Patent Application
20050250120
The subject of the present invention is the identification of nucleotide sequences which make it possible in particular to distinguish, in diagnostic terms, an immunization resulting from a BCG vaccination from an M. tuberculosis infection. The sequences in question are specific either to M. bovis BCG/M. bovis, or to M. tuberculosis. The subject of the present invention is also a method for detecting the sequences in question, a method for detecting antibodies generated by the products of expression of these sequences and the kits for carrying out these methods. Finally, the subject of the present invention is novel vaccines.
The high rate of mortality and morbidity caused by Mycobacterium tuberculosis, the etiological agent for tuberculosis, brings about the need to develop novel vaccines and ever shorter chemotherapeutic treatments. Indeed, the appearance of M. tuberculosis strains resistant to antituberculars and the increased risk in immunosuppressed patients, for example in AIDS patients, of developing tuberculosis, necessitates the development of rapid, specific and reliable methods for the diagnosis of tuberculosis and the development of novel vaccines. The conventional BCG vaccine is derived from a Mycobacterium bovis strain which was attenuated by repeated serial passages on bile potato-glycerinox agar (Calmette, 1927; Bloom and Fine, 1994). However, in spite of almost 50 years of worldwide use, the reason for the attenuation of M. bovis BCG is still unknown. Questions remain as regards the protection conferred by the vaccine against pulmonary tuberculosis, with an efficacy of between 0 and 80% (Fine, 1994). Furthermore, many BCG substrains exist and offer various levels of protection against tuberculosis in a mouse model (Lagranderie et al., 1996). The attenuation of the original M. bovis strain may have been caused by mutations in the genome of the bacillus which were selected during serial passages of the strain, which mutations remained stable in the genome. However, as the original M. bovis strain has been lost, direct comparison between it and M. bovis BCG is impossible. In spite of that, the identification of genetic differences between M. bovis, M. bovis BCG and M. tuberculosis is likely to reveal locations whose alteration may have led to the attenuation of M. bovis BCG.
The M. tuberculosis DNA has more than 99.9% homology with the DNA of the other members of the tuberculous complex (M. bovis, M. microtis, M. africanum). Although closely related, these strains may be differentiated on the basis of their host range, their virulence for humans and their physiological characteristics (Heifets and Good, 1994). As in the case of the attenuation of BCG, the genetic base for the phenotypic differences between the tubercle bacilli is mainly unknown. However, the wealth of information contained in the genomic sequence of M. tuberculosis H37Rv led to the thought that the genetic variations between the strains was going to be revealed (Cole et al., 1998). Genomic comparison presents a powerful tool for such research studies since the whole genomes may be studied in preference to the study of genes in their individual forms. A previous comparative study of M. bovis and M. bovis BCG by substractive genomic hybridization has shown that three regions, designated RD1, RD2 and RD3, were deleted in M. bovis BCG compared to M. bovis (Mahairas et al., 1996). However, the role, where appropriate, of these regions in the attenuation of M. bovis BCG has not been clearly established. Similarly, other studies of genomic differences between M. bovis, M. bovis BCG and M. tuberculosis have shown that many polymorphic locations existed between these strains (Philipp et al., 1996). Although the exact nature of these polymorphisms has not been elucidated, additional analyses have revealed that a polymorphism was due to the deletion of 12.7 kb in M. bovis and BCG compared to M. tuberculosis (Brosch et al., 1998). From that, it appears that there are two classes of deletion: those which are absent from BCG but present in M. bovis and M. tuberculosis and those which are absent from M. bovis and BCG but present in M. tuberculosis.
The bacterial artificial chromosome (BAC) library for M. tuberculosis H37Rv demonstrates complete knowledge of the genomic sequence of M. tuberculosis and presents a potential as a tool for postgenomic applications such as genomic comparisons (Brosch et al., 1998). To push the investigations into the genomic differences between M. tuberculosis and M. bovis BCG even further, the inventors prepared a BAC library from M. bovis BCG. This type of library indeed has certain advantages. Firstly, the BAC system can maintain large inserts of mycobacterial DNA, up to 120 kb. The 4.36 Mb of M. bovis BCG genome could therefore be represented in 50 to 60 clones, simplifying the storage and handling of the library. Secondly, the BAC system can allow, in complete confidence, replication of the inserts without genericing rearrangement or deletion in the clones. From that, alterations of the insert cannot be at the origin of an error for the duration in the genome. Thirdly, the positioning of the BAC clones on the M. bovis BCG chromosome is likely to generate a map of clones which overlap, which ought to allow direct comparison of the local segments on the M. tuberculosis and M. bovis BCG genome, while being a resource of interest for the sequencing of the M. bovis BCG genome.
The construction of a BAC library for M. bovis BCG-Pasteur (1-2049) is described below as well as its use, in conjunction with the BAC library for M. tuberculosis H37Rv (1-1945), as a tool for genomic comparison. With this approach, the inventors have been able to identify novel deletions and insertions between the tubercle bacilli, which makes it possible to have a picture in two genomes of the dynamics and differentiation in the M. tuberculosis complex.
The main route for extracting biological information from the genome is the comparison between the genomes. The technology of biochips or “DNA chips” (Chee et al., 1996; DeRisi et al., 1997) described, for example, in patents No. WO97/02357 and No. WO97/29212 makes it possible to make alignments and to select the sequences of interest. However, the availability of a minimum set of BAC clones for the genomes of M. bovis BCG and M. tuberculosis H37Rv has offered the inventors ready-to-use tools for the abovementioned comparative studies. The BAC library for M. bovis BCG contains more than 1500 clones with an average size of inserts of about 75 kb. 57 clones cover the BCG genome including a HindIII fragment of 120 kb which was absent from the M. tuberculosis BAC library. The construction of BAC chips from the M. bovis BCG library should allow the inventors to extend their comparative studies relating to the tubercle bacillus. These fragments can be hybridized with the genomic DNA from clinical isolates from M. tuberculosis or epidemic strains in order to identify other deletions or rearrangements, and from that, allow a novel picture relating to the plasticity of the genome as well as the identification of the genes and the gene products which may be involved in the virulence.
At the end of the experiments reported here, the inventors identified 10 locations or loci which are absent from M. bovis BCG compared to M. tuberculosis. Hybridizations with the genomic DNA of M. bovis revealed that 7 of these loci were also deleted in M. bovis compared to M. tuberculosis. Thus, in the text below, every time reference is made to the characteristics common to the genome of M. bovis BCG and to that of M. bovis it will be indicated that this means the “genome of M. bovis BCG/M. bovis”.
