FR2929958A1 - METHOD FOR SCREENING ANTI-TUBERCULAR COMPOUNDS - Google Patents

Abstract

The present invention relates to the use of a bacterial enzyme catalyzing the synthesis of a glucose polymer in which glucoses are linked to one another via alpha-1,4 and alpha-1,6 bonds, for the identification of compounds with antibiotic activity.

Description

Method for screening anti-tuberculous compounds

Field of the Invention The present invention relates, in particular, to a method of screening compounds for the prevention or treatment of tuberculosis.

BACKGROUND Tuberculosis remains a critical health problem in the world. It is estimated that one-third of the world's population is infected with Mycobacterium tuberculosis, the causative agent of tuberculosis, and that this disease kills more than 2 million people a year. The situation is such that the WHO has declared that the fight against this disease is a priority. Despite a treatment monitoring program in the most affected regions, the situation remains worrying. The aggravating factors are the impoverishment of the populations, the frequent co-infection of the tubercle bacillus and the AIDS virus, and the appearance of resistant strains. Current therapies rely mainly on the use of antituberculous drugs that inhibit the biosynthesis of constituents of the envelope (isoniazid, ethambutol), transcription (rifampin), protein synthesis (streptomycin) and nucleic acids (quinolones), and pyrazinamide. The appearance of multidrug-resistant strains (MDR) is further aggravated by the appearance of XDR (Extensive Drug Resistant) strains. It is therefore necessary to renew the arsenal available to doctors to fight against this disease.

Understanding the molecular mechanisms that allow the TB bacilli to multiply in a hostile environment, such as the macrophage, and survive in the host, is important in developing new strategies to combat the disease. The cellular envelope of pathogenic mycobacteria plays an important role in host-pathogen interactions and in the resistance of these microorganisms to chemotherapeutic treatments (Daffé & Draper (1998) Advances in Microbial Physiology 39 131-203, Jarlier & Nikaido (1990)). J. Bacteriol 172: 1418-1423). The envelope consists of a classical plasma membrane, surrounded by a complex wall of carbohydrates and lipids, which itself is surrounded by an outer layer (Daffé & Draper (1998) Advances in microbial physiology 39 131- 203). The unusual chemical nature of this envelope makes it difficult for the host to degrade.

The outermost compartment of the cellular envelope of pathogenic mycobacterial species consists of a loosely linked structure, called capsule (Chapman et al (1959) J. Bacteriol 77: 205-211; Daffé & Draper (1998) Advances in Microbial Physiology 39: 131-203, Hanks (1961) Int J. Leprosy 26: 172-174). It has previously been shown that the capsule is essentially composed of carbohydrates and proteins, with only a limited amount of lipids specific to the species or type of bacteria (Lemassu et al (1996) Microbiology 142: 1513-1520, Ortalo Magné et al., (1995) Microbiol., 141: 1609-1620). Although mycobacteria release some of this material into the culture medium during in vitro growth (Ortalo-Magné et al., (1995) Microbiol., 141: 1609-1620), it is clear that the capsule covers the bacteria that grow. multiply in vivo (Schwebach et al., (2002) Infect Immun 70: 2566-2575), presumably retained by the phagosome membrane (Daffé & Draper (1998) Advances in microbial physiology 39: 131-203). The main carbohydrate component of the M. tuberculosis capsule, which accounts for up to 80% of extracellular polysaccharides, is a high molecular weight α-glucan (> 100,000 Da) composed of a 4-aD-GIc-1 nucleus. - which is connected on average every 5 to 6 residues by oligoglucosides (Dinadayala et al., (2004) J. Biol Chem 279: 12369-12378; Lemassu & Daffé, (1994) Biochem J. 297: 351 -357, Ortalo-Magné et al (1995) Microbiol., 141: 1609-1620).

Since the true nature of the mycobacterial surface has been elucidated only very recently, there is currently little information available on the biological functions of its constituents.

