1 Unité de Génétique Moléculaire Bactérienne, Institut Pasteur, 2528 rue du Docteur Roux, 75724 Paris Cedex 15, France
2 Laboratoire de Génomique des Micro-organismes Pathogènes, Institut Pasteur, 2528 rue du Docteur Roux, 75724 Paris Cedex 15, France
3 Centre National de Référence des Mycobactéries, Institut Pasteur, 2528 rue du Docteur Roux, 75724 Paris Cedex 15, France
Correspondence
Roland Brosch
rbrosch{at}pasteur.fr
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ABSTRACT |
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Representative sequences of the junction regions reported in this article have been deposited in the EMBL database under accession numbers AJ583832, AJ583833 and AJ583834.
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INTRODUCTION |
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METHODS |
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In silico analyses of mycobacterial species.
The complete genome sequences of M. tuberculosis and M. leprae were shown to differ extensively in size and number of genes. The genome of M. tuberculosis comprises 4 411 532 bp and 3993 protein-coding genes (Cole et al., 1998; Camus et al., 2002
), whereas M. leprae contains 3 268 203 bp and only 1605 genes, but numerous pseudogenes (Cole et al., 2001
). In silico comparison of the predicted proteins shared by M. tuberculosis and M. leprae was done employing BLAST and FASTA alignment programs against public databases and partial genome sequences (available at web sites http://www.sanger.ac.uk/Projects/M_marinum/ and http://www.tigr.org). To be listed as a conserved mycobacterial protein, 40 % identity at the protein level between M. tuberculosis and M. leprae was used as the cut-off level (Cole, 2002
). The presence or absence of these genes in M. avium, M. marinum and M. smegmatis was determined in a similar manner by using 40 % identity over at least 70 % of the complete length of the tested protein. For selected cases, to determine if proteins corresponded to orthologous proteins in two species, the bi-directional best-hit method was applied, by comparing a given protein of M. tuberculosis with the sequence of another species, e.g. M. marinum. The protein sequence from M. marinum, which showed the highest similarity, was then compared back to the M. tuberculosis database, and, in the case of an orthologous protein, showed its best hit with the protein with which the initial comparison was started. This method was particularly useful when genes or proteins from multi-gene families were compared, as high scores due to cross-hybridization may appear.
PCR.
PCR amplification was used for making probes, for confirmation of the absence of genes that were suggested to be absent in certain tested strains by macro-array results, and for generating the DNA fragments of junction regions of deleted regions. According to the type of application, different volumes were used. For the production of probes, which were spotted on the macro-array, PCRs were performed in 96-well plates containing 12·5 µl of 10x PCR buffer [600 mM Tris/HCl pH 8·8, 20 mM MgCl2, 170 mM (NH4)2SO4, 100 mM -mercaptoethanol], 12·5 µl of 20 mM nucleotide mix, 25 µl each primer at 2 µM, 10 ng template DNA, 10 % DMSO, 2 U Taq polymerase (Gibco-BRL) and sterile water to 125 µl. Amplification of junction regions and evaluation of the presence or absence of genes that showed no or weak hybridization by macro-array experiments with genomic DNA from a given strain were performed in a total reaction volume of 12·5 µl, as described previously (Brosch et al., 2002
). Thermal cycling was performed on a PTC-100 amplifier (MJ) with an initial denaturation step of 90 s at 95 °C, followed by 35 cycles of 30 s at 95 °C, 1 min at 58 °C and 4 min at 72 °C.
Macro-arrays.