It was then found that 3 of the specific deletions which appeared in M. bovis BCG were identical to the RD1, RD2 and RD3 regions defined by the Stover team (Mahairas et al., 1996). Thus, by retaining the preceding nomenclature the inventors called the other 7 deletions of the M. bovis BCG/M. bovis genome, RD4, RD5, RD6, RD7, RD8, RD9 and RD10.
Other deletions have been found to be specific to the M. tuberculosis genome, it being understood that the “corresponding” sequences were present in M. bovis BCG/M. bovis; they were called RvD1 and RvD2 (tables 1 and 2).
The RD5-RD10, RvD1 and RvD2 deletions allowed the inventors to identify thoroughly the dynamics of the genome in the tubercle bacillus and gave information relating to the genetic bases of the phenotypic differentiation of the complex. The identification of RvD1 and RvD2 as deletions of the M. tuberculosis H37Rv genome shows that the deletion process does not function in a single direction, and the loss of information can therefore occur both in bovine strains and in human strains. It is observed that 8 of the 10 deletions detected are located in a region of the chromosome where termination of replication probably occurs.
The inventors then, within each deleted region, identified several ORFs (or open reading frames) or genes and they tried to determine the putative function of each of them (table 1).
The subject of the present invention is therefore nucleotide sequences deleted from the genome of M. bovis BCG/M. bovis and present in the genome of M. tuberculosis or conversely chosen from the following ORFs and genes: Rv2346c, Rv2347c, Rv2348c, plcC, plcB, plcA, Rv2352c, Rv2353c, Rv3425, Rv3426, Rv3427c, Rv3428c, Rv1964, Rv1965, mce3, Rv1967, Rv1968, Rv1969, lprM, Rv1971, Rv1972, Rv1973, Rv1974, Rv1975, Rv1976c, Rv1977, ephA, Rv3618, Rv3619c, Rv3620c, Rv3621c, Rv3622c, IpqG, cobL, Rv2073c, Rv2074, Rv2075, echA1, Rv0223c, RvD1-ORF1, RvD1-ORF2, Rv2024c, plcD, RvD2-ORF1, RvD2-ORF2, RvD2-ORF3, Rv1758.
The expression “nucleotide sequence” according to the present invention is understood to mean a double-stranded DNA, a single-stranded DNA and products of transcription of said DNAs.
More particularly, the nucleotide sequences listed above are grouped into nucleotide regions according to the following distribution:
Advantageously, 3 of the deletions (RD5, RD6 and RD8) contain 6 genes encoding PE and PPE proteins. As it has been suggested that these proteins have a possible role in antigenic variation (Cole et al., 1998), it can be deduced therefrom that these loci may represent sites of hypervariability between the tubercle strains.
At least 9 proteins capable of being exported or exposed at the surface are encoded by RD4 to RD10, which indicates that these polypeptides perhaps have a major role in the immune recognition of the bacillus. It has indeed been shown that secreted polypeptides can have a potential stimulatory role in the immune system and they are capable of playing a role of antigens known to become involved during the early stage of infection (Elhay et al., 1998; Horwitz et al., 1995; Rosenkrands et al., 1998).
The fact that RD5 and RD6 contain genes encoding proteins belonging to the ESAT-6 family, 14 of which are organized into 11 distinct loci, is particularly significant (F. Tekaia, S. Gordon, T. Garnier, R. Brosch, B. G. Barrell and S. T. Cole, submitted). ESAT-6 is a major T cell antigen which appears to be secreted by the virulent tubercle bacillus independently of the signal peptide (Harboe et al., 1996). It accumulates in the extracellular medium during the early phases of growth and its gene is located in RD1, a region which is deleted from the genome of M. bovis BCG (Mahairas et al., 1996; Philipp et al., 1996). 3 of the 10 RD regions thus contain genes of the ESAT-6 family, which indicates that other sites of ESAT-6 genes can also give rise to deletions or rearrangements.
The genomic sequence of M. tuberculosis H37Rv has moreover revealed the presence of 4 highly related genes encoding phospholipase C enzymes called plcA, plcB, plcC and plcD (Cole et al., 1998). Phospholipase C has been recognized as a major virulence factor in a number of bacteria including Clostridum perfringens, Listeria monocytogenes and Pseudomonas aeruginosa where it plays an intracellular role in the dissemination of bacterial cells, in intracellular survival and in cytolysis (Titball, 1993). The RD5 deletion includes 3 genes (plcA, plcB and plcC), this region being absent from M. bovis, M. bovis BCG and M. microti. The detection of the phospholipase activity in M. tuberculosis, M. microti and M. bovis but not in M. bovis BCG has been previously described in (Johansen et al., 1996; Wheeler and Ratledge, 1992) as well as the role of the enzymes encoded by plcA and plcB (also known under the name mpcA and mpcB) in the hydrolysis both of phosphatidylcholine and sphingomyelin. The levels of phospholipase C activity which are detected in M. bovis are considerably less than those observed in M. tuberculosis which are in agreement with the loss of plcABC, the sphingomyelinase activity still being detectable. The sequence data presented here show that full-length phospholipase is encoded by the plcD gene in M. bovis BCG-Pasteur and that its considerable sequence similarity with the products of plcA and plcB indicates that it is probably endowed both with phospholipase activity and with a sphingomyelinase activity. It is therefore probable that plcD may be responsible for the residual phospholipase C activity in strains exhibiting the RD5 deletion, such as M. bovis, although it is difficult to link this interpretation to the observed absence of phospholipase C in spite of the presence of sphingomyelinase in the M. bovis BCG strain used in other studies (Johansen et al., 1996; Wheeler and Raledge, 1992). Studies of expression with the cloned plcD gene ought to clarify this point.
The mce gene has been described by the Riley team as encoding a putative protein of M. tuberculosis of the invasin type, whose expression in E. coli allows the invasion of HeLa cells (Arruda et al., 1993). Three other Mce proteins have been identified as part of the genome sequencing project with their gene occupying the same position in the four large highly conserved operons comprising at least eight genes (Cole et al., 1998; Harboe et al., 1996). It is difficult to deduce the effects of the loss of mce3 (RD7) on M. bovis, M. microti and M. bovis BCG because of the fact that the remaining three copies of mce could complement any loss of activity, unless the operons are differently expressed. However, it is of interest to note that RD7 is absent from certain members of the M. tuberculosis complex which are not virulent for humans, suggesting that RD7 can play a specific role in human disease.