SUMMARY OF THE INVENTION The inventors have demonstrated that the genes glgC, glgA, Rv3032 and glgB encode proteins involved in glucan and / or glycogen biosynthesis of M. tuberculosis.

Moreover, the inventors have shown that, unexpectedly, the glg8 gene, encoding the branching enzyme, is essential for the survival of Mycobacterium tuberculosis (see Example 2). In addition, they could not observe any bacterial growth after concomitant inactivation of the genes gigA and Rv3032, encoding the elongation enzymes. This indicates that compounds that inhibit the function of the products of these genes will have antibacterial activity. Thus, the present invention relates to the use of a bacterial enzyme catalyzing the synthesis of a glucose polymer in which glucoses are linked to one another via α-1,4 and α-1,6 bonds; the identification of compounds with antibiotic activity. The present invention also relates to a method for screening compounds with antibiotic activity, comprising the following steps: - putting a compound whose antibiotic activity is to be determined in the presence of a bacterial enzyme catalyzing the synthesis of a glucose polymer in which the glucoses are linked together via α-1,4 and α-1,6 bonds; measuring the activity of the enzyme in the presence of the compound; comparing the activity of the enzyme in the presence of the compound to its activity in the absence of the compound; selecting the compound as an antibiotic if the activity of the enzyme in the presence of the compound is lower than in the absence of the compound. The present invention also relates to a bacterial enzyme inhibiting compound which catalyzes the synthesis of a glucose polymer in which the glucose is bound to each other via α-1,4 and α-1,6 bonds, for use as a antibiotic. The present invention also relates to the use of a bacterial enzyme inhibiting compound catalyzing the synthesis of a glucose polymer in which glucose is bound to one another via α-1,4 and α-1,6 linkages. , for the preparation of an antibiotic. The present invention also relates to a method of preventing or treating a bacterial infection in an individual, comprising administering to the individual a compound inhibiting a bacterial enzyme catalyzing the synthesis of a glucose polymer in which glucose is present. are linked together via α-1,4 and α-1,6 bonds, especially in a prophylactically or therapeutically effective amount. The present invention also relates to a vaccine comprising a bacterial enzyme catalyzing the synthesis of a glucose polymer in which glucoses are linked together via α-1,4 and α-1,6 bonds as a substance. active. Moreover, the inventors have unexpectedly shown that a mutant mycobacterial strain in which the gigA gene is inactive maintained significantly less well in infected organs than a wild type strain (see Example 4). Such a feature is advantageous for the preparation of a tuberculosis vaccine from this strain. The present invention therefore also relates to a vaccine comprising at least one bacterial strain of the genus Mycobacterium, in particular of the M. tuberculosis species, in which the gigA gene is inactivated.

Detailed Description of the Invention An antibiotic is a compound having antibacterial activity. It may especially be a bacteriostatic or a bactericidal.

Preferably, the antibiotics according to the invention target bacteria of the genus Mycobacterium and more preferably of the species Mycobacterium tuberculosis. The expression "glucose polymer" in which the glucoses are bonded to each other by means of α-1,4 and α-1,6 linkages comprises a combination of α-D-glucose units, preferably in pyranose form, which are linked together by means of a glycosidic bond formed from the hydroxy group borne by the carbon atom at the 1-position of the glucose molecule and the hydroxy group borne by the carbon atom in position 6 or by the carbon atom at the 4-position of the glucose molecule.

A representation of a molecule of α-D-glucopyranose (αD-Glcp) is given below: A representation of a glucose polymer in which glucoses are linked together via α-1 bonds , 4 and a-1.6 is given in Figure 1.

