 20M Glucose to 1000mL H ₂ 0 , pH 7.0, the glucose is autoclaved separately from the other components and added afterwards

LÖWENSTEIN

Ready-to-use Coletsos Ossein Slanting Tanks (Sanofi

Diagnostics Pasteur).

FEST MEDIA:

For the production of plates (90 mm, Greiner) of the nutrient media described above, the respective formulation 1, 5% agar was added.

ANTIBIOTICS

Antibiotics were added to the liquid media just before use from stock solutions. When making media, the addition was allowed to go until the solution was lukewarm after autoclaving. The antibiotics listed in Tab. 2.6 were used. Tab. 2.6: Antibiotics used and concentrations used

Antibiotic final concentration dissolved in

Ampicillin 100 μg / ml water

Chloramphenicol 20 μg / ml ethanol

Gentamicin 100 μg / ιnl ready for use (Sigma)

Kanamycin 30 μg / ml water

2.3.2 Buffer solutions

L-BUFFER: 50 mM Tris base, 10 mM EDTA, pH 6.8, autoclave

TE: 10 mM Tris base, 1 mM EDTA, pH 7.4, autoclave

TAE: 40 mM Tris-acetate, 1 mM EDTA, pH 8.0, autoclave

TBE: 89 mM Tris base, 89 mM boric acid, 2 mM EDTA, pH 8.0, autoclave

TBS: 50 mM Tris base, 137 mM NaCl, 3 mM KCl, pH 7.4, autoclave

TBS-TWEEN: TBS + 0.05% Tween-20

PBS: 137 mM NaCl, 3 mM KCl, 8 mM Na ₂ HPO ₄ , 2 mM KH ₂ PO ₄ , pH 7.0, autoclave 2.6.4 Alanine Dehydrogenase Assays

2.6.4.1 Quantitative Assay

The qualitative detection of AlaDH is based on a series of redox reactions, according to the following reaction scheme (Inagaki et al., 1986, Andersen et al., 1992):

L-alanine

AlaDH

P ruvat

Fig. 2.4: Principle of the alanine dehydrogenase assay

The violet end product can be seen very well with the naked eye. This assay was used to rapidly screen FPLC fractions and to demonstrate AlaDH activity in native protein gels.

This assay is based on a reaction mixture consisting of 1/2 vol. 0.5 M glycine-KOH, pH 10.2 and 1/8 vol. 0.5 M L-alanine, 6.25 mM NAD ⁺ , 2.4 mM NBT and 0.64 mM PMS.

For analysis of protein fractions, the substrate mix was mixed 1: 1 with the solution to be tested. Native gels were incubated directly in 10 ml of substrate mix after electrophoresis.

A positive reaction can be recognized after 5 minutes at the latest. 2.6.4.2 Semiquantitative Assay

This assay was used to study AlaDH activities in mycobacteria.

The mycobacteria were grown on Löwenstein's medium. Using the loop, bacteria were removed from the slant agar tubes, resuspended in water, and adjusted for turbidity equivalent to a McFarland Standard # 5. For the separation of cell aggregates, the suspensions were treated for 10 min in an ultrasonic bath.

The cells were then mixed 1: 1 with a reaction mixture (see 2.6.4.1) and incubated at RT for 10 min. After centrifugation at 20,000 g for 2 minutes, the absorbance of the supernatant against the blank was measured.

The reference measurement used was an approach to which no L-alanine was added.

An absorbance change of one unit per minute in this test corresponds approximately to an absorbance change of three units per minute in the quantitative assay (measurement at 340 nm, see 2.6.4.3).

2.6.4.3 Quantitative Assay

In this assay, the quantitative change in NADH content at 340 nm was measured directly.

The Standardre ^'action approaches had a volume of 1 ml. The composition is shown in Tab. 2.7. The absorbance was monitored for 10 minutes at 37 ° C and 340 nm. The extinction coefficient ε of NADH at 340 nm is 6.22 × 10 ⁶ cm ² / mol.

The standard approaches were varied to determine the biochemical properties of the enzyme as indicated in the text. Each measuring point shown represents the average of at least two, but usually three, independent measurements. An AlaDH unit is defined as the amount of enzyme that catalyses the formation of 1 μmol NADH in oxidative deamination in one minute.

Tab. 2.7: Composition of the quantitative AlaDH assay

The composition of the reaction mixture for the oxidative deamination is shown on the left, which is shown for the reductive amination on the right.

Oxidative deamination Reductive amination

125 mM glycine KOH, pH 10.2 1 M NH ₄ Cl / NH ₄ OH, pH 7.4

100 mM L-alanine 20 mM pyruvate

1.25 M NAD ⁺ 0.5 mM NADH

2.6.5 Densitometry

Densitometry was used to quantify the expression level of AlaDH. Furthermore, the signals of epitope mapping were quantified and correlated with each other.

The implementation was carried out with a Personal Densitometer (Molecular Dynamics) with the software Image Quant (Molecular Dynamics).

- 1? -

3.5 The spread of alanine dehydrogenase within the mycobacteria

At both gene and protein level, it should next be investigated in which mycobacteria an alanine dehydrogenase is present. Based on the virulence, the question was whether the AlaDH activity correlates with this property.

3.5.1 In vivo AlaDH activity

Since AlaDH activity is the exception rather than the rule in the microbial world, it was interesting to question whether this enzyme is ubiquitous within mycobacteria or whether it is restricted to certain species and strains. As a result, conclusions can be drawn on questions such as:

^" Do 'aDH producing strains have similarities in lifestyle?

Does a particular growth mode or phase induce AlaDH production?

^ How is AlaDH regulated?

Can other metabolic pathways catalyze the AlaDH-catalyzed reaction

replace?

^ What phenotype would AlaDH mutants have to show? All available strains were therefore tested for production of AlaDH activity. The repertoire included a total of 44 mycobacterial strains representing 29 different species. In addition, the two strains Nocardia asteroides and Rhodococcus equi, which are closely related to the mycobacteria, were tested.

In order to be able to compare the activities measured in the test system, all bacterial suspensions were adjusted to a density which corresponds to the turbidity of a McFarland Standard No. 5. The strains were in the late exponential phase at the time of measurement.

In addition to the AlaDH measurement, a measurement was also performed in which the reaction mixture lacked L-alanine. The activity of this approach is a measure of other parallel NAD ⁺ -reducing processes. The difference between this approach and the standard approach corresponds to the net AlaDH activity (ΔAsgs value).

According to the measured activities, the strains studied can be divided into three groups. The first group is that of the strong positive strains (Table 3.11). This group comprises the strains which have an AlaDH activity of more than 0.5 ΔA ₅ 95 units in the test system used.

Table 3.11: Strains with strong-positive AlaDH activity

The performance of this assay is described in 2.6.4.2.

Strain AlaDH activity [ΔA ₅₉₅ ]

M.marinum 3 2,327

M.chelonae 7 1, 842

M.microti 1 0,919

M. tuberculosis H37R _V 0.592 -> 9

The two pathogenic strains M.chelonae and M.marinum, which are also pathogenic strains M.microti and M.tuberculosis H37R _V , the latter a virulent tuberculosis reference strain, were classified as strongly positive.

The second group, that of the moderately positive strains, comprises those with an activity between 0.1 and 0.5 ΔAsgs units (Table 3.3).

Tab.3.12: Strains with moderately positive AlaDH activity

The performance of this assay is described in 2.6.4.2.

Strain AlaDH activity strain AlaDH activity IΔA ₅₉₅ ] [AA ₅₉₅ 1

M. smegmatis 3 0.375 M. tuberculosis 49 0.138

M. ulcerus i 0.369 M. tuberculosis 130 0J18

M.africanum 1 0.287 M.smegmatis 1 0.116

M. tuberculosis 118 0.210 M. tuberculosis 132 0.111

M. tuberculosis 145 0.190 M. tuberculosis 146 0.111

M. Intracellulare 1 0.155 M. tuberculosis 1 0.110

In this group, except M.smegmatis, only pathogenic clinical isolates of M. tuberculosis and other mycobacteria are found. However, both M.smegmatis strains tested also show very high NAD ⁺ -reducing activities in the absence of L-alanine. It is important to mention here that the strain M. smegmatis 1-2c (a derivative of M. smegmatis mc ² 6, Zhang et al., 1991, Garbe et al., 1994, by Dr. Peadar 0 Gaora , St.Mary's Hospital, London), a strain for genetic work in mycobacteria, shows no AlaDH activity but also has high background activity. Finally, in the last group all strains are listed which were found to be negative for AlaDH-activity, ie have an activity of less than OJ ΔA _5S5 units (Table 3J 3).

Table 3.13: strains without AlaDH activity

The performance of this assay is described in 2.6.4.2.

Strain AlaDH activity strain AlaDH activity [ΔA595] [ΔA595]

N. asteroides 1 0.048 M.bovis BCG 4 0.001

M.flavescens 1 0.042 M.terrae 2 0.001

M. tuberculosis H37R _a 0.032 M. tuberculosis 60 0

M.nonchromogenium 1 0,026 M. tuberculosis 163 0

M.fortuitum 11 0.022 M.gastπ 1 0

M. asiaticum 1 0,021 M.gordonae 3 0

M.bovis BCG 2 0,013 M.kansasii 1 0

M.lúfu 1 0.013 M. parafortuitum 1 0

R.equi 1 0.011 M.peπgrinυm 1 0

M.bovis 3 0,010 M.phlei 1 0

M.crofulaceum 1 0.009 M.phlei 4 0

M.intracellulare 5 0,007 M.scrofulaceum 8 0

M.thermosresistibile 1 0.006 M.simiae 1 0

M.avium 1 0,002 M.vaccae 3 0

M. triviale 1 0,002 M.xenopi 7 0

By far the largest group comprises opportunistic and non-pathogenic strains, as well as the two strains related to the mycobacteria, Nocardia asteroides and Rhodococcus equi. Exceptions were two clinical tuberculosis Isolates, as well as the causative agent of bovine Tb, M.bovis, but also the two vaccine strains of M.bovis BCG.

A graphical representation of the AlaDH activities is shown in Fig. 3J6 ordered by phylogenetic aspects.

MΛeπaβ turns ivUariυitυm verv.ε

M.tυbercjlcs! S Co-nplex MAIS comp ^ x Complex S-Smmθ Complex Sl ---

fast slow-growing S-äm-Te

Fig. 3.16: AlaDH activity in the kingdom of mycobacteria

The exact name of the individual strains is given in Tab. 2.3. The data rapidly growing or slowly growing may not be taken strictly, but rather represent a tendency within the groups shown.