The genome of M. tuberculosis H37Rv also encodes six proteins (Eph-A-F) which show similarity with epoxide hydrolases whereas at least 21 enoyl-CoA hydratases (EchA1-21) and multiple aldehyde dehydrogenases are present (Cole et al., 1998). The loss of ephA (RD8), echA1 and the aldehyde dehydrogenase encoded by Rv0223c (RD10) in M. bovis BCG/M. bovis can therefore be compensated by other enzymes although the substrate specificity of the M. tuberculosis enzymes is unknown. The epoxide hydrolases are generally considered as detoxifying enzymes; a recent report has again showed that they play a role in the activation of leukotoxins (Moghaddam et al., 1997), a toxic fatty acid produced by the leukocytes which are involved in respiratory distress syndrome in adults. However, the question of knowing if the M. tuberculosis epoxide hydrolases can chemically modify host chemokines is without response. Alternatively, they can play a role in lipid detoxification of the products of peroxidation which are generated by oxygen radicals from activated macrophages.
RD9 is a region deleted from the genomes of M. africanum, M. bovis, M. bovis BCG and M. microti compared to M. tuberculosis. Consequently, in contrast to the other RD regions, the location of M. africanum is close to M. bovis, which indicates the presence of this strain between M. tuberculosis and M. bovis (Heifets and Good, 1994). Similarly, the RD4 region can differentiate M. microti from the bovine strains (table 2).
Thus, the proteins encoded by RD4 to RD10 can therefore have antigens of interest, allowing discrimination between individuals vaccinated with BCG and patients infected with M. tuberculosis.
Thus, the subject of the present invention is also a method for the discriminatory detection and identification of M. bovis BCG/M. bovis or M. tuberculosis in a biological sample, comprising the following steps:
Preferably, in the context of the present invention, the biological sample consists of a fluid, for example human or animal serum, blood, a biopsy, bronchoalveolar fluid or pleural fluid.
Analysis of the desired sequences may, for example, be carried out by agarose gel electrophoresis. If the presence of a DNA fragment migrating to the expected site is observed, it can be concluded that the analyzed sample contained microbacterial DNA. This analysis can also be carried out by the molecular hybridization technique using a nucleic probe. This probe will be advantageously labeled with a nonradioactive (cold probe) or radioactive element.
Advantageously, the detection of the mycobacterial DNA sequences will be carried out using nucleotide sequences complementary to said DNA sequences. By way of example, they may include labeled or nonlabeled nucleotide probes; they may also include primers for amplification.
The amplification technique used may be PCR but also other alternative techniques such as the SDA (Strand Displacement Amplification) technique, the TAS technique (Transcription-based Amplification System), the NASBA (Nucleic Acid Sequence Based Amplification) technique or the TMA (Transcription Mediated Amplification) technique.
The primers in accordance with the invention have a nucleotide sequence chosen from the group comprising SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, and SEQ ID No. 18 with:
The amplified fragments may be identified by agarose or polyacrylamide gel electrophoresis by capillary electrophoresis or by a chromatographic technique (gel filtration, hydrophobic chromatography or ion-exchange chromatography). The specification of the amplification may be controlled by molecular hybridization using probes, plasmids containing these sequences or their product of amplification.
The amplified nucleotide fragments may be used as reagent in hybridization reactions in order to detect the presence, in a biological sample, of a target nucleic acid having sequences complementary to those of said amplified nucleotide fragments.
These probes and amplicons may be labeled or otherwise with radioactive elements or with nonradioactive molecules such as enzymes or fluorescent elements.
The subject of the present invention is also a kit for the discriminatory detection and identification of M. bovis BCG/M. bovis or M. tuberculosis in a biological sample comprising the following components:
Indeed, in the context of the present invention, depending on the pair of primers used, it is possible to obtain very different results. Thus, the use of primers which are internal to the deletion, are described in the present invention for RD4, RD5 and RD8, is such that no amplification product is detectable in M. bovis BCG. However, the use of primers external to the region of deletion does not necessarily give the same result, as regards for example the size of the amplified fragment, depending on the size of the deleted region in M. bovis BCG. Thus, the use of the pair of primers SEQ ID No. 5/SEQ ID No. 6 for the detection of RD6 is likely to give rise to an amplicon in M. bovis BCG of about 3 801 bp whereas the use of the pair of primers SEQ ID No. 11/SEQ ID No. 12 for the detection of RD9 will give rise in M. bovis BCG to an amplicon of about 1 018 bp.
The subject of the invention is also the use of at least one pair of primers as defined above for the amplification of DNA sequences of M. bovis BCG/M. bovis or M. tuberculosis.
The benefit of the use of several pairs of primers will be quite obviously to cross the results obtained with each of them in order to refine the result of the analysis. Indeed, when it is indicated, in the context of the present invention, that some deletions are specific to M. bovis BCG/M. bovis, that is not completely accurate since some of them are also found in M. microti OV254, in M. tuberculosis CSU#93 and in M. africanum as well as certain clinical isolates (table 2). Thus, the use of the pair of primers SEQ ID No. 1/SEQ ID No. 2 specific for the RD4 region will not give rise to amplicons of normal size with M. bovis BCG/M. bovis in the biological sample. On the other hand, if the pair of primers used is SEQ ID No. 5/SEQ ID No. 6 specific for RD6 and that amplicons of normal size are not found, it will not be possible, from this result only, to discriminate between the presence, in the biological sample, of M. bovis BCG/M. bovis, M. microti OV254 and M. tuberculosis CSU#93.
The discrimination will be more radical when it will involve determining if the mycobacterium present in the biological sample to be analyzed is M. bovis BCG/M. bovis or M. tuberculosis H37Rv because the pairs of primers SEQ ID No. 15/SEQ ID No. 16 and SEQ ID No. 17/SEQ ID No. 18 are specific only for M. tuberculosis H37Rv. Consequently, the absence of amplicon of normal size during the use of either of these pairs of primers may be considered as indicative of the presence of M. tuberculosis H37Rv in the biological sample analyzed.
The subject of the present invention is also the products of expression of all or part of the nucleotide sequences deleted from the genome of M. bovis BCG/M. bovis and present in M. tuberculosis or conversely as listed in table 1.
The expression “product of expression” is understood to mean any protein, polypeptide or polypeptide fragment resulting from the expression of all or part of the abovementioned nucleotide sequences and preferably exhibiting on least one of the following characteristics:
Indeed, the subject of the present invention is also a method for the discriminatory detection in vitro of antibodies directed against M. bovis BCG/M. bovis or M. tuberculosis in a biological sample, comprising the following steps:
The subject of the invention is also a method for the discriminatory detection of a vaccination with M. bovis BCG or an infection by M. tuberculosis in a mammal, comprising the following steps:
Cell proliferation may be measured, for example, by incorporating 3H-Thymidine.