Preferably, the glucose polymer is selected from the group consisting of glucan and glycogen. By glycogen is meant a high molecular weight glucose polymer, in particular having a molecular weight greater than 100,000 Da, composed of residues - 4-aD-Glc-1 - branched, about every 5-6 residues, on the hydroxyl group. located on the carbon at the 6-position of the glucose molecule by chains of αD-Glc units also linked together via α-1,4 linkages. In vivo, bacterial glycogen, in particular mycobacterial glycogen, is mainly located inside the cell. Glucan means a glucose polymer whose structural characteristics are similar to those of glycogen. In vivo, glucan, in particular mycobacterial, is mainly located on the surface of the bacterium. The term "bacterial enzyme catalyzing the synthesis of a glucose polymer in which the glucoses are linked together via α-1,4 and α-1,6 bonds, any enzyme of bacterial origin that catalyzes a reaction". extending a glucose polymer by adding one or more αD-Glcp units to a free hydroxy group located on the carbon at the 4-position or 6-position of the polymer glucose. Enzymes of bacterial origin are any enzyme which can be found naturally in a bacterium or which is derived from such an enzyme by a limited number of modifications. Preferably the enzyme is selected from the group consisting of? -1,4-glucosyltransferase and a branching enzyme.

Α-1,4-glucosyltransferase, also known as glycogen synthase, is defined by the EC 2.4.1.11 class in the International Union of Biochemistry and Molecular Biology classification. The branching enzyme is defined by the EC 2.4.1.18 class in the classification of the International Union of Biochemistry and Molecular Biology. Preferably, the enzyme belongs to the genus Mycobacterium and more preferably belongs to the species Mycobacterium tuberculosis. As used herein, an enzyme belongs to the genus Mycobacterium and the species Mycobacterium tuberculosis, if it is expressed in the natural state by such bacteria. Preferably, the enzyme is selected from the group consisting of GIgA, GIgB and Rv3032, and more preferably the enzyme is GIgB. GIgA is an α-1,4-glucosyltransferase which is encoded by the gigA gene. In M. tuberculosis gigA is also noted Rv1212c and is represented by SEQ ID NO: 21. M. tuberculosis GIgA is represented by SEQ ID NO: 22. GIgB is a branching enzyme which is encoded by the glg8 gene. In M. tuberculosis glg8 is also noted Rv1326c and is represented by SEQ ID NO: 23. M. tuberculosis GIgB is represented by SEQ ID NO: 24. Rv3032 is an α-1,4-glucosyltransferase which is encoded by the Rv3032 gene. . In M. tuberculosis Rv3032 is represented by SEQ ID NO: 25. The role of these enzymes in the synthesis of glycogen and glucan of M. tuberculosis is summarized in Figure 1. As used herein, the identification of compounds with antibiotic activity includes any activity leading to the detection of compounds with antibiotic activity. It can be experimental or modeling methods, in which the compounds are selected from libraries of molecules, physical or modeled, or are synthesized, in vitro or in silico. As used herein, screening refers to a method in which a compound having the characteristic of inhibiting a bacterial enzyme catalyzing the synthesis of a glucose polymer in which glucose is bound to one another via linkages is sought. α-1,4 and α-1,6 within a molecule library. Preferably, the screening is a high throughput screening.

The measurement of the enzymatic activity of such enzymes is well known to those skilled in the art and is described in particular by Takata et al. (1994) Applied and Env. Microbiol. 60: 3096-3104, Guan and Preiss (1993) Plant Physiol. 102: 1269-1273.

The vaccines of the invention are useful both for preventing bacterial infections and for treating them. The vaccine comprising a bacterial enzyme catalyzing the synthesis of a glucose polymer in which glucoses are linked to one another via α-1,4 and α-1,6 linkages makes it possible to induce the production of antibodies directed against the enzyme and therefore endowed with antibacterial activity. As regards the vaccine comprising at least one bacterial strain of the genus Mycobacterium in which the glgA gene is inactivated, it is particularly suitable for the production of live attenuated vaccines. Genes other than glgA can also be inactivated to modulate virulence of the strain. Many techniques are available and well known to those skilled in the art, including those presented in the Examples, for inactivating glgA.

DESCRIPTION OF THE FIGURES FIG. 1 represents the glycogen and glucan synthesis metabolic pathway in M. tuberculosis.