The selection of 500 genes for the focused macro-array included 219 genes common to both M. tuberculosis and M. leprae that code for proteins that did not show any similarity with proteins from other organisms in the public databases. Genes that were classified in the M. tuberculosis H37Rv genome as potentially involved in virulence (Cole et al., 1998) as well as genes that belonged to certain multi-gene families (Cole et al., 1998
; Tekaia et al., 1999
) were also included in the selection. Differences in the copy number of the insertion element IS1081 in the M. tuberculosis complex are almost entirely restricted to M. canettii. To determine if genes that flank IS1081 elements in the genome of M. tuberculosis H37Rv were conserved throughout the members of the M. tuberculosis complex, these genes were also selected for the construction of the macro-arrays. The selection also included genes that were variable between M. tuberculosis and the highly related vaccine strain M. bovis BCG (Mahairas et al., 1996
; Gordon et al., 1999
; Behr et al., 1999
). Several house-keeping genes and other genes for which oligonucleotides were available in the laboratory were used for control purposes. The sequences of selected genes from M. tuberculosis H37Rv and M. bovis BCG Pasteur were downloaded by using the complete genome sequence (http://genolist.pasteur.fr/TubercuList/) displayed by the ARTEMIS software (Rutherford et al., 2000
) or by using in-house databases. The design of primer pairs for the amplification of
500 bp portions of these genes was done using the PRIMER 3 software (available via http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). The oligonucleotides used for the amplification of the probes were designed to have annealing temperatures in the range 5860 °C. PCRs were performed in a final volume of 125 µl as described above. Fifty-five microlitres of the 125 µl of the PCR product were transferred from the 96-well plates in 384-well plates using a pipetting robot (Tecan). After estimation of the amount of amplification product by gel electrophoresis and ethidium-bromide staining, each PCR product was then deposited in duplicate on a 22x22 cm Q-filter N+222 mm membrane (Genetix) by a gridding robot (QPIX). Probes were fixed and denatured by putting the freshly spotted membranes on Whatman paper soaked with fixation solution (0·5 M NaOH, 1·5 M NaCl) and leaving them for 15 min. The reaction was stopped by using distilled water. Membranes were stored wet at -20 °C for further use. Membranes were pre-hybridized and hybridized in 10 ml of a solution containing SSPE buffer (750 mM NaCl, 50 mM NaH2PO4, 5 mM EDTA) with 1 % SDS, Denhardt's reagent composed of 0·01 % Ficoll, 0·01 % polyvinylpyrrolidone and 0·01 % BSA and sonicated salmon DNA at a final concentration of 100 µg ml-1. Pre-hybridization was performed during 1 h at 65 °C. Genomic DNAs from the various mycobacterial strains were labelled by random incorporation of [
-33P]dCTP into the synthesized complementary strand using the Prime-it II kit (Stratagene). Unincorporated nucleotides were removed by exclusion chromatography with the QIAquick Nucleotide Removal kit (Qiagen). Membranes were hybridized overnight at 65 °C with the labelled probe followed by four washing steps in 10 ml of 0·5x SSPE, 0·2 % SDS solution. The first two washes were done at room temperature for 5 min followed by two washes at 65 °C for 20 min. Membranes were sealed in Saran wrap, exposed to a screen for 2 days and scanned on a STORM phosphorimager (Molecular Dynamics); signals were quantified and visualized using the IMAGE QUANT (Molecular Dynamics) software. The hybridization signals of each spot hybridized with the genomic DNAs from the various strains were compared to a control membrane hybridized with the reference strain M. tuberculosis H37Rv using the ARRAY VISION software (Imaging Research). For normalization purposes, the intensities from the central and the surrounding area of each spot were calculated. The intensity from the surrounding area, due to non-specific background hybridization signals, was subtracted from the spot intensity. To compare spot intensities from different membranes, a mean background intensity was calculated for each membrane, which was then used for establishing a correction factor to normalize the spot intensities from individual membranes. The log10 ratios between the normalized intensities of each spot from a tested strain compared to the reference strain M. tuberculosis H37Rv were used to estimate whether a gene was present or absent in a given strain. The cut-off was determined using a Gaussian model involving the mean and the standard deviation. For confirmation purposes, the genes which were found absent by this approach were re-tested by PCR analysis in the corresponding strain.
Sequencing of junction regions.