The invention also relates to a kit for the in vitro diagnosis of an M. tuberculosis infection in a mammal optionally vaccinated beforehand with M. bovis BCG comprising:
The reagents allowing the detection of the antigen-antibody complexes may carry a marker or may be capable of being recognized in turn by a labeled reagent, more particularly in the case where the antibody used is not labeled.
The subject of the invention is also mono- or polyclonal antibodies, their chimeric fragments or antibodies, capable of specifically recognizing a product of expression in accordance with the present invention.
The present invention therefore also relates to a method for the discriminatory detection of the presence of an antigen of M. bovis BCG/M; bovis or M. tuberculosis in a biological sample comprising the following steps:
The invention also relates to the kit for the discriminatory detection of the presence of an antigen of M. bovis BCG/M. bovis or M. tuberculosis in a biological sample comprising the following steps:
The abovementioned reagents are well known to a person skilled in the art who will have no difficulty adapting them to the context of the present invention.
The subject of the invention is also an immunological composition, characterized in that it comprises at least one product of expression in accordance with the invention.
Advantageously, the immunological composition in accordance with the invention enters into the composition of a vaccine when it is provided in combination with a pharmaceutically acceptable vehicle and optionally with one or more immunity adjuvant(s) such as alum or a representative of the family of muramylpeptides or incomplete Freund's adjuvant.
The invention also relates to a vaccine comprising at least one product of expression in accordance with the invention in combination with a pharmaceutically compatible vehicle and, where appropriate, one or more appropriate immunity adjuvant(s).
Standard knowledge on the evolution of the M. tuberculosis complex is based on the hypothesis that M. tuberculosis is derived from M. bovis (Sreevatsan et al., 1997). However, a distribution of RD1 to RD10 among the tuberculous complex suggests that a linear evolution of M. tuberculosis from M. bovis is too simplistic. It appears, indeed, in a more probable manner, that the two bacilli are derived from a common strain, that the deletions therefore reflect the adaptation of the bacilli to their particular niche, that is to say that the loss of RD4 to RD10 probably helped M. bovis to become a more potent pathogenic agent for bovines than M. tuberculosis. Functional genomic studies will determine which role these deletions play in the phenotypic differentiation of the tuberculous complex.
Finally, the inventors have detected, still by comparing the BAC of M. tuberculosis H37Rv and the BAC of M. bovis BCG, two duplications in the genome of M. bovis BCG-Pasteur, called DU1 and DU2. They are duplications of regions of several tens of kilobases which appear to be absent both from the M. bovis and M. tuberculosis H37Rv type strain. The detection of these two duplications was made following digestion of the same clones for each BAC with HindIII and analysis on a pulsed-filled electrophoresis gel (PFGE). These observations have been confirmed by hybridization of the digested chromosomal DNA derived from M. bovis BCG, from the type strain of M. bovis and M. tuberculosis H37Rv with selected probes covering the duplicated regions. Primers specific for the rearranged regions were prepared and tested on the genomic DNA from additional isolates of M. bovis BCG and M. tuberculosis.
It was determined that DU1 and DU2 were present in three strains of M. bovis BCG including in M. bovis BCG-Pasteur and absent from three other substrains of M. bovis BCG.
These two duplications are also absent from the type strain of M. bovis and M. tuberculosis H37Rv.
Thus, still in the context of the present invention in relation to the discriminatory detection of M. bovis or M. tuberculosis, the subject of the invention is also a method for the discriminatory detection and identification of M. bovis BCG or M. tuberculosis in a biological sample comprising the following steps:
The digestion with the restriction enzyme may indeed be carried out either on the entire genome of the mycobacterium, or on one or more clones of the library produced from the genome in question.
Preferably, the restriction enzyme used in the context of the abovementioned method is HindIII.
As regards the analysis of restriction fragments, it may consist in counting said fragments and/or in determining their length. Indeed, as is explained below, HindIII digestion of M. bovis BCG gives rise to one fragment more than those obtained after HindIII digestion of the genome of M. tuberculosis H37Rv. The number of fragments thus obtained may also be complemented by the determination of their length. This may be carried out by means of techniques well known to persons skilled in the art, for example on a pulsed filled electrophoresis gel (PFGE). It has thus been possible to determine that the additional fragment appearing after HindIII digestion of the genome of M. bovis BCG-Pasteur had a size of about 29 kb.
Another way of analyzing the restriction fragments resulting from the enzymatic digestion of the genome of the mycobacterium as described above consists in bringing said fragments into contact with at least one appropriate probe, covering for example the duplicated region, under hybridization conditions so as to then identify the number and size of the fragments which have hybridized. The probes used for this purpose may be labeled or nonlabeled according to techniques well known to persons skilled in the art.
Map of the BAC of Mycobacterium bovis BCG-Pasteur superposed on the BAC of M. tuberculosis H37Rv and on the cosmid maps. The “X” clones correspond to the clones in pBeloBAC11 of M. bovis BCG, the “XE” clones correspond to the clones in pBACe3.6 of M. bovis BCG, the “Rv” clones correspond to the clones in pBeloBAC11 of M. tuberculosis, the clones “Y” correspond to the clones in the cosmid pYUB328 of M. tuberculosis and the “I” clones correspond to the clones in the cosmid pYUB412 of M. tuberculosis. The location of each deletion region is shown on the map. The scale bars indicate the position on the genome of M. tuberculosis.
The regions deleted from the genome of M. tuberculosis are delimited by arrows with a sequence flanking each deletion. The ORFs (open reading frames) are represented by “directed” boxes showing the direction of transcription as described above (Cole et al., 1998). The putative functions and the families of the ORFs are described in table 3. The stop codons are indicated by small vertical bars.
Digestions of the Rv143 clone of the BAC with the endonucleases EcoRI, PstI and StuI revealed that fragments of 1.5 kb (EcoRI), 1.5 kb (PstI), 1.3 and 2.7 kb (StuI) show no binding with M. bovis or M. bovis BCG DNA probes (the absent bands are indicated by arrows). The size in kilobases (kb) is indicated on the left.
A. Size polymorphism in amplicons generated by flanking primers (i) RvD1 and (ii) RvD2. PCR reactions were carried out using the GeneAmp XL PCR kit (Perkin Elmer) with DNA templates of M. tuberculosis H37Rv, M. bovis and M. bovis BCG-Pasteur in combination with primers described in table 3. The size in kilobases is indicated on the left of each image.
B. Structure of the ORFs of the loci of RvD1 and RvD2. The sequence of the two loci was determined from M. bovis BCG Pasteur, the flanking sequence in M. tuberculosis H37Rv being shown. The putative functions of the ORFs are described in table 1 with vertical barriers representing the stop codons.