Figure 2 Figure 2 shows the ratio Glc / (Glc + Ara + Man) (y-axis) for the strains indicated on the abscissa. The statistically significant differences from the H37Rv strain are indicated by two stars (**).

FIG. 3 represents the mass of glycogen relative to the mass of bacteria (amount of glycogen, ordinate axis) by taking strain H37Rv as base 100, for the strains indicated on the abscissa. The statistically significant differences from the H37Rv strain are indicated by two stars (**).

Figures 4 to 9 Figures 4 to 9 show the multiplication and the persistence (ordinate axis, log of the number of colony-forming units (CFU) per organ) of wild-type H37Rv (triangles), H37RvAtreZ (squares), H37RvAgIgC ( circles), H37RvARv3032 (diamonds), and H37RvAgIgA (cross) in the lungs (Figures 4, 6 and 8) and the spleen (Figures 5, 7 and 9) of BALB / c mice, as a function of time (axis of abscissa, number of days after infection). The results are expressed as the mean standard deviation of the number of CFUs for 5 infected mice. The mice were infected either intravenously (H37Rv, H37RvARv3032, H37RvAgIgA, Figures 6-9) or aerially (H37Rv, H37RvAtreZ, H37RvAgIgC, Figures 4 and 5). The star symbol (*) represents the statistically significant differences.

Examples

Materials and Methods Bacterial strains and culture conditions E. col / XL1-blue, the strain used for cloning experiments, was propagated in Luria Bertani (LB) medium (pH 7.5) (Becton Dickinson, Sparks, MD) . M. tuberculosis H37Rv was cultured at 37 ° C. in Middlebrook 7H9 medium supplemented with 10% of ADC enrichment medium (Difco) and 0.05% of Tween 80, and in a minimum medium of Sauton in the form of surface films, on a Middlebrook 7H11 agar medium supplemented with OADC (Difco). When necessary, the antibiotics were added at the following concentrations: kanamycin, 20 μg / ml; hygromycin B, 50 μg / ml. 2% sucrose was added to the solid medium for mutant selection by allele exchange.

Construction of the mutants treZ, glgB, glgA, glgC and glgA-Rv3032 The ts / sacB methodology described by Pelicic et al. (1997) Proc. Nat /. Acad. Sc /. USA 94: 10955-10960 was used to replace alleles at the TreZ (Rv1562c), glgC (Rv1213) and Rv3032 loci of M. tuberculosis H37Rv.