In summary, the distribution of AlaDH activity within the world of mycobacteria can be described as follows:

© By far the highest activity is shown by the two fish pathogenic strains M.chelonae and M.marinum. D Within the strains of M. tuberculosis there is a tendency for decreasing virulence to decrease AlaDH activity as well (H37R _V clinical isolates> 37R _a ).

3) All strains classified as positive are virulent. The only exception is M. smegmatis, which is easily distinguished by its high background activity.

© Not all virulent strains are AlaDH positive.

© M. tuberculosis can be differentiated from the vaccine strain M.bovis BCG by AlaDH activity.

3.5.2 The gene for alanine dehydrogenase

3.5.2Λ The first PCR fragments

Now that the AlaDH activities within the different strains had been quantified, the next question was why some strains produce the enzyme but others do not. Also, the extent of expression differs even significantly between closely related species.

The lack of measurable activity can be explained to some extent by the fact that not all strains were in exactly the same growth phase, as it is very difficult to attract all strains in parallel, at the same stage. But the lack of activity could also be one of the reasons why genetic changes affect the expression of the gene. These changes could have occurred in the coding or regulatory area.

In order to verify this fact, the AlaDH gene from various strains was tried to be amplified in whole or in part by PCR. As a primer served this purpose oligonucleotides based on the sequence of M. tuberculosis H37R _V (Andersen et al., 1992, see Section 2.2.2, Table 2.5).

The primer pairs used for the detection of the AlaDH, the expected length of the products in each case and the Aπnea // t? G temperatures of the PCR used are summarized in Table 3.14.

Table 3.14: Primer pairs for the detection of AlaDH in mycobacteria

The sequences of the primers are given in Tab. 2.5.

Name Primer # 1 Primer # 2 Product Temperature

Annabel AlaDH-F1 AlaDH-RM 433 bp 65 ° C

Beatrice AlaDH-F1 AlaDH-R2 1102 bp 45 ° C

Claudette AlaDH-F1 AlaDH-R3 1120 bp 55 ^β C

Dέsirέe AlaDH-F1 AlaDH-R6 1072 bp 45 ^β C

Eleonore AlaDH-F1 + AlsDH-R1 1099 bp 55 ° C

Francoise AlaDH-F1 + AlaDH-R2 1117 bp 50 ° C

Giselle AlaDH-F2 AlaDH-R7 757 bp 35 ° C

Helen AlaDH-F4 AlaDH-RM 1080 bp 55 ° C

Isabelle AIaDH-F4 A! ADH-R6 1050 bp 55 ° C

Jeanette AlaDH-F5 AlaDH-R1 507 bp 45 ° C

Karen AlaDH-F5 AlaDH-R4 834 bp 45 ° C

Laπssa AlaDH-F6 AlaDH-R4 786 bp 55 ° C

Melanie AlaDH-F6 AlaDH-R5 405 bp 55 ° C

The first attempts to detect the gene for the AlaDH in various mycobacterial species was carried out with the primer pair Annabel. The result obtained was somewhat surprising. All strains of M. tuberculosis complex - showed the expected fragment of 433 bp. In addition, an additional fragment of approximately 900 bp had been amplified in all these strains (Fig.3.17).

1 2 3 4 5 6 7 8 10

Fig. 3.17: PCR of different strains with the primer pair Annabel.

Were driven in these ^PCR's are each 40 cycles of the sequence: melting for 2 min at 96 ° C, annealing 2 min at 65 ^C and exlension C 3 min at 72 ° C. The MgCl ₂ concentration was 1.5 mM.

Lane 1: M. tuberculosis H37R _V lane 6: M.bovis BCG 4 lane 2: M. tuberculosis H37R _a Eahn 7: M.africanum 1 lane 3: M. tuberculosis 1 Eahn 8: M.microti 1 lane 4: M. bovis 3 Train 9: M.marinum 3 Train 5: M.bovis BCG 2 Train 10: M.chelonae 7

As it turned out, this second fragment was also part of the AlaDH gene, which was formed by attachment of the primer AlaDH-RM at a more C-terminal point. By increasing the annealing temperature in the PCR from 65 to 69 ° C this second fragment could be suppressed (see Fig. 3.18, lanes 2 and 3).

However, what was actually amazing was the appearance of the amplified fragment in all strains of the M. tuberculosis complex, regardless of the presence of AlaDH activity. In some other strains one or more fragments could be amplified with the primer pair Annabel. However, the amplified bands were usually not particularly strong, so they can be considered as background in view of the 40 cycles of PCR. These are probably weak non-specific reactions. However, it can not be ruled out that due to lack of homology between the different species, the PCR primers could not optimally bind to the target sequence.

The two fish pathogenic strains with strong AlaDH activity, M.marinum and M.chelonae, showed a distinctly different behavior in the PCR with the primer pair Annabel. While M.marinum gave a product of about 540 bp, no fragment was obtained at the selected conditions with the primer pair Annabel at M.chelonae (Figure 3.17, lanes 9 and 10).

3.5.2.2 The AlaDH Gene of the M.tuberculosis Complex

Since the presence of the gene for the AlaDH was now detected in all strains of the M.tuberculosis Complex, the question arose as to how the discrepancy to the measured activities should be explained.

For this reason, larger fragments of the gene were started to be amplified. From M. tuberculosis H37R _V all fragments listed in Table 3.15 could be amplified (part of these fragments is shown in Fig. 3.18). Of the other strains of the M. tuberculosis complex, all the PCR reactions from Table 3.15 that were tried were also positive. However, it was not understood with every strain every reaction.

Fig. 3.18: PCR products of strain M.tuberculosis H37R _V

Dangers were indicated in these PCR's 40 cycles each as shown in Fig. 3J7. The spin-on temperatures, with the exception of lanes 2 and 3, are shown in Table 3J4. The MgC! Concentration of the primer pair Annabel was 1.5 mM, that of all other reactions was 3 mM.

Train 1: KBL Train 7: Giselle Train 2: Annabel, 65 ^e C Train 8: Helen Train 3: Annabel, 69 ^C C Train 9: Isabelle Train 4: Dέsirέe Train 10: Larissa Train 5: Eleonore Train 11: Melanie Train 6 : Francoise Rail 12: KBL

The area amplified by all strains of the M. tuberculosis complex comprises 1260 bp. It contains the complete coding section for the AlaDH, as well as further 75 bp υpstream and 63 bp downstream. This area was completely sequenced by all strains of the M. tuberculosis complex (Fig. 3J 9). Only in the last about 20 bases inaccuracies creep in. The complete remaining region ^', however, is protected by multiple sequencing.

It should be noted that all but three site sequences are completely identical to the published sequence of the AlaDH of the λAA65 clone (Andersen et al, 1992). - *? -