The present application is not limited to the above description and will be understood more clearly in the light of the examples below which should not in any manner be considered as limiting the present invention.
1. Procedures and Results
Construction of an M. bovis BCG-Pasteur BAC Library
Recent attempts for cloning very large inserts of mycobacterial DNA (120-180 kb) into the vector pBeloAC11 have resulted in failure (Brosch et al., 1998). To establish if this size determination was due to the vector pBeloBAC11, the inventors have tested in parallel the vector pBACe3.6 from BAC which uses the selection system sacB (Lawes and Maloy, 1995; Pelicic et al., 1996). Ligations carried out with fragments in the size ranges from 50 to 125 kb gave 5 to 10 times fewer transformants in pBACe3.6 than the control ligations using pBeloBAC11 (clones X). The size of an insert in the clones pBACe3.6 was approximately between the interval 40-100 kb, similar to what was observed for pBeloBAC11. This suggests that a size of about 120 kb is indeed the upper size limit for the feasibility of the cloning of mycobacterial DNA.
Definition of the Minimum Set of BCG BACs
100 clones randomly selected from pBeloBAC11 and pBACe3.6 libraries were sequenced at the ends to determine their position relative to the M. tuberculosis H37Rv chromosome (Cole et al., 1998). This gave a minimum network of clones on the genome but with a preferential group in the vicinity of the sole operon rrn, which was also observed during the construction of the M. tuberculosis BAC map (Brosch et al., 1998). To fill the holes between the positioned clones, PCR primers were prepared, on the basis of the sequence of the complete M. tuberculosis genome, so as to screen the BAC pools for specific clones. Using this methodology, clones covering more than 98% of the genome were isolated and positioned on the sequence of the M. tuberculosis genome.
A minimum set of 57 M. bovis BCG clones was necessary to cover the genome (
Use of BAC Chips for Detecting Deletions in the M. bovis BCG Genome
This involves the detection, from the M. tuberculosis H37Rv BAC library, of 63 clones covering 97% of the genome (Brosch et al., 1998). Analysis in silico of the sequence of the M. tuberculosis genome revealed that the digestion of these clones with either PvuII or EcoRI gave rise to a reasonable number of restriction fragments for each clone. The digested fragments migrated through agarose gels, gave rise to spots on membranes and were then hybridized with the 32P-labeled genomic DNA of M. bovis BCG and M. bovis. The restriction fragments which did not hybridize with the DNA probes were considered to be absent from the genomes of M. bovis or BCG. As the initial screening used only two enzymes, it is possible that other deletions passed unnoticed. However, it is probable that all the important deletions (>5 kb) were detected by this approach.
From an analysis of the entire genome, 10 loci were identified which appeared to be absent from M. bovis BCG compared with M. tuberculosis. Hybridizations with the M. bovis genomic DNA revealed that 7 of these loci were also deleted in M. bovis compared with M. tuberculosis. Closer analysis revealed that the three deletions specific to M. bovis BCG were identical to the RD1-RD3 regions defined by the Stover team (Mahairas et al., 1996). Retaining the previous nomenclature, the 7 M. bovis/BCG deletions were designated RD4, RD5, RD6, RD7, RD8, RD9 and RD10 (
RD4
RD4 is a 12.7 kb deletion previously characterized as a region absent from M. bovis and M. bovis BCG of the Pasteur, Glaxo and Denmark substrains (Brosch et al., 1998). Among the proteins encoded by the 11 ORFs, some show resemblance with the enzymes involved in the synthesis of the lipopolysaccharides. To determine if RD4 was deleted only in the bovine strains, M. africanum, M. microti, M. tuberculosis CSU#93 and 27 clinical isolates of M. tuberculosis were examined for the presence of the locus (table 2). PCR reactions using primers internal to RD4 (table 3) generated only products in nonbovine strains.
RD5
RD5 has a size of 8964 bp located between the genomic positions 2626067-2635031 (
RD6
RD6 was mapped at the level of the insertion sequence IS1532, an IS element which is absent in M. microti, M. bovis and M. bovis BCG (Gordon et al., 1998) (table 1). The delimiting of the size of the deletion was complicated by the presence of repeat regions directly flanking the IS element and requiring the use of primers outside the repeat region (table 3). These primers amplified the products in M. bovis and M. bovis BCG which are about 5 kb smaller than the M. tuberculosis amplicon. Primer walking was used to precisely locate the junctions of deletions and revealed a deletion of 4 928 b in M. bovis and M. bovis BCG (genomic position of M. tuberculosis 3846807-3841879). Like the IS1532 element, it was determined that RD6 contained two genes encoding PPE proteins (Rv3425 and Rv3426) and part of Rv3424c whose function is unknown (table 1).
RD7
The RD2 deletion described in Mahairas et al. (Mahairas et al., 1996) was mapped in the M. tuberculosis Rv420 clone and the results obtained by the inventors have suggested the existence of an additional deletion in M. bovis BCG which is very close to RD2. Hybridizations were repeated using the M. bovis genomic DNA as probe since this strain contains RD2 sequences, thus simplifying the identification of other deleted fragments. This analysis (
RD8
RD8 covers a region of 5895 bp positions on the genomic sequence of M. tuberulosis at 4556836-4062731. The deletion contains 6 ORFs (
RD9 and RD10
The 2030 bp deletion spanned by RD9 covers 2 ORFs, Rv2037c and Rv2074, which probably encode an oxidoreductase and an unknown protein respectively (table 1). 2 additional ORFs are truncated by RD9: Rv2075c encodes a putative exported protein whereas cobL encodes a precorrin methyltransferase involved in the synthesis of cobalamin. PCR analysis with flanking primers (table 3) revealed that RD9 is also present in M. africanum and M. microti (table 2). RD10 is a 1903 bp deletion which truncates 2 ORFs, echA1 and Rv0223, which encode an enoyl-CoA hydratase and an aldehyde dehydrogenase respectively (table 1). PCR reactions revealed that RD10 was absent from M. microti as well as from M. bovis and BCG.