The gigA gene (Rv1212c) was invalidated using a two-step homologous recombination procedure (Jackson et al., (2001) In Mycobacterium tuberculosis protocols, Vol 54, Parish, T. and Stocker, NG (eds), Totowa NJ. Humana Press, pp. 59-75). The essential character of glg8 (Rv1326c) was studied following a classical strategy based on the construction of a merodiploid strain (Jackson et al., Cit.). Conventional PCR strategies were used to amplify the treZ, glg8, gigA, Rv3032 and glgC genes of M. tuberculosis H37Rv. gigA and its flanking regions were amplified using primers glgA.1 (5'-gcggaattccgcggtcgcattttcacgtgg-3 ', SEQ ID NO: 1) and glgA.2 (5'-gcggaattcggatggtcgacgaccatatcc-3', SEQ ID NO: 2). The invalidated gigA gene allele was constructed by cloning the kanamycin resistance gene (Km) from pUC4K (Amersham Pharmacia Biotech) in the gigA NcoI sites. Ncol digestion resulted in the deletion of 253 bp of the gigA coding sequence. glgA :: Km was then cloned into pJQ200-xylE (Stadthagen et al., (2007) J Biol Chem 282: 27270-27276), to give pJQgIgAKX, the plasmid used for allelic replacement. glgB and its flanking regions were amplified using primers B130.2 (5'-gcatttggtcctgaagaagctacg-3 ', SEQ ID NO: 3) and B130.4 (5'-ggggatatccacagcgccgaagtgggcgg-3', SEQ ID NO: 4). The invalidated allele of the glgB gene was constructed by cloning the Km cassette into the SalI sites of glgB. Cleavage with SalI led to the deletion of 740 bp of the coding sequence of glgB. glgB :: Km was then cloned with the xylE color marker (Pelicic et al., op cit) into the BamHI site of pJQ200 (Quandt & Hynes (1993) Gene 127: 15-21) to give pJQgIgBKX, the construct was used for allelic replacement at the glgB locus. glgC and its flanking regions were amplified using primers C364.1 (5'-cccgaattcggctgggatagacccgcaac-3 ', SEQ ID NO: 5) and C364.2 (5'-cccgaattcggcgtttcatcagcagttcg-3', SEQ ID NO: 6), then inserted into the vector pGEM -T easy (Promega), to give pGEMTgIgC. The invalidated allele of the glgC gene was then constructed by cloning the Km cassette into the BamHI site of glgC. glgC :: Km was finally cloned with the xylE gene into the BamHI site of pPR27 (Pelicic et al op cit), to give p27glgCKX. treZ was amplified using primer pair B48.2 (5'-gcttcctgggcggcgcataccatc-3 ', SEQ ID NO: 7) and B48.3 (5'-cccgaattcggccggctccgcagcccgcag-3', SEQ ID NO: 8). The invalidated allele of the treZ gene was constructed by cloning the Km cassette into the XhoI site of treZ treZ :: Km was then cloned with the xylE gene into the BamHI site of pPR27, to give p27treZKX. The Rv3032 gene and its flanking regions were amplified by PCR from M. tuberculosis H37Rv genomic DNA using primers Rv3032.1 (5'-gggctgcagatcgccggcgcgctggcc-3 ', SEQ ID NO: 9) and Rv3032.2 (5'-tgagccatgtcgcctccctg-3 ', SEQ ID NO: 10). The invalidated allele, Rv3032 :: Str, was obtained by inserting a streptomycin resistance cassette into the SmaI restriction site of Rv3032. Rv3032 :: Str was then cloned into NotI cut pPR27-xylE and blunt ended to obtain pPR27Rv3032SX, the construct used for allelic replacement in H37RvAgIgA.

Complementation of the glgA, glgB, glgC and treZ mutants Wild copies of the M. tuberculosis H37Rv gigA, glg8, glgC and treZ genes carried by the mycobacterial expression plasmid pVV16 (Jackson et al., 2000), respectively in pVVgIgA, pVVglgBtb , pVVglgC and pVVtreZ, were used for complementation studies. The primers used to amplify glgC were glgC.1 (5'-gggctôcaôccatgagagaagtgccgcac-3 ', SEQ ID NO: 11) and glgC.2 (51-cccaagcttgatccaaacacccttgcccacg-3', SEQ ID NO: 12). glgZ.2 (5'-tggtgggcatatgcctgaattccgagtatgggc-3 ', SEQ ID NO: 13) and glgZ.3 (51-gggaaoccggctccgcagcccgcag-3', SEQ ID NO: 14) were used to amplify treZ glgA.5 (5'-ccgcgcgcatatgcgggtggcgatgttgactc- 3 ', SEQ ID NO: 15) and glgA.6 (5'-cccaagcttcgcgcacaccttccggtagatg-3', SEQ ID NO: 16) were used to amplify gigA. glgB.1 (5'-cccccccccatatôagtcgatccgagaaactcacc-3 ', SEQ ID NO: 17) and glgB.2 (5'-gggaaöcttggcgggcgtcagccacagcgc-3', SEQ ID NO: 18) were used to amplify glg8. In addition, the glg8 gene of E. col / was amplified by PCR using primers glgBc.5 (5'-ggccgggcatatgtccgatcgtatcgatagagac-3 ', SEQ ID NO: 19) and glgBc.6 (5'-cccaagcttttctgcctcccgaaccagccag-3', SEQ ID NO: 20) and then cloned in pVV16, to give pVVglgBcoli. The primers were designed to generate PCR products corresponding to the entire genes lacking their STOP codons and carrying the NdeI or PstI and HindIII restriction sites (underlined in the above primer sequences), allowing direct cloning in pVV16. In this vector, the genes are constitutively expressed under the control of the transcription and translation signals of hsp60 and the produced recombinant proteins carry a tag of six histidines at their C-terminus. The production of the recombinant proteins in the complemented mutants was analyzed by immunolabeling with the Qiagen Penta-His monoclonal antibody as previously described (Jackson et al., (2000) J. 8iol Chem 275: 30092-30099).