For genes that were missing from certain strains, PCR confirmation was done as described above. Then, new primers that were situated in the flanking regions of the missing gene(s) were designed. After amplification of the fragment containing the junction region of a given new deleted region, the fragment was purified by using a QIAquick PCR purification kit. For sequencing, we used 500 ng purified amplification product, 3 µl Big Dye sequencing mix (Applied Biosystems), 2 µl (2 µM) flanking primer and 3 µl of 5x buffer (5 mM MgCl2, 200 mM Tris/HCl pH 8·8). Thermal cycling was performed on a PTC-100 amplifier (MJ), with an initial denaturation step of 1 min at 96 °C, followed by 35 cycles of 30 s at 96 °C, 15 s at 56 °C and 4 min at 60 °C). The products were then precipitated with 80 µl of 76 % ethanol, centrifuged, washed with 70 % ethanol and dried. Then, 2 µl of formamide/EDTA buffer were added and, after denaturation, the samples were loaded onto 4 % polyacrylamide gels (48 cm). Electrophoresis lasted for 1012 h on a model 377 automated DNA sequencer (Applied Biosystems). Obtained sequences were compared to the genome sequence of M. tuberculosis H37Rv using the TubercuList server at the Institut Pasteur, allowing the size and exact location of deleted regions to be determined. Representative sequences of the junction regions were deposited in the EMBL database under accession numbers AJ583832, AJ583833 and AJ583834.
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RESULTS |
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Similarly, we identified a specific deletion, RD2seal, for strains which were isolated from infected seals in different parts of the world. Hybridization results suggested that genes Rv1978 and Rv1979 were absent from the seal isolates. Sequence analysis of the junction region in the four tested seal isolates confirmed this finding and showed that in these strains a 1941 bp deletion has removed parts of genes Rv1978 and Rv1979 (Fig. 4a). We named this region RD2seal as it overlaps the 10·7 kb RD2 region, which is missing from some but not all BCG substrains (Mahairas et al., 1996
; Behr et al., 1999
; Gordon et al., 1999
). In addition, strains isolated from seals were deleted for regions RD7, RD8, RD9 and RD10, whereas regions RD4, RD5, RD6, RD11, RD12 and RD13, usually missing from M. bovis (Brosch et al., 2002
; Mostowy et al., 2002
), were present. As the RD2seal junction regions in the four seal isolates were identical, but different from the RD2 deletion of BCG strains, it appears that this deletion is a specific evolutionary marker for strains prevalent in seals and sea lions (Fig. 4d
).
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Micro-deletions in gene pks15/1
In a recent study, Guilhot and colleagues showed that several well characterized M. tuberculosis strains (H37Rv, Erdman, CDC1551 and MT106) lack a particular phenolglycolipid (PGL) that is produced by M. tuberculosis 210 (Beijing type) and M. canettii 14000059, and they linked this observation to a deletion of 7 bp that introduces a frameshift in a gene encoding a polyketide synthase (pks15/1) in these strains (Constant et al., 2002). Interestingly, in M. bovis AF2122/97 and M. bovis BCG, a 6 bp deletion was observed at the same locus of the pks15/1 gene. As M. bovis and M. bovis BCG both produce PGL, it seems likely that this 6 bp deletion, which does not cause a frameshift in the pks15/1 gene, does not influence the enzymic activity of the resulting gene product for the synthesis of the particular PGL (Constant et al., 2002
). However, as this interesting polymorphism has direct phenotypic consequences, we were interested to determine at what stage of the phylogenetic diversification the deletion of 7 or 6 bp occurred. Therefore, we sequenced PCR products from the polymorphic locus in the pks15/1 gene (Fig. 4c
) in 15 strains from the present study and in 21 additional strains used previously (Brosch et al., 2002
). The results of this approach showed that the 7 bp deletion only occurred in a particular subgroup of M. tuberculosis strains that show the katG463 mutation CGG and, according to the nomenclature of Sreevatsan and colleagues, belong to genetic group 2 or 3 (Sreevatsan et al., 1997
). All these strains had region TbD1 deleted and also lacked spacers 3336 in their spoligotype, which is a characteristic feature of these genetic groups (Brosch et al., 2002
; Soini et al., 2000
). In contrast, no M. tuberculosis strains of genetic group 1 (katG463 CTG), including strains of the ancestral type that have the TbD1 region present as well as strains of the Beijing type cluster, which lack the TbD1 region (Brosch et al., 2002
), showed a deletion in the polymorphic locus of the pks15/1 gene.