40kD -60 ATCTTGCAGA TTAATCGAAC TTTCTTCACA CTGAAGCGTA CAGTATCGAG AGGGGTAATC - 1

Tubl -60 ATCTTGCAGA TTAATCGAAC TTTCTTCACA CTGAAGCGTA CAGTATCGAG AGGGGTAATC -1

K37Rv -60 ATCTTGCAGA TTAATCGAAC TTTCTTCATA CTGAAGCGTA CAGTATCGAG AGGGGTAATC -

H37Ra-60 ATCTTGCAGA TTAATCGAAC TTTCTTCATA CTGAAGCGTA CAGTATCGAG AGGGGTAATC -1

BCG4 -60 ATCTTGCAGA TTAATCGAAC TTTCTTCACA CTGAAGCGTA CAGTATCGAG AGGGGTAATC - τ_

BCG2 -60 ATCTTGCAGA TTAATCGAAC TTTCTTCACA CTGAAGCGTA CAGTATCGAG AGGGGTAATC -1

Bov3 -60 ATCTTGCAGA TTAATCGAAC TTTCTTCACA CTGAAGCGTA CAGTATCGAG AGGGGTAATC -1

Afrl -60 ATCTTGCAGA TTAATCGAAC TTTCTTCACA CTGAAGCGTA CAGTATCGAG AGGGGTAATC - 1

Kiel -60 ATCTTGCAGA TTATCGAAC TTTCTTCACA CTGAAGCGTA CAGTATCGAG AGGGGTAATC -1

Begin ******

40 D 1 ATGCGCGTCG GTATTCCGAC CGAGACCAAA AACAACGAAT TCCAATTCCG GGTGGCCATC eo

Tbl 1 ATGCGCGTCG GTATTCCGAC CGAGACCAAA AACAACG AA.TTCCG GGTGGCCATC 60

H37Rv 1 ATGCGCGTCG GTATTCCGAC CGAGACCAAA AACAACG AATTCCG GGTGGCCATC 60

H37Ra 1 ATGCGCGTCG GTATTCCGAC CGAGACCAAA AACAACG AATTCCG GGTGGCCATC 60

BCG4 1 ATGCGCGTCG GTATTCCGAC CGAGACCAAA AACAACG AATTCCG GGTGGCCATC 60

BCG2 1 ATGCGCGTCG GTATTCCGAC CGAGACCAAA AACAACG AATTCCG GGTGGCCATC 60

Bov3 1 ATGCGCGTCG GTATTCCGAC CGAGACCAAA AACAACG AATTCCG GGTGGCCATC 60

Afrl 1 ATGCGCGTCG GTATTCCGAC CGAGACCAAA AACAACG AATTCCG GGTGGCCATC 60

Micl 1 ATGCGCGTCG GTATTCCGAC CGAGACCAAA AACAACG AATTCCG GGTGGCCATC 60

40kD 61 ACCCCGGCCG GCGTCGCGGA ACTAΛCCCGT CGTGGCCATG AGGTGCTCAT CCAGGCAGGT 120

Tubl 61 ACCCCGGCCG GCGTCGCGGA ACTAACCCGT CGTGGCCATG AGGTGCTCAT CCAGGCAGGT 120

K37Rv 61 ACCCCGGCCG GCGTCGCGGA ACTAACCCGT CGTGGCCATG AGGTGCTCAT CCAGGCAGGT 120

H37Ra 61 ACCCCGGCCG GCGTCGCGGA ACTAACCCGT CGTGGCCATG AGGTGCTCAT CCAGGCAGGT 120

BCG4 61 ACCCCGGCCG GCGTCGCC-GA ACTAACCCGT CGTGGCCATG AGGTGCTCAT CCAGGCAGGT 120

BCG2 61 ACCCCGGCCG GCGTCGCGGA ACTAACCCGT CGTGGCCATG AGGTGCTCAT CCAGGCAGGT 120

Bov3 61 ACCCCGGCCG GCGTCGCGGA ACTAACCCGT CGTGGCCATG AGGTGCTCAT CCAGGCAGGT 120

Afrl 61 ACCCCGGCCG GCGTCGCC-GA ACTAACCCGT CGTGGCCATG AGGTGCTCAT CCAGGCAGGT 120

Micl 61 ACCCCGGCCG GCGTCGCGGA ACTAACCCGT CGTGGCCATG AGGTGCTCAT CCAGGCAGGT 120

40kD 121 GCCGGAGAGG GCTCC-GCTAT CACCGACGCG GATTTCAAGG CGGCAGGCGC GCAACTGGTC ISO

Tubl 121 GCCGGAGAGG GCTCGGCTAT CACCGACGCG GATTTCAAGG CGGCAGGCGC GCAACTGGTC ιεo

H37Rv 121 GCCGGAGAGG GCTCGGCTAT CACCGACGCG GATTTCAAGG CGGCAGGCGC GCAACTGGTC lεo

H37Ra 121 GCCGGAGAGG GCTCGGCTAT CACCGACGCG GATTTCAAGG CGGCAGGCGC GCAACTGGTC lcO

BCG4 121 GCCGGAGAGG GCTCGGCTAT CACCGACGCG GATTTCAAGG CGGCAGGCGC GCAACTGGTC iso

BCG2 121 GCCGGAGAGG GCTCGGCTAT CACCGACGCG GATTTCAAGG CGGCA.GGCC-C GCAACTGGTC 160

Bov3 121 GCCGGAGAGG GCTCGGCTAT CACCGACGCG GATTTCAAGG CGGCAGGCGC GCAACTGGTC 1E0

Afrl 121 GCCGGAGAGG GCTCGGCTAT CACCGACGCG GATTTCAAGG CGGCAGGCGC GCAACTGGTC 180

Micl ^• 121 GCCGGAGAGG GCTCGGCTAT CACCGACGCG GATTTCAAGG CGGCAGGCGC GCAACTGGTC leo

40kD 181 GGCACCGCCG ACCAGGTGTG C-GCCGACGCT GATTTATTGC TCAAGG7CAA AGAACCGATA 240

Tubl 181 GGCACCGCCG ACCAGGTGTG GGCCGACGCT GATTTATTGC TCAAGGTCAA AGAACCGATA 240

H37Rv 181 GGCACCGCCG ACCAGGTGTG C-GCCGACGCT GATTTATTGC TCAAGGTCAA AGAACCGATA 240

H37Ra 181 GGCACCGCCG ACCAGGTGTG GGCCGACGCT GATTTATTGC TCAAGGTCAA AGAACCGATA 240

BCG4 181 GGCACCGCCG ACCAGGTGTG C-GCCGACGCT GATTTATTGC TCAAGGTCAA AGAACCGATA 240

BCG2 181 GGCACCGCCG ACCAGGTGTG C-GCCGACGCT GATTTATTGC TCAAGGTCAA AGAACCGATA 240

Bov3 181 GGCACCGCCG ACCAGGTGTG GGCCGACGCT GATTTATTGC TCAAGGTCAA AGAACCGATA 240

Afrl 181 GGCACCGCCG ACCAGGTGTG GGCCGACGCT GATTTATTGC TCAAGGTCAA AGAACCGATA 240

Micl 181 GGCACCGCCG ACCAGGTGTG C-GCCGACGCT GATTTATTGC TCAAGGTCAA AGAACCGATA 240

Fig. 3.19 (1/5): Alignment of the AlaDH gene and the flanking regions of different strains of the M.tuberculosis complex

See legend page 101. 40kD 241 GCGGCGGAAT ACGGCCGCCT GCGACACC-GG CAGATCTTGT TCACGTTCTT GCATTTGGCC 300

Tubl 241 GCGGCGGAAT ACGGCCGCCT GCGACACC-GG CAGATCTTGT TCACGTTCTT GCATTTGGCC 3C0

H37Rv 241 GCGGCGGAAT ACGGCCGCCT GCGACACC-GG CAGATCTTGT TCACGTTCTT GCATTTGGCC 300

H37Ra 241 GCGGCGGAAT ACGGCCGCCT GCGACACGGG CAGATCTTGT TCACGTTCTT GCATTTGGCC 300

3CG4 241 GCGGCGGAAT ACGGCCGCCT GCGACACC-GG C-GATCTTGT TCACGTTCTT GCATTTGGCC 300

BCG2 241 GCGGCGGAAT ACGGCCGCCT GCGACACC-GG C-GA.TCTTGT TCACGTTCTT GCATTTGGCC 300

3ov3 2 1 GCGGCGGAAT ACGGCCGCCT GCGACACC-GG C-GATCTTGT TCACGTTCTT GCATTTGGCC 300

Afrl 241 GCGGCGGAAT ACGGCCGCCT GCGACACC-GG CAGATCTTGT TCACGTTCTT GCATTTGGCC 300

Micl 241 GCGGCGGAAT ACGGCCGCCT GCGACACC-GG CAGATCTTGT TCACGTTCTT GCATTTGGCC 300

40kD 301 GCGTCACGTG CTTGCACCGA TGCGTTGTTG GATTCCGGCA CCACGTCAA TGCCTACGAG 360

Tubl 301 GCGTCACGTG CTTGCACCGA TGCGTTGTTG GATTCCGGCA CCACGTCAAT TGCCTACGAG 3S0

H37RV 301 GCGTCACGTG CTTGCACCGA TGCGTTGTTG GATTCCGGCA CCACGTCAAT TGCCTACGAG 360

K37Ra 301 GCGTCACGTG CTTGCACCGA TGCGTTC-TTG GATTCCGGCA CCACGTCAAT TGCCTACGAG 360

BCG4 301 GCGTCACGTG CTTGCACCGA TGCGTTGTTG GATTCCGGCA CCACGTCAAT TGCCTACGAG 360

BCG2 301 GCGTCACGTG CTTGCACCGA TGCGTTGTTG GATTCCGGCA CCACGTCAAT TGCCTACGAG 360

Bov3 301 GCGTCACGTG CTTGCACCGA TGCGTTGTTG GATTCCGGCA CCACGTCAAT TGCCTACGAG 360

Afrl 301 GCGTCACGTG CTTGCACCGA TGCGTTGTTG GATTCCGGCA CCACGTCAAT TGCCTACGAG 360

Micl 301 GCGTCACGTG CTTGCACCGA TGCGTTGTTG GATTCCGGCA CCACGTCAAT TGCCTACGAG 360

40kD 361 ACCGTCCAGA CCGCCGACGG CGCACTACCC CTGCTTGCCC CG TGAGCGA AGTCGCCGGT 420

Tubl 361 ACCGTCCAGA CCGCCGACGG CGCACTACCC CTGCTTGCCC CGATGAGCGA AGTCGCCGGT 420

H37Rv 361 ACCGTCCAGA CCGCCGACGG CGCACTACCC CTGCTTGCCC CGATGAGCGA AGTCGCCGGT 420

H37Ra 361 ACCGTCCAGA CCGCCGACGG CGCACTACCC CTGCTTGCCC CGATGAGCGA AGTCGCCGGT 420

BCG4 361 ACCGTCCAGA CCGCCGACGG CGCACTACCC CTGCTTGCCC CGATGAGCGA AGTCGCCGGT 420

BCG2 361 ACCGTCCAGA CCGCCGAAGG CGCACTACCC CTGCTTGCCC CGATGAGCGA AGTCGCCGGT 420

Bov3 361 ACCGTCCAGA CCGCCGAAGG CGCACTACCC CTGCTTGCCC CGATGAGCGA AGTCGCCGGT 420

Afrl 361 ACCGTCCAGA CCGCCGACGG CGCACTACCC CTGCTTGCCC CGATGAGCGA AGTCGCCGGT 420

Micl 361 ACCGTCCAGA CCGCCGACGG CGCACTACCC CTGCTTGCCC CGATGAGCGA AGTCGCCGGT 420

40kD 421 CGACTCGCCG CCCAGGTTGG CGCTTACCAC CTG.ATGCGAA CCCAAGGGGG CCGCGGTGTG 480

Tubl 421 CGACTCGCCG CCCAGGTTGG CGCTTACCAC CTGATGCGA.A CCCAAGGGGG CCGCGGTGTG 4c0

K37Rv 421 CGACTCGCCG CCCAGGTTGG CGCTTACCAC CTGATGCG.AA CCCAAGGGGG CCGCGGTGTG 480

H37Ra 421 CGACTCGCCG CCCAGGTTGG CGCTTACCAC CTG.ATGCGAA CCCAAGGGGG CCGCGGTGTG 480

ECG4 421 CGACTCGCCG CCCAGGTTGG CGCTTACCAC CTG.ATGCGAA CCCAAGGGGG CCGCGGTGTG 480

BCG2 421 CGACTCGCCG CCCAGGTTGG CGCTTACCAC CTGATC-CC-AA CCCAAGGGGG CCGCGGTGTG 480

3ov3 421 CGACTCGCCG CCCAGGTTGG CGCTTACCAC CTGATGCGAA CCCAAGGGGG CCGCGGTGTG 460

Afrl 421 CGACTCGCCG CCCAGGTTGG CGCTTACCAC CTGATGCGAΛ CCCAAGGGGG CCGCGGTGTG 480

Micl 421 CGACTCGCCG CCCAGGTTGG CGCTTACCAC CTGATGCGAA CCCAAGGGGG CCGCGGTGTG 480

40kD 4SI CTGATGGGCG GGGTGCCCGG CGTCGAACCG GCCGACGTCG TGGTGATCGG CGCCGGCACC 540

Tubl 481 CTGATGGGCG GGGTGCCCGG CGTCGAACCG GCCGACGTCG TGGTGATCGG CGCCGGCACC 540

H37Rv 481 CTGATGGGCG GGGTGCCCGG CGTCGAACCG GCCGACGTCG TGGTGATCGG CGCCGGCACC 540

H37P.a 481 CTGATGGGCG GGGTGCCCGG CGTCGAACCG GCCGACGTCG TGGTGATCGG CGCCGGCACC 540

BCG4 481 CTGATQGGCG GGGTGCCCGG CGTCGAACCG GCCGACGTCG TGGTGATCGG CGCCGGCACC 540

BCG2 481 CTGATGGGCG GGGTGCCCGG CGTCGAACCG GCCGACGTCG TGGTGATCGG CGCCGGCACC 540

3ov3 481 CTGATGGGCG GGGTGCCCGG CGTCGAACCG GCCGACGTCG TGGTGATCGG CGCCGGCACC 540

Afrl 481 CTGATGGGCG GGGTGCCCGG CGTCGAACCG GCCGACGTCG TGGTGATCGG CGCCGGCACC 540

Micl 481 CTGATGGGCG GGGTGCCCGG CGTCGAACCG GCCGACGTCG TGGTGATCGG CGCCGGCACC 540

Fig. 3.19 (2/5): Alignment of the AlaDH gene and the flanking regions of different strains of the M.tuberculosis complex