Growth and persistence of M. tuberculosis in mice BALB / c female mice (6 to 8 weeks old) purchased from CERJanvier (Genest St Isle, France) were infected by air with wild-type M. tuberculosis H37Rv or with mutant strains treZ and glgC as previously described (Raynaud et al (2002) Mol Microbiol 45: 203-217). Mice were infected intravenously with 105 CFU of wild-type M. tuberculosis or by the mutants glgA and Rv3032 as described by Jackson et al. (1999) Infect. Immun. 67: 2867-2873. Five mice were used per experimental point and per strain.

Extraction, purification and analysis of mycobacterial glucan and glycogen The strains of M. tuberculosis were cultured in sail on Sauton's medium. The polysaccharides released into the culture medium, including glucan, were extracted and purified according to Lemassu & Daffé (1994) op. cit. The glucan content was determined after gas chromatography analysis of the trimethylsilated hydrolysis products, observing the glucose (from glucan) released relative to that of mannose and arabinose constituting other exopolysaccharides (mannan and arabinomanane). The glucans were purified according to Lemassu and Daffé (1994) op. cit.

Intracellular glycogen was extracted from sterilized bacteria, washed, weighed, and then broken with French press (140 bar). After removal of unbroken cells and walls by centrifugation, the glycogens were purified according to Bueding & Orrell (1964) J Biol Chem. 239: 4018-20. The glycogen content was given by the ratio of the mass of purified glycogen back to the mass of bacteria. Glycogens and glucans were analyzed by NMR and by DLS according to Dinadayala et al., (2004) J. Biol. Chem. 279: 12369-78. Example 1 Identification of M. tuberculosis H37Rv genes involved in the biosynthesis of α-D-glucan and glycogen The glucans of the M. tuberculosis capsule and the M. bovis BCG strain are composed of repeating units of residues - 4-aD-Glcp-1 - and some 4-aD-Glcp units substituted in the 6-position with one to several aD-Glcp residues, indicating a highly branched glycogen-like structure. Given the structural similarities between α-D-glucan and glycogen, it is believed that the synthesis of both polysaccharides involves similar catalytic reactions. The two polysaccharides may even share, at least partially, a common biosynthetic pathway. In addition to the Rv3032 gene of α-1,4 glucosyltransferase, which has recently been shown to be involved in the synthesis of 6-O-methylglucosyl lipopolysaccharide (MGLP) and glycogen (Stadthagen et al., 2007). J. Biol.

Chem. 282: 27270-27276), four genes were identified as potentially involved in the synthesis of? -D-glucan and / or glycogen in the M. tuberculosis H37Rv genome. Rv1213 encodes a pyrophosphorylase ADPGIc. The product of this gene shares 35% amino acid identity with E. coli ADPGIc pyrophosphorylase (GlgC). col / which is responsible for the synthesis of the glucosyl donor, ADP-Glc. Rv1213 is immediately adjacent to the putative glycogen synthase gene, gIgA (Rv1212c), whose orthologue to Corynebacterium glutamicum has been implicated in glycogen synthesis (Tzvetkov et al (2003) Microbiology 149: 1659-1673).