As for the 6 bp deletion previously observed for M. bovis AF2122/97 and M. bovis BCG (Constant et al., 2002), in the present study we found the same 6 bp deletion in the pks15/1 gene of all tested M. bovis strains, seal isolates, M. microti and M. africanum lacking regions RD7RD10. Only M. africanum strains that lack the RD9 region but have retained regions RD7, RD8 and RD10 did not show the 6 bp deletion, suggesting that it occurred after the RD9 deletion, at about the same period as deletion of regions RD7, RD8 and RD10 occurred in the M. africanum
M. bovis lineage (Fig. 4d
). These findings fit well with the proposed evolutionary scenario of the M. tuberculosis complex (Brosch et al., 2002
) and suggest that two independent deletion events have occurred in the pks15/1 gene in two distinct branches of the phylogenetic tree of the M. tuberculosis complex. The 7 bp deletion, which inactivated the pks15/1 gene, occurred in the branch of TbD1-deleted modern M. tuberculosis strains at about the same time-range as the katG463 mutation (CTG
CGG), whereas the 6 bp deletion occurred after the RD9 and before the RD10 deletion in the M. africanum
M. bovis lineage. Considering a clonal structure (Supply et al., 2003
; Fleischmann et al., 2002
) of the M. tuberculosis complex, it seems that this 6 bp deletion in gene pks15/1 was then inherited by the other members of this branch, and can therefore be found in M. microti, seal isolates, M. bovis and BCG strains (Fig. 4d
).
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DISCUSSION |
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Among the few exceptions, members of the ESAT-6 family were most prominent. The members of this family are characterized by a small size (100 aa) (Cole et al., 1998
), common amino acid motifs (Tekaia et al., 1999
), and several of them are organized in genomic loci with similar organization, suggesting that the neighbouring genes may have some function in the transport of these proteins out of the bacterial cell (Cole et al., 1998
; Tekaia et al., 1999
; Pallen, 2002
). The first experimental proof for this hypothesis was recently obtained for the RD1 region of M. tuberculosis (Fig. 2
), which is absent from BCG and M. microti (Pym et al., 2003
). In the same study, it was shown that recombinant vaccine strains that appropriately exported ESAT-6 and CFP10 induced better protection against tuberculosis in animal models. This finding may be linked to the highly immunogenic character that was demonstrated in several studies for ESAT-6 and other members of this family (Skjot et al., 2002
). Indeed, most ESAT-6 proteins, deleted from one or more strains as identified in the present study, are strongly recognized by the immune system of the host (Skjot et al., 2002
). Furthermore, two additional members of the ESAT-6 family (Rv3809c, Rv3905c) are reported to be altered in the sequenced M. bovis AF2122/97 strain (Garnier et al., 2003
). Taken together, it seems plausible that variation of ESAT-6 family proteins in strains of M. tuberculosis and/or members of the M. tuberculosis complex could contribute to antigenic variation, eventually helping the bacteria to escape immune recognition by the host. To elucidate the biological function of this protein family, further studies are necessary. The finding that the RD1 region is highly conserved in gene content and gene order in several pathogenic and non-pathogenic mycobacterial species (Fig. 2
) suggests that ESAT-6 systems may play a fundamental role in survival in specific environments. This knowledge, together with appropriate cosmid and BAC libraries from these species (Brosch et al., 1998
), should enable now very focused studies on the role of these proteins in the various mycobacteria.
In the tight-knit M. tuberculosis complex, where single nucleotide substitutions do not seem to be a substantial source of genetic diversity between strains, the presence or absence of certain regions of difference may play important roles in the varying phenotypes, host range and virulence of these bacteria (Pym et al., 2002; Lewis et al., 2003
). Analyses of these RD regions in well-defined strains from the M. tuberculosis complex have allowed us to describe distinct phylogenetic lineages within the M. tuberculosis complex (Brosch et al., 2002
) that have evolved from a common ancestor. In this study, we describe RD regions that are characteristic for certain subpopulations of the M. tuberculosis complex. One of the regions (RD2seal) is restricted to strains that were isolated from seals. In the past, seals have been described to be susceptible to tuberculosis, but it was not always clear if the infections in seals were caused by M. tuberculosis and/or M. bovis (Zumarraga et al., 1999
). However, by the use of macro-arrays and sequencing strategies we show here that the tested strains, which were isolated from seals in different geographical regions (Argentina, France), lack RD7, RD8, RD9 and RD10 and the particular region RD2seal that seems to be specific for tubercle bacilli hosted by seals. The analysis of all available genetic markers (RDs, mmpL6 polymorphism, pks15/1 polymorphism and spoligotype) showed that the seal isolates are phylogenetically more closely related to M. bovis than to M. tuberculosis. Their position in the established evolutionary scheme (Fig. 4d
) is somewhere close to M. microti, which also lacks RD7RD10, shares the mmpL6 codon 551 single nucleotide polymorphism (SNP) of M. bovis (AAG) and presents a particular deletion (RD1mic) that is restricted to this subspecies. The position of the seal isolates in the phylogenetic scheme of the members of the M. tuberculosis complex shown in Fig. 4(d)
is in good agreement with a recent SNP analysis by Musser and colleagues (Gutacker et al., 2002
), who also placed these isolates as intermediate between M. tuberculosis and M. bovis. In a very recent study, the seal isolates were considered as sufficiently distant from M. bovis and M. tuberculosis to place them in a separate subspecies of the M. tuberculosis complex (Cousins et al., 2003
). In this respect, the marker RD2seal is a valuable tool for the rapid identification of such strains.