Other Differences Between M. tuberculosis and BCG
Given the fact that the genomes of tubercle bacilli are highly conserved (Sreevatsan et al., 1997), direct local comparison may be undertaken in a simple and targeted manner by examining the restriction enzyme profiles generated from M. tuberculosis and M. bovis BCG BAC clones which cover the same regions. Comparative mapping of the region covered by the clone X318 has identified this region as being very different from the corresponding M. tuberculosis clones. The data relating to the terminal sequences from the clone X066 revealed that if its terminal sequence SP6 made it possible to position about 2380 kb on the M. tuberculosis template, the terminal sequence T7 would not generate any significant similarity with any sequence of H37Rv, indicating that one end of X066 was internal to the DNA segment present in BCG but absent from H37Rv. Sequencing primers were used to walk along the BCG BAC clone X318 (
An ORF encoding a phospholipase, plcD, is interrupted by IS6110 in M. tuberculosis H37Rv (Cole et al., 1998). To determine if plcD was intact in other members of the tuberculous complex, primers flanking the insertion site IS6110 (table 3) were used in PCR reactions with M. bovis, M. bovis BCG and M. tuberculosis H37Rv. This revealed polymorphism at the locus plcD where the M. bovis and M. bovis BCG amplicons were about 5 kb larger than the product of H37Rv (
II. Experimental Data
Bacterial Strains and Plasmids
The strains of the M. tuberculosis complex (Mycobacterium africanum, Mycobacterium microti, Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium bovis BCG) and substrains of M. bovis BCG (Danemark, Glaxo, Russe, Japonais, Pasteur and Moreau) were obtained from laboratory stalks (Unite de G. M. B., Institut Pasteur). Mycobacterium tuberculosis CSU#93 was received from John BELISLE, Department of Microbiology, Colorado State University, Fort Collins, Co 80523. Nonepidemic clinical isolates of M. tuberculosis were provided by Beate HEYM, Ambroise Paré hospital, 9 avenue Charles de Gaulle, 92104 BOULOGNE CEDEX, FRANCE. The BAC vectors pBeloBAC11 (Kim et al., 1996) and PBACe3.6 (Genbank accession No. U80929) were given by H. SHIZUYA, Department of Biology, California Institute of Technology, Pasadena, Calif., and P. de JONG, Roswell Park Cancer Institute, Human Genetics Department, Buffalo, N.Y., respectively. The vectors and the derived recombinants were maintained in E. coli DH10B.
Preparation of the Genomic DNA
The preparation of the genomic DNA in agarose cubes from M. bovis BCG Pasteur was carried out as previously indicated (Philipp et al., 1996; Philipp et al., 1996) but with two proteinase K digestions for 24 h each, rather than one digestion of 48 h. The cubes were stored in 0.2 M EDTA at 4° C. and washed twice in 50 ml of Tris-EDTA (pH 8)/Triton X-100 (0.1%) at 4° C. for 1 h, and then washed twice in 50 ml of a buffer of restriction enzyme Triton X-100 (0.1%) for 1 h at room temperature before use.
Construction of the BAC Library
A DNA vector was prepared as previously indicated (Woo et al., 1994). Partial HindIII and EcoRI digestions of the DNA in agarose, for cloning into pBeloBAC11 and pBACe3.6 respectively, and then contour-clamped homogeneous electric field (CHEF) migration were carried out as previously described (Brosch et al., 1998). 5 zones, 50-75 kb, 75-100 kb, 100-125 kb and 150-170 kb were excized from agarose gels and stored in TE at 4° C. Ligations with the vectors pBeloBAC11 and pBACe3.6 and transformation in E. coli DH10B were carried out as previously described (Brosch et al., 1998). The pBeloBAC11 transformants were selected on LB agar containing 12.5 μg/ml of chloramphenicol, 50 μg/ml of X-gal and 25 μg/ml of IPTG, and were screened with white recombinant colonies. The pBACe3.6 transformants were selected on LB agar containing 12.5 μg of chloramphenicol and 5% of sucrose. The recombinant clones were subcultured, in duplicates, in 96-well microtiter plates containing a 2×YT medium with 12.5 μg of chloramphenicol and were incubated overnight at 37° C. An equal volume of glycerol at 80% was then added to the wells and a plate was stored at −80° C. as master plate. The remaining plate was used to make sets of clones for screening purposes (see above).
Preparation of DNA from Recombinants and Examination of the Size of the Inserts
A recombinant carrying a DNA plasmid was prepared from 40 ml of culture and was grown on the 2×YT medium containing 12. 5 μg of chloramphenicol as previously described (Brosch et al., 1998). 100-200 ng of DNA were digested with DraI (Gibco-BRL) and the restriction products were separated on a pulsed-field electrophoresis gel (PFGE) with an LKB-Pharmacia CHEF apparatus using a 1% (weight/volume) and a pulse of 4 seconds for 15 h at 6.25 V/cm. PFGE markers of average low size (New England Biolabs) were used as size standard. The sizes of the inserts were estimated after ethidium bromide staining and visualization with UV light.
Sequencing Reactions
Sequencing reactions were carried out as previously indicated (Brosch et al., 1998). For clones isolated from the pBeloBAC11 library, the primers SP6 and T7 were used to sequence the ends of the inserts whereas for the clones pBACe3.6, the primers derived from the vector were used. The reactions were loaded onto 6% polyacrylamide gels and electrophoresis was carried out with a 373A or 377 automated DNA sequencer (Applied Biosystems) for 10 to 12 h. The reactions generally gave between 300 and 600 bp of readable sequences.
BAC Chips
The overlapping clones from the pBeloBAC11 library of M. tuberculosis H37Rv (Brosh et al., 1998) were selected so that 97% of the M. tuberculosis genome was represented. The DNA prepared from these clones was digested with EcoRI (Gibco-BRL) or PvuII (Gibco-BRL) and was run on 0.8% agarose gels 25 cm in length, at a low voltage for 12 to 16 h. After staining and visualization under UV, the agarose gels were treated by the standard Southern method and the DNAs were transferred onto Hybond-C Extra nitrocellulose membranes (Amersham). The DNA was fixed on the membrane by heating at 80° C. for 2 h. The genomic DNA of M. tuberculosis H37Rv, Mycobacterium bovis ATCC 19210 and M. bovis BCG Pasteur was labeled with [α-33P]dCTP using the Prime-It II kit (Stratagene). The probes were purified on a P10 column (Biorad) before use. Hybridizations were carried out at 37° C. in formamide at 50% as previously described (Philipp et al., 1996). The membranes were washed for 15 min at room temperature in 2×SSC/0.1% SDS and then in 1×SSC/0.1% SDS and finally in 0.1×SSC/0.1% SDS. The results were interpreted from autoradiograms. In general, it was difficult to visualize on the autoradiograms the fragments of less than 1 kb, especially after repeated use of the membranes. The fragments larger than 1 kb gave clearer results. The clones which appeared to contain fragments with no counterpart in M. bovis BCG were subcultured for subsequent analyses. The genomic sequence allowed the establishment of restriction maps with the aim of determining the suspected regions of deletion, making it possible to select enzymes giving the best resolution of the regions. Clones could thus be digested with a second range of enzymes (generally PstI and StuI, with EcoRI included as a control) and hybridized in order to obtain a more accurate size of the deletion. The sequencing primers flanking the deletions were thus designated and used in the sequencing reactions with the corresponding BAC of M. bovis BCG used as template.