See legend page 101. 40kD 541 GCCGGCTACA ACGCAGCCCG CA.TCGCCAAC GGCATGGGCG CGACCGTTAC GGTTCTAGAC eco

Tubl Ξ41 GCCGGCTACA ACGCAGCCCG CATC3CCAA.C GGCATGGGCG CGACCGTTAC GGTTCTAGAC 6C0

H37Rv 541 GCCGGCTACA ACGCAGCCCG CA.TCGCCAAC GGCATGGGCG CGACCGTTAC GGTTCTAGAC 6C 0

K37Ra 541 GCCGGCTACA ACGCAGCCCG GGCATGGGCG CGACCGTTAC GGTTCTAGAC eco

BCG4 541 GCCGGCTACA ACGCAGCCCG CA7CGCCAA.C GGCATGGGCG CGACCGTTAC GGTTCTAGAC 600

BCG2 541 GCCGGCTACA ACGCAGCCCG CATCGCCAAC GGCATGGGCG CGACCGTTAC GGTTCTAGAC 600

Eov3 541 GCCGGCTACA ACGCAGCCCG CA.TCGCCAAC GGCATGGGCG CGA.CCGTTAC GGTTCTAGAC 600

Afrl 541 GCCGGCTACA ACGCAGCCCG CA.TCGCCAAC GGCATGGGCG CGACCGTTAC GGTTCTAGAC 600

Micl 541 GCCGGCTACA ACGCAGCCCG CATCGCCAAC GGCATGGGCG CGACCGTTAC GGTTCTAGAC 600

4CkD 601 ATCAACATCG ACAAACTTCG C-CAACTCGAC GCCGAGTTCT GCGGCCGG.AT CCACACTCGC 660

Tubl 601 ATCAACATCG ACAAA.CTTCG GCAACTCGAC GCCGAGTTCT GCGGCCGG.AT CCACACTCGC 660

H37Rv 601 ATCAACATCG ACAAACTTCG GCAACTCGAC GCCGAGTTCT GCGGCCGG.AT CCACACTCGC 660

H37Ra 601 ATCAΛCATCG ACAAACTTCG GCAACTCGAC GCCGAGTTCT GCGGCCGG.AT CCACACTCGC 660

BCG4 601 ATCAACATCG ACAAACTTCG GCAACTCGAC GCCGAGTTCT GCGGCCGG.AT CCACACTCGC 660

BCG2 601 ATCAACATCG ACAAACTTCG GCAACTCGAC GCCGAGTTCT GCGGCCGG.AT CCACACTCGC 660

Bov3 601 ATCAACATCG ACAAACTTCG C-CAACTCGAC GCCGAGTTCT GCGGCCGG.AT CCACACTCGC 660

Afrl 601 ATCAACATCG ACAAACTTCG GCAACTCGAC GCCGAGTTCT GCGGCCGGAT CCACACTCGC 660

Micl 601 ATCAACATCG ACAAACTTCG GCAACTCGAC GCCGAGTTCT GCGGCCGC-AT CCACACTCGC 660

40kD 661 TACTCATCGG CCTACGAGCT CGAC-C-GTGCC GTCAAΛCGTG CCGACCTGGT GATTGGGGCC 720

Tubl 651 TACTCATCGG CCTA.CGAGCT CGAGG TGCC GTCAAACGTG CCGACCTGGT GATTGGGGCC 720

K37Rv 661 TACTCATCGG CCTACGAGCT CG.AC-GGTGCC GTCAAACGTG CCGACCTGGT GATTGGGGCC 720

H37Ra 661 TACTCATCGG CCTA.CGAGCT CGAC-GGTGCC GTCAAACGTG CCGACCTGGT GATTGGGGCC 720

BCG4 661 TACTCATCGG CCTACGAGCT CGAGGGTGCC GTCAAACGTG CCGACCTGGT GATTGGGGCC 720

BCG2 661 TACTCATCGG CCTACGAGCT CGAGGGTGCC GTCAAACGTG CCGACCTGGT GATTGGGGCC 720

Bov3 661 TACTCATCGG CCTACGAGCT CGAGC-GTGCC GTCAAACGTG CCGACCTGGT GATTGGGGCC 720

Afrl 651 TACTCATCGG CCTACGAGCT CGAC-C-GTGCC GTCAAACGTG CCGACCTGGT GATTGGGGCC 720

Micl 661 TACTCATCGG CCTA.CGAGCT CGAGGGTGCC GTCAAACGTG CCGACCTGGT GATTGGGGCC 720

40kD 721 GTCCTGGTGC CAC-GCGCCAA GGCACCCAAA TTA.GTCTCGA ATTCACTTGT CGCGCATATG 750

Tubl 721 GTCCTGGTGC CAGGCGCCAA GGCACCCAAA TTAGTCTCGA ATTCACTTGT CGCGCATATG 780

K37 v 721 GTCCTGGTGC CAC-GCGCCAA GGCACCCAAA TTA.GTCTCGA ATTCACTTGT CGCGCATATG 780

K37Ra 721 GTCCTGGTGC CAGGCGCCAA GGCACCCAAA TTA.GTCTCGA ATTCACTTGT CGCGCATATG 7S0

BCC-4 721 GTCCTGGTGC CAC-GCGCCAA GGCACCCAAA TTA.GTCTCGA ATTCA.CTTGT CGCGCATATG 7S0

BCG2 721 GTCCTGGTGC CAC-GCGCCAA GGCACCCAAA TTAGTCTCGA ATTCACTTGT CGCGCATATG 780

3ov3 721 GTCCTGGTGC CAGGCGCCAA GGCACCCAAA TTAGTCTCGA ATTCACTTGT CGCGCATATG 780

Afrl 721 GTCCTGGTGC CAC-GCGCCAA GGCACCCAAA TTA.GTCTCGA ATTCACTTGT CGCGCATATG 7 = 0

Micl 721 GTCCTGGTGC CAGGCGCCAA GGCACCCAAA TTAGTCTCGA ATTCACTTGT CGCGCATATG 780

40kD 781 AAACCAGGTG CGGTACTGGT C-GATATAGCC ATCGACCAGG GCGGCTGTTT CGAAGGCTCA 840

Tubl 781 AAACCAGC-TG CGGTACTGGT C-GATATAGCC ATCGACCAGG GCGGCTGTTT CGAAGGCTCA 840

H37Rv 781 AAACCAGGTG CGGTACTGGT C-GATATAGCC ATCGACCAGG GCGGCTGTTT CGAAGGCTCA 840

H37? .A 781 AAACCAGGTG CGGTACTGGT C-GATATAGCC ATCGACCAGG GCGGCTGTTT CGAAGGCTCA 840

BCG4 781 AAACCAGGTG CGGTACTGGT GG.ATATAGCC ATCGACCAGG GCGGCTGTTT CGAAGGCTCA 840

BCG2 761 AAACCAGGTG CGGTACTGGT C-GATATAGCC ATCGACCAGG GCGGCTGTTT CGAAGGCTCA 840

Bov3 781 AAACCAGGTG CGGTACTGGT GGATATAGCC ATCGACCAGG GCGGCTGTTT CGAAGGCTCA 840

Afrl 781 AAACCAGGTG CGGTACTGGT GG TATAGCC ATCGACCAGG GCGGCTGTTT CGAAGGCTCA 840

Micl 781 AAA.CCAGG G CGGTACTGGT GG.ATATAGCC ATCGACCAGG GCGGCTGTTT CGAAGGCTCA 840

Fig. 3.19 (3/5): Alignment of the AlaDH gene and the flanking regions of different strains of the M.tuberculosis complex