The analysis of the sequence polymorphism in the pks15/1 gene, which abolishes production of a particular phenolglycolipid in a large group of M. tuberculosis strains (Constant et al., 2002), showed excellent agreement of the observed polymorphism with all other evolutionary markers available and confirmed the phylogenetic position of the strains used in this study (Fig. 4c, d
). These results suggest that the 6 bp deletion in the pks15/1 gene in the M. africanum
M. bovis lineage arose independently from the 7 bp deletion observed for M. tuberculosis strains of Sreevatsan's group 2 and 3. Closer inspection of the flanking sequences of this polymorphic locus (Fig. 4c
) in the pks15/1 gene showed that this genomic region is very GC-rich. In genes that code for PE and PPE proteins, such GC-rich regions have previously been associated with increased sequence polymorphism between strains (Cole et al., 1998
; Banu et al., 2002
). Interestingly, the pks15/1 sequence polymorphism is not the only example where independent deletion events have occurred in different evolutionary lineages of the tubercle bacilli in the same genomic regions. Other examples are the RD1 region of BCG (9·7 kb) and M. microti (14 kb), the RD2 region of BCG (10·7 kb) and seal strains (2 kb), or the RD12 region in M. bovis (2·7 kb) and M. canettii (12·4 kb). The size of the deletions, as well as the junction sequences of these regions, are clearly distinct from each other, indicating that no direct phylogenetic relationship exists between them. This observation raises an important point for the interpretation of micro- and macro-array data and implies that sequencing of the junction regions of thereby identified deleted regions (Fig. 4a, b
) is necessary before the presence/absence of these marker genes can be used in the construction of evolutionary schemes. From a practical point of view, the pks15/1 polymorphism may serve as an important additional marker for the identification and classification of members of the M. tuberculosis complex, as well as for the characterization of mycobacterial DNAs amplified from mummified human remains. Recent studies have shown that, according to their spoligotype and their katG463 SNP, in former human populations M. tuberculosis strains were present that resembled TbD1-deleted M. tuberculosis strains of Sreevatsan's genetic group 2 and 3 (Zink et al., 2003
; Fletcher et al., 2003
). As shown in the present study, it appears that a strict correlation exists between these characteristics and the frameshift mutation (deletion of 7 bp) in the pks15/1 gene.
The situation of mycobacterial research has considerably changed in the last few years due to the information contained in the whole-genome sequence of M. tuberculosis H37Rv, the paradigm strain of tuberculosis research. However, genomic variation may exist among different strains and, for the mycobacteria, only very few studies have addressed this question by the use of DNA arrays and then for a limited number of strains (Behr et al., 1999; Kato-Maeda et al., 2001
). We therefore evaluated the extent of the conserved gene pool relative to the flexible gene pool in a collection of strains from the M. tuberculosis complex and for some other mycobacterial species; this has led to a better understanding of the genetic criteria that may have played a role in the selection of the most successful M. tuberculosis strains during the evolution of the pathogen. This information is of importance for the development of new therapeutic and preventive strategies in the fight against tuberculosis.
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ACKNOWLEDGEMENTS |
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Received 22 July 2003;
revised 30 September 2003;
accepted 2 October 2003.