Rv1326c encodes an α-1,4-glucan branching enzyme potentially responsible for the branching of oligoglucosyl units on the glucoside backbone (Garg et al (2007) Protein Expr Purif 51: 198-208). Rv1326c shares 48% amino acid identity (63% similarity) with E-branching enzyme. al. This M. tuberculosis glgB gene is arranged in operon with gIgE (Rv1327c), a gene encoding a glucanase whose orthologue in M. smegmatis is required for the degradation and recycling of glycogen (Belanger & Hatfull (1999) J Bacteriol 181: 6670-6678). It is also adjacent to another catabolic gene, glgP (Rv1328), probably encoding glycogen phosphorylase (Schneider et al (2000) Mol Gen. Genet 263: 543-553). Finally, Rv1562c (TreZ) is the third gene of an operon that has been implicated in trehalose metabolism (De Smet et al (2000) Microbiology 146: 199-208, Woodruff et al (2004) J. Biol. Chem 279: 28835-28843). The sequence similarity that Rv1562c shares with bacterial α-1,4-glucan branching enzymes (26% identity with E. coli GIgB protein / over a 241 amino acid overlap) suggested that it could also catalyze the branching of glycogen and / or α-D-glucan. However, the inventors have shown that it is not involved in this activity while it is now known to be involved in the synthesis of trehalose. The metabolic pathway of glycogen and glucan synthesis in M. tuberculosis is summarized in Figure 1.

Example 2 Invalidation of glgA, glgB, glgC and treZ genes by allele exchange in M. tuberculosis H37Rv glgA, glgC and treZ were invalidated by homologous recombination in M. tuberculosis H37Rv as indicated above. Allelic replacement at glgA, glgC and treZ loci was confirmed in each mutant by Southern hybridization. A mutant glgA, a mutant treZ and a mutant glgC were selected for the remainder of the study and are respectively designated H37RvAgIgA, H37RvAtreZ and H37RvAgIgC. Unexpectedly, despite several attempts to invalidate the gene glgB gene in M. tuberculosis H37Rv, no allelic exchange candidate could be isolated. However, it was possible to perform a simple crossover at the glgB locus. In order to determine whether the impossibility of invalidating the glgB gene was due to its essentiality, the inventors then performed an allelic exchange experiment using a merodiploid strain of M. tuberculosis. This strain was constructed by transforming the single crossover clone at the glgB locus with pVVglgBtb. Gene replacement was then performed following standard procedures (Jackson et al., Op.cit.). PCR analysis and Southern hybridization of 8 clones corresponding to potentially cross-linked recombinants showed that allelic exchange occurred at the chromosomal locus glgB of 5 of them. Thus, expression of glgB from pVVglgBtb is sufficient for the survival of an invalid mutant of M. tuberculosis, indicating that this gene is essential for growth. Allelic replacement at the M. tuberculosis glgB locus could also be performed in the presence of a wild-type copy of the E. coli glg8 gene. col / expressed from pVVglgBcoli, suggesting that the two orthologues of glg8 have a similar function. Finally, although it is possible to obtain simple knockout mutants for gigA and Rv3032 (Stadthagen et al, op cit), two independent experiments to disrupt the Rv3032 gene in H37RvAgIgA in order to create a mutant deficient for both a-1,4 glucosyltransferases were unsuccessful. Thus, a functional copy of at least one of these two genes is necessary for the growth of M. tuberculosis H37Rv.

EXAMPLE 3 Qualitative and Quantitative Analysis of GlgA-, GlgB-, GlgC- and TreZ Mutants in M. tuberculosis H37Rv The analysis of glucan production is given by the ratio of glucose (Glc) versus Glc + arabinose (Ara) + mannose ( Man) (Figure 2), glucose being the only constituent glucan monosaccharide, and arabinose and mannose the constituents of manan and arabinoman.The analysis of glycogen production is given by the glycogen mass ratio versus the mass of bacteria (Figure 3).

Activation step: Figures 2 and 3 show that the inactivation of glgC modifies the biosynthesis of intracellular glycogen, but also that of surface glucan. It should be noted that this quantitative difference is accompanied by a qualitative difference, since the estimated glucan radius by DLS decreases from 28 to 22 nm. The phenotype is restored in the complemented mutant. The GIgC protein is therefore involved in the biosynthesis of the two polysaccharides.