PCR Analysis
The primers used in the PCR reactions are listed in table 1. The reactions for expected products of less than 3 kb were carried out with a standard Taq polymerase (Boehringer Mannheim). The reactions used 5 μl of 10×PCR buffer (100 mM β-mercaptoethanol, 600 mM Tris-HCl, pH 8.8), 20 mM MgCl2, 170 mM (NH4)2SO4, 5 μl of nucleotide mixture at 20 mM, 0.2 μM of each primer, 10-50 ng of DNA template, DMSO at 10%, 0.5 unit of Taq polymerase and sterile distilled water to 50 μl. The heat cycles were carried out with a PTC-100 amplifier (MJ Inc.) with an initial denaturation step of 90 seconds at 95° C. followed by 35 cycles of 30 seconds at 95° C., 1 min at 55° C. and 2 min at 72° C.
The PCR reactions capable of giving rise to products greater than 3 kb were carried out using the PCR GeneAmp XL kit (Perkin Elmer). The reactions were initiated according to the manufacturer's instructions, with 0.8 mM Mg(OAc)2, 0.2 μM of each primer and 10-30 ng of DNA template per reaction. The heat cycles were carried out at 96° C. for 1 min, then followed by 15 cycles in 2 stages at 94° C. for 15 seconds and 70° C. for 7 min, followed by 20 cycles in 2 stages at 94° C. for 15 seconds and 70° C. for 8 min plus 15 seconds per cycle.
Computer Analysis
The data relating to the sequences were transferred from the automated ABI373A sequencer to the Sun or Digital work station and edited using the TED software from the Staden package. The edited sequences were compared with the inventors' database relating to M. tuberculosis (H37Rv.dbs) to determine the relative positions of the terminal sequences on the sequence of the M. tuberculosis genome. With this method, a map of the M. bovis BCG BAC clones was constructed using the M. tuberculosis H37Rv sequence as template.
To make the genomic comparison, digestions in silico using restriction enzymes were carried out with the NIP (Nucleotide Interpretation Program) software using the Staden package. The Display and Analysis program (DIANA) of the Sanger Centre, Cambridge, UK, was used to interpret the sequence data.
Accession Numbers for the DNA Sequences
The nucleotide sequences which flank each RD locus in M. bovis BCG have been deposited in the EMBL database. The accession numbers for RD5, RD6, RD7, RD8, RD9 and RD10 are AJ007300, AJ131209, AJ007301, AJ131210, Y181604 and AJ132559, respectively. The sequences of RvD1 and RvD2 in M. bovis BCG have been deposited under the Nos. Y18605 and U18606 respectively.
Detection of the Duplicated Region DU1
DU1 was the first depleted region observed when the bands for HindIII digestion of the clone X038 of the BCG BAC and of the clone Rv13 of the H37Rv BAC were compared. The two clones X038 and Rv13 had identical terminal sequences, extending from position HindIII˜4367 kb to the HindIII site ˜0027 kb (via 4411529 b) on the sequence of the genome of M. tuberculosis H37Rv (MTBH37RV), spanning the replication origin.
Analysis in silico of the HindIII restriction sites for the region given between ˜4367 kb and ˜0 027 kb revealed a HindIII site at position ˜4 404 kb. Consequently, digestion of these clones should show two restriction fragments plus the band specific for the vector at about 7 kb. That was the case for the H37Rv Rv13 clone. By contrast, the clone X038 of the BCG BAC showed three bands plus the band specific for the vector at about 7 kb, two of them were identical to the Rv13 scheme. The additional band has a size of about 29 kb. Additional PFGE analyses using DraI revealed that X038 is indeed 29 kb longer than Rv13. For PCR screening of the BCG BAC pools using selected oligonucleotides, the inventors were able to identify three further clones X covering the parts of this genomic region in BCG: X585, X592, X703. The terminal sequence and the PFGE analysis showed that each of these clones contains an insert of a different size, corresponding to the three bands observed in the results of digestion of X038 (
The terminal sequences are: X585 (˜4367-4404 kb); X592 (˜4404-4404 kb); X703 (˜4404-0027 kb). The sequences were repeated twice with the same results. The strange result according to which the clone X592 has T7 and SP6 and in the same genomic region could be explained by duplication of this genomic region in BCG and also give information on the extent of the rearrangement. Additional comparative restriction analyses of the clones X585, X592, X703 and X038 with EcoRI revealed that X592 and X703 have the same restriction pattern with the exception of a 10 kb band present in X703 but absent from X592. On the basis of these results, primers were prepared for the amplification of the joining region where the duplicated DNA segment joins the unique region.
PCR analysis with primers at 16.000 and at 4398.700 R gave a product of an expected size from the clone X592 and also on the BCG-Pasteur genomic DNA. Sequencing of the PCR products obtained directly on the BAC DNA of the clone X592 revealed that the junction was indeed located at bases 16.732/4398.593 compared with the genomic sequence of H37Rv. Additional proof of the rearrangement was obtained using a PCR fragment of 500 bp spanning the oriC region of H37Rv as 32P-labeled probe in order to hybridize the products of digestion of the genomic DNA of M. tuberculosis, M. bovis and M. bovis BCG-Pasteur. Whereas in M. bovis and M. tuberculosis a band having an average size of about 35 kb was detected, in M. bovis BCG-Pasteur two bands hybridized, one of approximately 35 kb and the other of 29 kb.
PCR analysis using primers at 16.000F or 16.500F (sense primers) and at 4398.770R (reverse primer) on the genomic DNA of various BCG strains (Pasteur, Glaxo, Copenhagen, Russia, Prague, Japan) have revealed that products were only obtained from three strains, including M. bovis BCG-Pasteur. The other three substrates always gave negative results despite the confirmation of the positive controls.
As expected, the M. bovis and M. tuberculosis H37Rv type strains were also always negative. However, it cannot be excluded that PCR analyses give false-positive results or false-negative results from time to time. The final confirmation of these data ought to be given on spots of genomic DNA cleaved with HindIII from a variety of BCG substrains. Thus, investigations are under way to try to obtain DNA from the BCG substrains (Glaxo, Prague, Russia, Copenhagen and Japan) suitable for digestion with HindIII. A summary of the mapping data is shown in
Detection of the Duplicated Region DU2
The second big genomic rearrangement observed in the M. bovis BCG-Pasteur chromosome was found by analyzing several BCG BAC clones covering a genomic region of about 200 kb (3550-3750 kb). Their sizes, evaluated by PFGE, did not conform to those expected from the H37Rv genome and data relating to the terminal sequences.