See legend page 101. 40kD 8 1 CGACCGACCA CCTACGACCA CCCGA.CGTTC GCCGTGCACG ACACGCTGTT TTA.CTGCGTG 500

Tubl 8 1 CGACCGACCA CCTACGACCA CCCGA.CGTTC GCCGTGCACG ACACGCTGTT TTACTGCGTG 500

H37Rv 841 CGACCGACCA CCTACGACCA CCCGACGTTC GCCGTGCACG ACACGCTGTT TTACTGCGTG 900

K37Ra 6 1 CGACCGACCA CCTACGACCA CCCGACC-TTC GCCGTGCACG ACACGCTGTT TTA.CTGCGTG SOO

BCG4 841 CGACCGACCA CCTACGACCA CCCGACGTTC GCCGTGCACG ACACGCTGTT TTA.CTGCGTG 900

BCG2 841 CGACCGACCA CCTACGACCA CCCGACGTTC GCCGTGCACG ACACGCTGTT TTA.CTGCGTG 900

Bov3 6 1 CGACCGACCA CCTACGACCA CCCGACGTTC GCCGTGCACG ACACGCTGTT TTA.CTGCGTG 900

Afrl 841 CGACCGACCA CCTACGACCA CCCGACC-TTC GCCGTGCACG ACACGCTGTT TTA.CTGCGTG 900

Micl 841 CGACCGACCA CCTACGACCA CCCGACGTTC GCCGTGCACG ACACGCTGTT TTA.CTGCGTG 900

40kD SOI GCGAA.CATGC CCGCCTCGGT GCCGAJAGACG TCGACCTACG CGCTGACCAA CGCGACGATG 960

Tubl SOI GCGAA.CATGC CCGCCTCGGT GCCGAAGACG TCGACCTACG CGCTGACCAA CGCGACGATG 960

H37Rv SOI GCGAACATGC CCGCCTCGGT GCCGAA .ACG TCGACCTACG CGCTGACCAA CGCGACGATG 960

H37Ra 90 GCGAACATGC CCGCCTCGGT GCCGAAGACG TCGACCTACG CGCTGACCAA CGCGACGATG 960

BCG4 SO GCGAACATGC CCGCCTCGGT GCCGAAGACG TCGACCTACG CGCTGACCAA CGCGACGATG 960

3CG2 SOI GCGAACATGC CCGCCTCGGT GCCGAAGACG TCGACCTACG CGCTGACCAA CGCGACGATG 960

3ov3 SOI GCGAACATGC CCGCCTCGGT GCCGAAGACG TCGACCTACG CGCTGACCAA CGCGACGATG 960

Afrl SOI GCGAACATGC CCGCCTCGGT GCCGAAGACG TCGACCTACG CGCTGACCAA CGCGACGATG 960

Micl SOI GCGAACATGC CCGCCTCGGT GCCGAAGACG TCGACCTACG CGCTGACCAA CGCGACGATG 960

40kD 561 CCGTATGTGC TCGAGCTTGC CGACCATC-GC TGGCGGGCGG CGTGCCGGTC GAATCCGGCA 1020

Tubl S61 CCGTATGTGC TCGAGCTTGC CGACCATGGC TGGCGGGCGG CGTGCCGGTC G.AATCCGGCA 1020

K37Rv S61 CCGTATGTGC TCGAGCTTGC CGACCATGGC TGGCGGGCGG CGTGCCGGTC GAATCCGGCA 1020

H37Ra S61 CCGTATGTGC TCGAGCTTGC CGACCATC-GC TGGCGGGCGG CGTGCCGGTC GAATCCGGCA 1020

BCG4 S61 CCGTATGTGC TCGAGCTTGC CGACCATC-GC TGGCGGGCGG CGTGCCGGTC GAATCCGGCA 1020

BCG2 S61 CCGTATGTGC TCGAGCTTGC CGACCATGGC TGGCGGGCGG CGTGCCGGTC GAATCCGGCA 1020

Bov3 S61 CCGTATGTGC TCGAGCTTGC CGACCATC-GC TGGCGGGCGG CGTGCCGGTC G.AATCCGGCA 1020

Afrl S61 CCGTATGTGC TCGAGCTTGC CGACCATGGC TGGCGGGCGG CGTGCCGGTC G.AATCCGGCA 1020

Micl 561 CCGTATGTGC TCGAGCTTGC CGACCATGGC TGGCGGGCGG CGTGCCGGTC GAATCCGGCA 1020

40kD 1021 CTAGCCAAAG GTCTTTCGAC GCA.CGAAGGG GCC-TTACTGT CCG.AA.CGGGT GGCCACCGAC 1080

Tubl 1021 CTAGCCAAAG GTCTTTCGA.C GCACGAAC-GG GCGTTACTGT CCGAACGC-GT GGCCACCGA.C 1080

H37Rv 1021 CTAGCCAAAG GTCTTTCGAC GCA.CGAA.C-GG GCGTTACTGT CCG.AACC-GGT GGCCACCGAC 1080

K37Ra 1021 CTAGCCAAAG GTCTTTCGA.C GCACGAAC-GG GCGTTACTGT CCGAACGC-GT GGCCACCGAC 1080

3CG4 1021 CTAGCCAAAG GTCTTTCGAC C-CACGAAGGG GCGTTACTGT CCG.AA.CGGGT GGCCACCGAC 1080

3CG2 1021 CTAGCCAAAG GTCTTTCGAC GCACGAAC-GG GCGTTACTGT CCGAACGGGT GGCCACCGAC 1080

3ov3 1021 CTAGCCAAAG GTCTTTCGAC GCACGAAC-GG GCGTTACTGT CCGAACGGGT GGCCACCGAC 1080

Afrl 1021 CTAGCCAAAG GTCTTTCGA.C GCACGAAC-GG GCGTTACTGT CCGAACGGGT GGCCACCGAC 1080

Micl 1021 CTAGCCAAAG GTCTTTCGA.C GCACGAAC-GG GCGTTACTGT CCGAACGC-GT GGCCACCGAC 1080

stop

40kD 1081 CTGGGGGTGC CGTTCACCGA GCCCGCCAGC GTGCTGGCCT GACTCTCGGC CGCTCGTTAC 1140

Tubl 1081 CTGGGGGTGC CGTTCACCGA GCCCGCCAGC GTGCTGGCCT GACTCTCGGC CGCTCGTTAC 1140

H37Rv 1081 CTGGGGGTGC CGTTCACCGA GCCCGCCAGC GTGCTGGCCT GACTCTCGGC CGCTCGTTAC 1140

H37Ra 1081 CTGGGGG'TGC CGTTCACCGA GCCCGCCAGC GTGCTGGCCT GACTCTCGGC CGCTCGTTAC 1140

BCG4 1081 CTGGGGGTGC CGTTCACCGA GCCCGCCAGC GTGCTGGCCT GACTCTCGGC CGCTCGTTAC 1140

BCG2 1081 CTGGGGGTGC CGTTCACCGA GCCCGCCAGC GTGCTGGCCT GACTCTCGGC CGCTCGTTAC 1140

Bov3 1081 CTGGGGGTGC CGTTCACCGA GCCCGCCAGC GTGCTGGCCT GACTCTCGGC CGCTCGTTAC 1140

Afrl 1081 CTGGGGGTGC CGTTCACCGA GCCCGCCAGC GTGCTGGCCT GACTCTCGGC CGCTCGTTAC 1140

Micl 1081 CTGGGGGTGC CGTTCACCGA GCCCGCCAGC GTGCTGGCCT GACTCTCGGC CGCTCGTTAC 1140

Fig. 3.19 (4/5): Alignment of the AlaDH gene and the flanking regions of different strains of the M.tuberculosis complex

See legend page 101. 40kD 1141 GCCGAGCACA CGTCGGGAGT AAC-GGAAGCG ATGATGTCGG CCGCG

Tubl 11 1 GCCGAGCACA CNTCGGGAGT AANG-GAAGCG ATGATGTCGN C 1185

H37Rv 11 1 GCCGAGCA.CA CGTCGGGAGT AAC-G AAGCG ATGATGTCGG CCG 1185

H37Ra 11 1 GCCGAGCACA CGTCGGGAGT AAGC-G.AAGCG ATGA 11S5

BCG4 1141 GCCGA CACA CGTCGGGAGT AAC-GGAAGCG ATGATGTCGG CC 11S5

BCG2 11 1 GCCGAGCACA CGTCNGGAGT AAC-GGAAGCG ATGATG 1185

Bov3 11 1 GCCGAGCACA CGTCGGGAGT AAC-C-GAAGCG ATGATGTCGG CC 1185

Afrl 1141 GCCGAGCNCA CGTCG 11S5

Micl 11 1 GCCGAGCACA CGTCGGC-AGT AAGC-GAAGCG ATGATGTCGG CC I S 5

Fig. 3.19 (5/5): Alignment of the AlaDH gene and the flanking regions of different strains of the M.tuberculosis complex

The line labeled "40kD" gives the sequence of Andersen et al. (1992) again. Sequence differences are each marked with a "*" above the sequence. The start and stop codons are also marked above the sequence. The bold bases at the end of the sequence are sequencing inaccuracies.

The first place where the sequences differ is base -32, that is upstream of the translation start signal. Interestingly, the sequences of M. tuberculosis H37R _V and H37R _a determined in this work differ from the sequence of Andersen et al. (Andersen et al., 1992). All other sequences examined in this work, including those of the third strain of M. tuberculosis tested, are consistent with the Andersen sequence.

This comes as surprising as the originally published sequence based on the clone of a lambda gt11 library, which had been prepared from strain M. tuberculosis H37R _v. Therefore, the question of whether an error was introduced by the PCR was investigated. This was not confirmed. However, it may also be possible that the strain of M. tuberculosis H37R _V used in this work has a different origin than that of Andersen. Similar small variances are also known in different strains of different origin of M.bovis BCG.

In the second place, all strains of the M.tuberculosis complex differ from the published sequence of AlaDH of M.tuberculosis H37R _V. It is a matter of the region of bases 38 to 49. Within these twelve bases, the sequence AATTCC- is repeated, ie bases 44 to 49 represent a direct repeat of bases 38 to 43. However, in all eight sequenced strains, this pattern is found only once , It can therefore be assumed that in the case of Andersen et al. (1992), a particular sequence has undergone a sequencing or reading error. The gene sequence and the deduced amino acid sequence thereby changes as follows:

Andersen et al., 1992:

Gene sequence A A C G A A T C C A A T C C G G G T G Protein sequence Asn Glu Phe Gin Phe Arg Val

This work :

Gene sequence A A C G A A T C C G G T G

Protein sequence Asn Glu Phe - - Arg Val

It is effectively the 'loss' of the two amino acids glutamine and phenylalanine. After this deletion, the sequence continues as described by Andersen et al. (1992).

This fact was confirmed by the N-terminal sequencing of the protein. Neither in the native protein of M. tuberculosis H37R _V , nor in the recombinant protein from E. coli, the two amino acids were found.

The third distinct site is Base 272. At this point, with the exception of three strains, there is an adenine residue. In these three strains, M.bovis and two strains of M.bovis BCG, this base is deleted. This deletion results in a frameshift that affects the entire following portion of the resulting protein. By this reading frame shift occurs at the bases 404 to 406 an opa / stop signal. Thus, the product of this gene is only about one-third the size of the functional AlaDH of the other strains.

The key to this third aberration in the gene sequence is the fact that it occurs exactly in the three strains that do not show AlaDH activity. M.bovis and M.bovis BCG are the only strains of M. tuberculosis complex that show no activity. All other strains were classified as moderate or strong positive. The observed deletion is therefore the reason for the lack of a functional AlaDH. However, since HBT-10 mAb can not detect the truncated protein (the epitope of HBT-10 is in the range before the frameshift), it can be assumed that the truncated protein does not occur at all, or only in very small amounts. with the mAb HBT-10 undetectable amounts is produced.

3.5.2.3 The AlaDH Gene of M.marinum

In addition to the members of the M.tuberculosis complex, it was primarily the M.marinum strain that was able to recover a fragment in the PCR with the primer pair Annabel (FIG. 3J7), which proved to be AlaDH in subsequent sequencing. As it turned out, this was a stroke of luck in that most of the other PCRs with the primer pairs from Table 3J5 gave no, or at least not the expected, fragments with this strain. Only the PCR with the primer pair Giselle was also positive. As it turned out later, the reason for this was the lack of homology between the genes within the different species. 50 60 70