Elongation step The glucan biosynthesis is affected in the same way in the glgA- mutant, but not in the Rv3032 and TreZ mutants Moreover, the Rv3032 gene, whose role in glycogen biosynthesis is already known, complements the phenotype of the mutant gigA- which indicates a functional redundancy between the two enzymes encoded by these genes.The GIgA protein is therefore involved in the glucan biosynthesis while the protein encoded by the Rv3032 gene is known for its involvement in the biosynthesis of the glycogen.

Branching step: Since the glgB gene is essential, the implication of this gene has been studied on the merodiploid strain H37RvAgIgB / pVVglgBtb. Since glucan and glycogen production represent only 57% and 37% of that in the wild strain, and DLS and polarimetry analyzes show that the structure of the polysaccharides is affected, the GIgB protein is involved. in the glucan and glycogen biosynthetic pathways. Example 4 Multiplication and persistence of glgA, glgC and treZ mutants in mice A mouse infection model was used to analyze the effects of the invalidation of glgA, Rv3032, glgC or treZ on the virulence of M. tuberculosis H37Rv. The ability of wild-type and mutant strains to multiply and persist in the lungs and spleen of BALB / c mice was studied by CFU analysis. As shown in Figures 4 to 9, organ bacterial loads in H37RvAtreZ, H37RvAgIgC and H37RvARv3032-infected mice were comparable to those of H37Rv parental strain-infected mice over a period of 90-100 days. In comparison, the ability of H37RvAgIgA to maintain itself in both organs during late stages of infection was significantly reduced. At 42 and 90 days post-infection, the lungs of mice infected with the glgA mutant carried approximately 10-fold less CFU than those of control-infected mice, M. tuberculosis H37Rv.30

Claims (14)

REVENDICATIONS1. Use of a bacterial enzyme catalyzing the synthesis of a glucose polymer in which glucoses are linked together via α-1,4 and α-1,6 bonds, for the identification of compounds with antibiotic activity . 2. Use according to claim 1, for the identification of compounds targeting bacteria of the genus Mycobacterium. 3. Use according to claim 1 or 2, for the identification of compounds targeting bacteria of the Mycobacterium tuberculosis species. 4. Use according to one of claims 1 to 3, wherein the glucose polymer is selected from the group consisting of glucan and glycogen. 5. Use according to one of claims 1 to 4, wherein the enzyme is selected from the group consisting of a-1,4-glucosyltransferase and a branching enzyme. 20 6. Use according to one of claims 1 to 5, wherein the enzyme belongs to the genus Mycobacterium. 7. Use according to one of claims 1 to 6, wherein the enzyme belongs to the species Mycobacterium tuberculosis. Use according to one of claims 1 to 7, wherein the enzyme is selected from the group consisting of GlgA, GlgB and Rv3032. 9. Use according to one of claims 1 to 8, wherein the enzyme is GlgB. 10. A method for screening compounds with antibiotic activity, comprising the steps of: placing a compound whose antibiotic activity is to be determined in the presence of a bacterial enzyme catalyzing the synthesis of a glucose polymer in which the glucoses are linked together via α-1,4 and α-1,6 linkages; measuring the activity of the enzyme in the presence of the compound; comparing the activity of the enzyme in the presence of the compound to its activity in the absence of the compound; selecting the compound as an antibiotic if the activity of the enzyme in the presence of the compound is lower than in the absence of the compound. 11. The method of claim 10 for screening compounds targeting bacteria of the genus Mycobacterium. 12. The method of claim 10 or 11 for screening compounds targeting bacteria of the Mycobacterium tuberculosis species. The method according to one of claims 10 to 12, wherein the glucose polymer is selected from the group consisting of glucan and glycogen. 14. Process according to one of Claims 10 to 13, in which the enzyme is as defined in one of Claims 5 to 9.