The terminal sequences of BAC X495 were both located around the HindIII site at 3593 kb, whereas the PFGE results showed that the clone has a size of about 100 kb, containing three HindIII fragments, of about 39 kb, about 37 kb and about 35 kb. While the bands of about 39 kb and 37 kb were also present in the corresponding H37Rv Rv3 clone, the 23 kb band was about 2 kb longer than the HindIII fragment of 21 kb in Rv403 (the X1070 clone also showed this BCG-specific fragment of 23 kb). This finding led to the hypothesis that the genomic region at around 3593 kb must have been duplicated, giving rise to the introduction of a novel HindIII site at the point where the clone X495 ends. To show this, several primers in the chromosomal region of 3589 kb to 3593 kb were tested for the sequencing of the BAC X495 or X1070 DNA and a junction was therefore identified at bases 3690124/3590899 relative to the genomic sequence of H37Rv.
This explains why the HindIII fragment of 21 kb in H37Rv which extended from 3669 kb to 3691 kb in the Rv403 clone is about 2 kb shorter than the corresponding fragment in M. bovis BCG. In M. bovis BCG, the HindIII site has 3691 kb was displaced in the 3′ direction by the insertion of a large DNA fragment at base 3690.124. Therefore the HindIII fragment extended from 3669 kb to 3690.124/3590.899 up to the 3593 kb HindIII site, making the fragment about 2 kb longer than in H37Rv (
These results were confirmed by hybridization experiments on the genomic DNA of M. bovis, M. bovis BCG-Pasteur and M. tuberculosis H37Rv digested with HindIII and using 32P-labeled DNA of the clone X1070. Whereas in M. bovis and in M. tuberculosis one of the hybridized bands had a size of about 21 kb, the corresponding band in M. bovis BCG-Pasteur had a size of about 23 kb, similar to the situation observed with the BAC clones. This also explains the strange terminal sequencing data for the X495 clone whose SP6 end is located at the 3593 kb site of HindIII and whose T7 end is also located at the 3593 kb site of HindIII. In fact, the clone spans the genomic region 3593 to 3690.124 and thus ends about 2 kb further in the duplicated region at the 3593 site of HindIII. Likewise, the estimation of the size by PFGE of about 100 kb is correct. This insertion of the large duplication at the base 3690.124 interrupts the lpda gene (Rv3303), but the PCR results show that a second (probably intact) copy of this gene is present in the duplicated region, covered by the X094 or X1026 clones.
These surrounding clones X094 and X1026 have identical terminal sequences (3593 kb and 3749 kb) which are also strange. In accordance with the data relating to the terminal sequences, the clones appear to have a length of 155 kb while the estimation of the size by PFGE of these clones showed that the clones were about 100 kb long. The inventors therefore assumed that the clones X094 and X1026 did not extend from 3749 kb to the “original” site of HindIII at 3593 kb which is about 155 kb in the 5′ direction but extended only up to the duplicated site of HindIII, where the X495 clone ends (see above).
The hybridization results showed that a band of about 35 kb in the HindIII profile of the clone X094 hybridized with the genomic DNA of the clone X1070, suggesting that the clones X094 and X1026 contained the duplicated DNA derived from the 3690 kb region. An additional proof was obtained by PCR analyses and DNA sequencing, with various primers obtained from the 3675, 3680 and 3690 kb regions. However, by PCR, the primers for the 3669 region never gave products from X094, suggesting that there was another rearrangement junction between 3669 and 3675. This junction was determined by sequencing and it is located at position 3608.471/3671.535. The PCR analyses were also positive for X094 with 3692 and 3707 primers, suggesting that X094 contains chromosomal regions from the 3691 HindIII site up to 3749 as indicated by the data relating to the terminal sequences. However, when the PFGE fragments of the BAC H37Rv clones Rv403 and X094 were compared, it proved obvious that the new HindIII site ought to have been introduced into BCG-Pasteur in the region between 3691 to 3749 as a HindIII digest of the clone Rv403, a band of about 48 kb is present (corresponding to the 3691 to 3749 region) whereas in BCG, two bands of about 20 and about 30 kb are present. It would appear that M. tuberculosis H37Rv has lost a fragment of about 2 kb at position 3710 kb. This fragment is designated RvD3 in
Experiments with multiple sets of primers (3689.500F (SEQ ID No. 22) or 3689.900F (sense) 3591.000R, 3591.500R or 3592.000R (reverse)) to amplify the joining region at the level of the base 3690124/3590899 (described above) in various M. bovis BCG strains revealed that amplicons could only be obtained from M. bovis BCG-Pasteur and from two other BCG substrates, whereas the other BCG substrates gave no amplicon. However, as for DU1, the final conclusions will have to be made only after confirmation of the results on HindIII spots hybridized with labeled DNA derived from the 3689500F-3690.000R region which ought to give rise to bands with rearranged BCG strains, one of them has a size of about 23 kb, about 2 kb more than the corresponding band in the genomic digestions of M. bovis and M. tuberculosis. The second band of about 35 kb ought to be present only in the rearranged strains and not in M. tuberculosis H37Rv or the M. bovis type strain (
However, the fact that M. bovis BCG-Pasteur and two other substrains of M. bovis BCG have a duplicated complement set of genes responsible for major processes such as, inter alia, cell division and signal translation, comprising two replication origins, is one of the surprising aspects revealed to the inventors by this approach to genetic comparisons.
Since the biological material is subject to changes, and given that BCG vaccination trials highly varied protection results (0-80%), it could be important to evaluate if this variation in the efficacy of protection may be partly attributed to the choice of the BCG substrain used.
Thus it is advisable to investigate even further in order to determine if a correlation exists between the genomic findings and the phenotypic variations among the various BCG substrains.
NA: Not applicable
* As defined by Cole et al., Nature, 1998, 393, pages 537-544
M. tuberculosis
M. tuberculosis
M. bovis
M. microti
M. tuberculosis
M. africanum
M. bovis
ND: Not determined:
✓ = the region is present,
X = the region is deleted
*Number of clinical isolates positive for the presence of a region
* = Primers internal to the deletion
† = Size including a copy of IS6110 not present in BCG
| Number | Date | Country | Kind |
|---|---|---|---|
| 9903250 | Mar 1999 | FR | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 09936523 | Sep 2001 | US |
| Child | 10980194 | Nov 2004 | US |