Mar3 AATTCCGGTTAGCGATCACCCCGGCCGGCG

IIIMMI I II MIMIIIIMIIIII

H37RV TTCCGACCGAGACCA-AAAACAACGAΛTTCCAATTCCGGGTGGCCATCACCCCGGCCGGCG

80 90 100 110 120 130

Mar3 TCGCCGCCTTGACCAAGCGCGGCCACGAGGTGCTGATCCAGGCCGGTGCCGGAGAAGGCT

MM II IM II MIM IIIMMI MUMM MM

H37RV TCGCGGACTA-ACCCGTCGTGGCCATGAGGTGCTCATCCAGGCAGGTGCCGGAGAGGGCT

140 150 160 170 180 190

Mar3 CCGCCATCTCCGACGCCGACTTCAAGGCCGCCGGTGCCCAGCTGATCAGCACCGCCGACC

I II IM IIIMM II MUMM II II II II III II MINI II II 11

H37RV CGGCTATCACCGACGCGGATTTCAAGGCGGCAGGCGCGCAACTGGTCGGCACCGCCGACC

200 210 220 230 240 250

Mar3 AGGTGTGGGCGGATGCGGACCTGCTGCTCAAGGTCAAGGAACCGATCGAGTCCGAGTACG

MIIMIMI II II II I MUMM I I I II MM

H37RV AGGTGTGGGCCGACGCTGATTTATTGCTCAAGGTCAAAGAACCGATAGCGGCGGAATACG

260 270 280 290 300 310

Mar3 GCCGGCTGCGCCGGGGCCAGACCCTGTTCA.CCTACCTGCACCTGGCCGCCTCGCGCCCCT

MM MIM II MM I IIIMM I I MM II II I I

H37RV GCCGCCTGCGACACGGGCAGATCTTGTTCACGTTCTTGCATTTGGCCGCGTCACGTGCTT

320 330 340 350 360 370

Mar3 GCACCGATGCCCTGCTGAAGTCCGGCACCACGTCCATCGCCTACGAGACGGTGCAGACCG

MIIMIMI II II I MIMMMIMM II IMIIMMII II IIIMM

H37RV GCACCGATGCGTTGTTGGA-TTCCGGCA-CCACGTCAATTGCCTACGAGACCGTCCAGACCG

380 390 400 410 420 430

Mar3 CCGACGGCGCATTGCCGCTGCTGGCCCCCATGAGCGAGGTCGCCGGGCGCCTGTCCGCCC I II MIM MIMMUM MUMM II II

H37RV CCGACGGCGCACTACCCCTGCTTGCCCCGATGAGCGAAGTCGCCGGTCGACTCGCCGCCC

440 450 460 470 480 490

Mar3 AAGCTGGGGCCTACCACCTGATGCGCACCCACGGCGGTCGCGGCGTGCTGATGGGCGGCG

I IM II MMMMIIMM MIM II II MIM IMIIIMIIMM I

H37RV AGGTTGGCGCTTACCACCTGATGCGAACCCAAGGGGGCCGCGGTGTGCTGATGGGCGGGG

500 510 520 530 540 550

Mar3 TCCCCGGCGTCA-AGCCTGCCGACGTCGTGGTGATCGGCGCGGGCACGGCCGGATACAACG

I II IM II II I II MIMMMMMMIMIM MIM

H37Rv TGCCCGGCGTCGAACCGGCCGACGTCGTGGTGATCGGCGCCGGCACCGCCGGCTACAACG

Fig.3.20 (1/2): AlaDH gene alignment of M.marinum and M. tuberculosis H37R _V

The screening of the database and the alignment were carried out with the FASTA software {http://www.embl-ebi.ac.uk (sensitivity ktup = 6). A "|" indicates an identical pair of Eases. - 3S -

560 570 ≡SO 590 600 610 Mar3 CCGCCCGCGTCGCCAACGGCATGC-C-CGCGATGGTCACCGTGCTGGA7GTCAACATCAACA.

I M I N I I M I I I I I I M I M I I I I I I I I I I l l l l l l M

H37RV CAGCCCGCATCGCCAACGGCATGG3CGCGACCGTTACGGTTCTAGACATCAACATCGACA

620 630 640 650 660 670

Mar3 AGCTCCGCCAGATCGACGCGGAGTTCGGCGGTCGCGTCCGGACCCGCTACTCGTCGACCC

I II II II MM II MI II IM II

H37RV AACTTCGGCAACTCGACGCCGAGTTCTGCGGCCGGATCCACACTCGCTACTCATCGGCCT

680 690 700 710 720 Mar3 TCGACCTCGAGGATGCGGCAGTCCACGCCGACATGGTGATCGGGGCCGTCCT

IM IM I

H37Rv ACGAGCTCGAGGGTGCCGTCAAACGTGCCGACCTGGTGATTGGGGCCGTCCTGGTGCCAG

Fig. 3.20 (2/2): AlaDH gene alignment of M.marinum and M. tuberculosis H37R _V

The screening of the database and the alignment were carried out with the FASTA software (http: //www.embl- ebi.ac.uk/) (sensitivity ktup = 6). A "|" denotes an identical base pair.

A total of 682 bases of this gene could be determined (Fig. 3.20), which represents about 60% of the complete gene. These 682 bases encode the N-terminal region of the protein. Only the first 35 bases after the start codon are missing.

The sequenced section of the AlaDH from M.marinum shows 80.4% identity with the corresponding section of M. tuberculosis H37R _V. The sequence differences are distributed relatively uniformly over the entire section.

The deduced peptide of the M.marinum gene sequence (Figure 3.21) shows 85.3% identity and 92.0% similarity to the M. tuberculosis H37R _V protein. Homologies to other proteins of the Swiss Prot database are given in Tab. 3J5 (as of 9/1996, Release 33). For comparison, it should be noted that the AlaDH of M.tuberculosis ^{'' has} 53% identity to the enzymes of B.sphaericus and B.stearothermophilus (Andersen et al., 1992). In the AlεDH of M.marinum, an identity of 51% with the enzyme of B.sphaericus or of 50% with the enzyme of B.stearothermophilus can be ascertained (Table 3.15). ** ** _****** * _{* ***************} * _t *** _* ****** _* *** _{****** ****} *

Mar3 14 FRLAITPAGVA.ALTKP.GKΪVLICAGAGΞGSAISDA-DF KAAGAQLISTADQVΛADAD LK 73

H37RV 14 FRVAITPAGVAΞLTRRGHEVLIQAGAC-ΞGSAITDA-DriAAGAQ VGTADQVft "A - DADL L 73

***** _ * + **** ** + **, * + ***** ****** ****************** ***** Mar3 74 VKΞPIESΞYC-Λ RHC-QTLFTY H AΛSHPCTDA LKSGTTSIAYETVQTA-DC.ALPLLAPM 123

H37RV 74 VKEPIAA-EYGRLF-rGQI rTFLKLAASΛACTDALLDSGTTSIAYETVQTA GALPL APM 123

******* _ * + ******** ************** ** + ********* + + _ *** + ** Mar3 134 SEVAGR SAQAGA ^: iJ-STHGGRGVI-J-: GGVPGVKPADVA / IGAGTAGY ^ AA.Vv; A - NG GAl 183 H37RV 134 SEVAGRIJAQVGA ^: U ^ TQGGRGVIJ-: 3GV? LOVE? ADVVVIGAGTAGYNA -A-RIANGMGAT 183

***** ** _ **** _ *** + ** _ ***** _ ** * ** _# *****

Mar3 184 V VLD \ miΥ RQIDAΞFGGRVRTRYSST DLΞDAAVKADI ^> IVIGAV 229 H37Rv 184 VTVLDINIDXLRQLDAΞrCGRIKTRYESAYELΞGAVK-RA-D VIGAV 229

Fig.3.21: AllaDH's alignment of M.marinum and M. tuberculosis H37R _V

An "*" indicates an identical, a "." a functionally related amino acids. The screening of the database and the alignment were performed with the FASTA software (http: //www.embl-ebi.ac.ulv) (sensitivity ktup = 2).

Table 3.15: Proteins with homology to AlaDH of M.marinum

The Swiss Prot database was searched using the FASTA software (http: /ΛwΛV.embl- ebi.sc.uk) (as of 9/1996, Release 33).

Protein organism identity similarity

AlaDH M. tuberculosis 85.3% 92.0%

AlaDH B.sphaericus 50.9% 69.0%

AlaDH B.stearothermophilus 50.0% 67.7%

AlaDH B.subtilis 48.7% 68.6%

PNT beef, mi'ochondrial 33.7% 51.4%

PNT E.coli 30.7% 50.0%

PNT Haemophilus influenzae 28.8% 43.2%

The amino acid residues Gly165, Gly167, Gly170 and Asp198 are considered to be essential for the binding of the cofactor of AlaDH of M. tuberculosis H37R _V. These four amino acid residues are characteristic of the βαβ secondary structure of NAD (H) binding sites (Wierenga et al, 1986; Bork & Grunwald, 1990). In M.marinum's enzyme, as in all other AlaDH's in Table 3.14, these four residues are conserved.

The corresponding section of the mAb HBT-10 mAb epitope of M.marinum AlaDH has the sequence AISDADFKAAG, thus differing only in third place from the M. tuberculosis H37R _V sequence. Based on the consensus epitope of the antibody determined in this work (Table 3.9), it would also have to react with the AlaDH of M.marinum. In fact, this strain is also the only one with which a cross-reaction could be observed (Andersen et al., 1992). The determined sequence is also consistent with this observation.

AlaDH activity in mycobacteria. The measured AlaDH activities allow some interesting observations regarding the lifestyle of the organisms with positive activity.

The strains with high activity are all pathogenic. Interestingly, two of the four strains that belong to this group are pathogenic for fish (Austin & Austin, 1987). However, both of them, M.marinum and M.chelonae, can also be humans

infect (Wallace et al, 1983; Johnston & Izumi, 1987). In contrast to tuberculosis, however, they usually cause pathological infections of the upper layers of the skin, which are usually relatively unproblematic to treat.

M.chelonae is a comparatively fast-growing, non-chromogenic bacterium. Infections in humans often occur as secondary wound infections after surgery (Cooper et al, 1989). M.marinum is a slow-growing organism that forms a yellow pigment when grown in light. In more than 50 warm-blooded species (reptiles, amphibians, fish) infections with M.marinum were detected (Clark & Shepard, 1963). In humans, the bacterium usually manifests itself in the elbow or knee area.

The other two strains with strong-positive AlaDH activity are representatives of the M. tuberculosis complex. These are the tuberculosis reference strain M.tuberculosis H37R _V , as well as the strain M.microti, which is considered to be a phylogenetic link between M.tuberculosis and M.bovis.

Apart from M. smegmatis, all strains classified as moderately positive are also pathogenic. The majority of these strains include clinical isolates of M. tuberculosis. Pathogenic variants of tuberculosis strains thus generally have AlaDH activity. However, two isolates were found that do not have AlaDH activity. The only non-pathogenic organism with AlaDH activity is the rapidly growing M.smegmatis strain. However, M.smegmatis has an unusually strong background NAD ⁺ -reducing activity and is therefore very easily distinguishable from all other strains with AlaDH activity. Furthermore, in the strain M.smegmaits 1-2c, a mycobacterial expression strain, no AlaDH activity was found. Within the 44 strains of mycobacteria tested, and this is by far the majority of all known strains, the conclusion is therefore:

πn ^ - A slow-growing mycobacterium with positive AlaDH activity is virulent.

The inverse of this statement is wrong. Among the strains without AlaDH activity are quite a few virulent. Nevertheless, one can not help but notice a tendency, albeit not solid, for AlaDH activity to increase with increasing pathogenicity of a strain. In particular, by the activities of different strains of M. tuberculosis this thesis is underlined. The highest activity by far has the strain H37R _V , which serves as a reference strain for all tuberculosis laboratories, and has a known high infectivity. At the very end, the avirulent derivative of H37R _Vl ranks H37R _a . Between these two poles rank the clinical isolates of tubules, which sometimes show slightly more and sometimes less activity.

The AlaDH gene in mycobacteria. The gene for alanine dehydrogenase could be identified in all strains investigated in the M.tuberculosis complex and in the M.marinum strain.

The crucial point in comparing the sequences within the M. tuberculosis complex is the deletion of the base 272, which leads to frameshifting of the M.bovis and M.bovis BCG strains studied and ultimately to a truncated, nicKt-functional protein. In these strains, no AlaDH activity could be detected in cell extracts. These data also agree with the results of Andersen et al. (1992), who received signals with these strains in Southern blots but were unable to detect protein in Western blots.

Through the amplification and sequencing of the gene, the cause could be found in this work. However, it must also be considered that further changes in the regulatory gene segments may be responsible for the absence of the truncated protein. This could be a measure of the cell not to invest energy in a non-functional protein. Generally, little is known about regulatory gene sequences in mycobacteria (Dale & Patki, 1990, Gupta et al., 1993). It seems, however, that, according to the principle of enhancers, even more distant parts can influence the gene expression considerably. The mutations needed to stop production of the protein do not necessarily have to be within the range sequenced in this work.

The other identified AlaDH gene, that of M.marinum, is significantly different at the DNA level from the genes of the M.tuberculosis complex. However, four out of five bases (80.4%) are on average still identical when comparing these sequences. This value is even higher at the protein level (85.3 o identity, 92.0% similarity). However, since AlaDH activity was also found in a number of other species, it can be assumed that the corresponding genes, lacking homology to the primers used, can not be amplified under the conditions used. A more detailed study on this point would have to find these genes as well. A comparison of all these sequences could allow further conclusions about the role of the enzyme.

Furthermore, it is conceivable that, based on such a sequence comparison, it should be possible to develop a PCR method with which it is possible to distinguish mycobacteria which have an AlaDH gene from one another. And as shown in this work, it is the important strains that have an AlaDH gene. In particular, the possibility of using such a PCR assay to distinguish the M. tuberculosis pathogen from the M.bovis BCG vaccine strain makes such a project interesting. Outlook. The 40 kD antigen treated in this work is in many ways a worthwhile object for further investigations. One point that was not considered in this work is the possible application of this enzyme in medical diagnostics. For example, assays based on AlaDH have been described for the enzymes dipeptidase (Ito et al, 1984), γ-glutamyltransferase (Kondo et al, 1992), and γ-glutamyl cyclotransferase (Takahashi et al, 1987). All three of these enzymes are found in various diseases in altered urine, serum or blood concentrations.

However, the main focus is on the use of the 40 kD antigen in tuberculosis. Starting points here are conceivable in several places.

In diagnostics alone, several options are conceivable, such as the use of the 40 kD antigen or its underlying gene. Since the recombinant protein can now be readily recovered from the overproducing E. coli strain, it seems worthwhile to review the usefulness of this protein in serology. In addition, diagnostic procedures could be developed which rely on the direct detection of AlaDH activity or, as previously mentioned, on the amplification of specific parts of the gene. The deletion of the base 272 in the strains M.bovis and M.bovis BCG can serve as a starting point for the discrimination of these two strains of M. tuberculosis.

A PCR assay would also have to be established for the M.marinum strain, which differs considerably at the gene level from the M. tuberculosis complex. To date, a PCR assay based on the amplification of a portion of the gene sequence encoding the JδS rRNA has heretofore been used (Knibb et al, 1993). This is of great importance given the increasing number of M.marinum infections in fish farms in recent years (Knibb et al., 1993). Also

Human infections have been reported more frequently in recent years (Harris et al, 1991, Kullavanijaya et al, 1993, Siosarek et al, 1994). The observation that the virulence of a strain of M. tuberculosis correlates very well with its AlaDH activity again raises the question of whether the enzyme has a Represents virulence factor. To answer this question, approaches such as knockout of the gene in M. tuberculosis or overexpression of the gene in a low virulence strain are conceivable. In both cases, the virulence can be checked in the animal model.

The disclosure of the present application also includes the disclosure of the attached EP 97 101 33g. e, in particular the literature cited therein. The disclosure also includes all conceivable combinations of disclosed individual features.

Claims

5. SummaryTuberculosis is an infectious disease that affects more than 3 million people every year. Although there is a vaccine as well as various diagnostic and therapeutic procedures, the effectiveness of all these measures, in view of the increasing number of cases, is in urgent need of improvement. One focus of research is the characterization of antigens secreted during an infection because they make the first contact of the immune system with the pathogen. The 40 kD antigen treated in this work is present in vivo as a hexamer, and despite the high molecular weight and lack of signal sequence, it can be found extracellularly after just a few days of growth. It is functionally an L-alanine dehydrogenase and reacts with the monoclonal antibody HBT-10 directed against this protein. HBT-10 was the first known antibody specific for a M. tuberculosis protein that does not cross-react with the M.bovis BCG vaccine strain.

1. The gene coding for the 40 kD antigen had already been cloned into a plasmid vector for E. coli and the expression of the resulting plasmid optimized. In this work, a completely new method was established, which makes it possible to purify the hexameric protein in a two-way reaction in soluble and active form.

2. All essential biochemical parameters of the recombinant enzyme were determined. The determined properties are consistent with those of other described L-alanine dehydrogenases. The enzyme of M. tuberculosis is alanine Dehydrogenase with the lowest known pH optimum for the reductive amination.

3. The determined biochemical data strongly support a role of the enzyme in an early step in peptidoglycan synthesis. It provides L-alanine, which is also the precursor of D-alanine, for the synthesis of the penta or tetrapeptide chains of Λ / -glycolyimuramic acid. This is the first description of this task for an L-alanine dehydrogenase.

4. A postulated cross-reaction of the monoclonal antibody HBT-10 with the PNT was refuted. The thesis that the 40 kD antigen intervene in the critical balance between NADH and NADPH was thereby removed from the basis. Also, the ability of intracellular survival in macrophages, the 40kD antigen, at least in the absence of other mycobacterial proteins, has no influence.

5. Over 40 mycobacterial strains were tested for the presence of AlaDH activity. All strains exhibiting AlaDH activity are virulent. M.bovis BCG shows no activity. In addition, the level of activity appears to correlate with the degree of virulence of a M. tuberculosis slamτnes.

6. The gene for alanine dehydrogenase was completely sequenced from eight strains of the M. tuberculosis complex. In the strains M.bovis and M.bovis BCG a deletion was found which leads to a frameshift and is responsible for the lack of measurable activity in these strains.

7. The M.marinum AlaDH gene was identified and much of the sequence determined. The gene has about 80% identity with the M. tuberculosis gene.

7. Attachments

List of abbreviations

A pre-exponential factor or impact factor

A _xxx absorption at a wavelength of xxx nm

AlaDH L-alanine dehydrogenase (E.C. 1.4J.)

AMC Academic Medical Center, Amsterdam, Netherlands

Ap ampicillin

AP alkaline phosphatase app. Apperent

AS amino acid

ATCC American Type Culture Collection, Rockville, USA

ATP adenosine triphosphate

BCG Bacille Calmette Guerin

BCIG 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside

BCIP 5-bromo-4-chloro-3-ndolyl phosphate

Boc / erf-butoxycarbonyl bp base pair (s)

cfu colony forming units

Cm Chloramphenicol

Conc concentration

DMEM Dulbecco's Modified Eagle Medium

DMF dimethylformamide

DMSO dimethyl sulfoxide

DNA deoxyribonucleic acid

DTNB Dithiobisnitrobεnzoesäure

DTT dithiothreitol

E _a activation energy

EDTA ethylenediaminetetraacetate

Eth ethionamide ~ 4V -

Farad

FBS fetal bovine serum

FCS fetal calf serum

Fmoc 9-fluoro-methoxycarbonyl

FPLC Fast Protein Liquid Chromatography frag. fragment

9 Fall acceleration

GBF Society for Biotechnological Research mbl

GlcNAc N-acetylglucosamine

Gm gentamicin

GOGAT glutamine-oxoglutarate aminotransferase

GS glutamine synthetase

GST glutathione-S-transferase

h hour (s)

HBSS Hank Balanced Salt Solution

HIV Human Immunodeficiency Virus

HOBt hydroxybenzotriazole

HRP horseradish peroxidase

Hsp heat shock proteins

ig immunoglobuiin

IL interleukin

INH isonicotinic acid hydrazide, isoniazid

IPTG isopropyl-β-D-thiogalactoside

k conversion rate of an enzyme kb kilobases

KBL Kilobasenleiter kD, kDa Kilodalton

KIT Royal Tropical Institute, Amsterdam, Netherlands

K _M Michaelis Constant

Km kanamycin

MΦ macrophage (mAb) monoclonal antibody

MAIS M.avium - M.intracellulare - M.scrofulaceum Com MBP maitosis binding protein

MCAC metal chelate affinity chromatography mesoDAP meso diaminopimelic acid min. (M.o.i. multiplicity of infection

MRC Medical Research Council, Tuberculosis and Related Infections Unit, London, England

MTT thiazolylbluetetrazolium bromide

MurNAc A / acetylmuramic acid

MurNGl Λ / -glycolylmuramic acid

NAD ⁺ nicotinamide adenine dinucleotide, oxidized form

NADH nicotinamide adenine dinucleotide, reduced form

NADP ⁺ nicotinamide adenine dinucieotide phosphate, oxidized form

NADPH nicotinamide adenine dinucleotide phosphate, reduced form n.b. not determined

NBT nitroblue tetrazole chloride

Number

NTP any nucleoside in the form of a triphosphate

and oxidative deamination

ORF open reading frame, open reading frame

OtBu terf butyl ester

PAGE polyacrylamide gel electrophoresis pac protein antigen c, old name for the 40 kD antigen

PCR polymerase chain reaction, polymerase chain reaction

Pfp pentaflourophenyl

PMA phorbol myristate acetate

Pmc pentamethylchroman

PMS phenazine methosulfate

PNT pyridine nucleotide transhydrogenase

PPD Purified protein derivative

PVDF polyvinylidene difluoride

R Rydberg constant or resistance (if superscript) rA reductive amination rec recombinant Rha rhamnose $ -4 -

Rif rifampicin

RIV National Institute of Public Health and the Environment, Bilthoven, Netherlands

RNA ribonucleic acid

RNI reactive nitrogen intermediates, reactive nitrogen intermediates

ROI reactive oxygen intermediates, reactive oxygen intermediate rpm rounds per minute, RPM rRNA ribosomal ribonucleic acid

RT room temperature

SDS sodium dodecyl sulfate see second (s) Str streptomycin

TB tuberculosis

TEMED Λ / .Λ / .ΛT.N-Tertamethylethylenediamino

TIR translation initiation region

Tris tris (hydroxymethyl) aminomethane

TU Trityl ts temperature-sensitive

Tween polyoxyethylene sorbitan monolaurate

U Unit (s) over night unopened unpublished

^v max maximum reaction rate

VMDC Veterinary Microbiological Diagnostic Center, Utrecht, Netherlands

Vol. Volume

WHO World Health Organization, World Health Organization WKZ Academic Ziekenhuis, Utrecht, The Netherlands

eg for analysis, of the highest degree of purity Abbreviations for amino acids and nucleotides

Amino acid 3-letter £

Alanin Ala A

Arginine Arg R

Asparagine Asn N

Aspartate Asp D

Cysteine Cys C

Glutamine Gin Q

Glutamate Glu E

Glycine Gly G

Histidine His H

Isoleucine He

Leucine Leu L

Lysine Lys K.

Methionine Met M

Pheny-alanine Phe F

Proline Pro P

Serin Sεr S

Threonine Thr T

Tryptophan TΦ w

Tyrosine Tyr Y

Valin Va! V

Base nucleoside / nucleotide abbreviation

Adenine Adenosine A

Cytosine cytidine C

Guanin Guanosin G

Uracil uridine U

Thymine thymidine T

DNA Sequence of Mycobacterium marinum and Use

P A T E N T A N S P R E C H E

1. DNA sequence (I) of Mycobacterium marinum of the following formula, DNA sequence complementary thereto, double-stranded DNA sequence from the DNA sequence (I) and its complementary strand, their partial sequences and with them preferably at a temperature of at least 20 C and in particular at a concentration of 1 M NaCl and a temperature of at least 25 C hybridizable DNA sequences:

50 60 70

Mar3 AATTCCGGTTAGCGATCACCCCGGCCGGCG

IIIIMII I II

-1137Rv- - ^ TGeGA € G & AGAeGA -AAA.e AeGAA-τ-τe € -AATτe ssβτGGeeAτeAeeeeGΘeeGGeα-

80 90 100 110 120 130

Mar3 TCGCCGCCTTGACCAAGCGCGGCCACGAGGTGCTGATCCAGGCCGGTGCCGGAGAAGGCT

-H ^ y- σ - Tea sGAA- ^ AAeeeGTeGTGβeeATΘAGG βe-peATeeAGΘeAGOTGeeGGAGAΘΘΘe '- ¹ -

140 150 160 170 180 190

Mar3 CCGCCATCTCCGACGCCGACTTCAAGGCCGCCGGTGCCCAGCTGATCAGCACCGCCGACC

II37RV GGGGTAΦ € AΘGGAeGeG & AT-T-TGAAGGGGGeAGGGGeGeA-A € -TG & T- € GGAGGGGeGAΘ € -

200 210 220 230 240 250

Mar3 AGGTGTGGGCGGATGCGGACCTGCTGCTCAAGGTCAAGGAACCATCGAGTCCGAGTACG II II II I I I II MM

H37RV AGGΦ ΦGGG GAβGGΪG ^ TΨA ^ TG TGAAGGTGAAAGAGS & AΦAGGGG ^ GA- ^ TAGG-

260 270 280 290 300 310

Mar3 GCCGGCTGCGCCGGGGCCAGACCCTGTTCACCTACCTGCACCTGGCCGCCTCGCGCCCCT

MM MIM I! Ill I I I MM II II I I

-i & rf & v - GeGGe «FGe & AeAGGGGGAGATeT-TGTTGAGGT-TG-TTGGA-l P-TGGGCGGGTGAGGTGfrT-Φ

320 330 340 350 360 370

Mar3 GCACCGATGCCCTGCTGAAGTCCGGCACCACGTCCATCGCCTACGAGACGGTGCAGACCG II II I II II IIIMM

• H37R- - GGAGG & A-TGβGT-TGΦ5GGAT-TGGGGGAGGAGGΦGAA-T.TGGG-TAGGAGAGGG-T-CCAGACCG

380 390 400 410 420 430

Mar3 CCGACGGCGCATTGCCGCTGCTGGCCCCCATGAGCGAGGTCGCCGGGCGCCTGTCCGCCC

MIMIIMM I II MIM MIM II II in 7 R - eeGAeGQeGe ETAeeeeTGeTTG € eee & ATGAGe A GTGGeeGGT-eG -TGGGGG

440 450 460 470 480 490

Mar3 AAGCTGGGGCCTACCACCTGATGCGCACCCACGGCGGTCGCGGCGTGCTGATGGGCGGCG

-H3-3-RV - GGΦ-TGG € GEAeeAeeTΘA Θe & AfteeeAAGGGGGCt-GCGGTGTσCTGATGGGCGGGG "

500 510 520 530 540 550

Mar3 TCCCCGGCGTCAAGCCTGCCGACGTCGTGGTGATCGGCGCGGGCACGGCCGGATACAACG

I IIIIIMM I II MIMIMMMIMMMMII MIM MIM

-ji3 "7Rv - -TΘeΘGGΘeGϊ € GAAeGGGeeG eG eG'P G GatEGGeGeeβGeAgeGeeGG eT eAAeG"

560 570 580 590 600 610 Mar3 CCGCCCGCGTCGCCAACGGCATGGGCGCGATGGTCACCGTGCTGGATGTCAACATCAACA

I 11111 II 11 II II 11! 11111 II II II II MUMM IM

-Η-3-7-R ^« P AGe €€ βeA-TeGeeAAGGΘGA ¹ ? GGGeΘeΘAeeG ^c F'PA S © TGT-AGAeA- AAe e A A '

680 690 700 710 720

Mar3 TCGACCTCGAGGATGCGGCAGTCCACGCCGACATGGTGATCGGGGCCGTCCT f £)

1137Rv A € GAβe ⁱ ϊ ^ & AβGβ-rGe € β-j ^ -AJ ^^ Θ-rGe € - & Aee? EG-θAΥ-Υ ^β G6GgεGTCCroΘ-Tβeeftβ ^{^}

2. DNA sequence according to claim 1, characterized in that they produce or win with their help Alanindehydrogenase and

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

(ii) can be used to diagnose other mycobacterial infections in humans and animals, especially those caused by Mycobacterium marinum.

3. RNA as a transcript of a DNA sequence according to claim 1, in particular for use

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

(ii) in the diagnosis of other mycobacterial infections in humans and animals caused in particular by Mycobacterium marinum.

4. protein which is encoded by a DNA sequence according to claim 1 and in particular corresponds to alanine dehydrogenase and

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

(ii) can be used to diagnose other mycobacterial infections in humans and animals, especially those caused by Mycobacterium marinum.

5. Protein of the following sequence (II)

** _ ******** ** _# **************** _# *********** _# ****** ********

MAR3 14 FR AITPAGVAALT RGHEV IQAGAGEGSAISDADF AAGAQ ISTADQVWADAD LLK 73 --H-57RY 14 FRVAITPAΘVASLTRRGKSV IQftgftSBΘSftg ^ -SJ ^ Pigti ^ AQLVGTADQVWftBftDL LK 73 ^■

***** _ ****** ** *** ******* ****** ******************* ****

Mar3 74 VKEPIESEYGRLRRGQTLFTY HL-AASRPCTDA LKSGTTSIAYETVQTADGALPLLAPM 123 • -K37RV 71 VKEPIAΛEYGR nHσQI PT-FLHI - ΛasftΛCTDΛ ^ 123

******* ** ******** ************** _# ****************** _ ******

Mar3 134 SEVAGRLSAQAGAYHLMRTHGGRGVLMGGVPGVKPADVWIGAGTAGYNAARVANGMGAM 183 1137Rv 134-CΞVAGRI-ι- ^ QVGAYHI-MRTQCCRCVkMGGVPGVS ^^

***** ** ****, **** ** _, ***** _# ** * ** _ ***** Mar3 184 VTV DVNINKLRQIDAΞFGGRVRTRYSST D ΞDAAVHADMVIGAV 229 «37Rv 18d _" VTV PI I rJiRQI ^ _l A5gCGR KΦR ¥ SSA "5 ^ SCA, y ^j UPL.V.IGΛV 238 .. and oligopeptide which is encoded by a DNA sequence comprising a DNA sequence encoding the protein of formula (II), preferably at a temperature of at least 20 C and in particular at a concentration of 1 M NaCl and a temperature of at least 25 ° C, hybridizable, as well as oligopeptide which is encoded by a partial sequence of the hybridizable DNA sequence.

6. A specific and sensitive method for the identification of mycobacteria in a medium, characterized in that

(i) isolating cells, strains or species of mycobacteria, (ii) recovering crude or purified genomic DNA or RNA, (iii) identifying the genomic DNA or RNA or a cDNA fragment whose sequence matches that of the alanine dehydrogenase gene of Mycobacterium tuberculosum (Figure 3.20) is identical or practically identical, preferably by amplifying with the aid of a primer sequence based on the nucleotide sequence according to claim 1, (iv) after which the DNA is digested with a restriction enzyme, preferably BglII, and subjected to gel electrophoresis (v ) and / or the DNA sequence of the amplified DNA.

7. Use of alanine dehydrogenase from Mycobacterium marinum (wild type) for the diagnosis of tuberculosis by measuring the

Enzyme activity or by an immunological method.

8. Verf hren for the preparation of recombinant alanine dehydrogenase, characterized in that the recombinant alanine dehydrogenase is formed using a DNA sequence according to claim 1 in bacteria, yeast, fungi, higher eucaryotes or cell lines and win.

9. Use of the recombinant alanine dehydrogenase obtained according to claim 8 for the diagnosis of mycobacterial infections. tuberculosis and swimmer's disease in humans and animals.

10. Use of a DNA sequence according to claim 1 and / or of native or recombinant alanine dehydrogenase of Mycobacterium marinum for the direct detection and diagnosis of tuberculosis and other mycobacterial infections in humans and animals in clinical samples.

11. Use of a DNA sequence according to claim 1 and / or of native or recombinant alanine dehydrogenase of Mycobacterium marinum

(i) in the control of epidemics and / or

(ii) after vaccination (follow-up) of humans and animals.

12. Use of a DNA sequence according to claim 1 and / or of native or recombinant alanine dehydrogenase of Mycobacterium marinum for the detection, testing, obtaining and production of substances which inhibit pathogens of tuberculosis or other mycobacterial diseases in humans and animals.

13. Use of a DNA sequence according to claim 1 and / or native or recombinant alanine dehydrogenase of Mycobacterium marinum for biotransformation reactions specific for L-alanine.