Molecular Evolutionary History of Tubercle Bacilli Assessed by Study of the Polymorphic Nucleotide within the Nitrate Reductase (narGHJI) Op
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微生物临床杂志 2005年第8期
Unite de la Tuberculose et des Mycobacteries, Institut Pasteur de Guadeloupe, Pointe-a-Pitre, Guadeloupe
Division of International Medicine and Infectious Diseases, Department of Medicine, Joan and Sanford I. Weill Medical College, Cornell University, New York, New York
ABSTRACT
A well-characterized collection of Mycobacterium tuberculosis complex (MTC) isolates, representing all known subspecies as well as some relevant genotypic families of M. tuberculosis, was analyzed for the newly discovered narGHJI –215 C-to-T promoter single-nucleotide polymorphism (SNP). This point mutation has been shown in earlier studies to be responsible for the differential nitrate reductase activity of M. tuberculosis versus M. bovis. As previously defined by the presence or the absence of the TbD1 genetic locus, the group included both the "modern" W-Beijing, Haarlem, and Central-Asian1 (CAS1) families as well as the "ancestral" East-African-Indian (EAI) clade. Interestingly, among "modern" M. tuberculosis isolates, those previously classified as Principal Genetic Group 1 (PGG1) organisms by katG463-gyrA95 polymorphism analysis did not present the two-banded narGHJI restriction fragment length polymorphism analysis of PCR products pattern common to the other PGG1 MTC members, including the "ancestral" M. tuberculosis isolates. Instead, they showed a one-banded pattern, aligning them with other evolutionarily recent M. tuberculosis isolates of the PGG2 and PGG3 groups, such as Haarlem, Latin-American and Mediterranean (LAM), and X families. The presence of a nitrate reductase producer phenotype in "Mycobacterium canettii" and some "ancestral" M. tuberculosis isolates, despite a two-band –215C genotype, argues in favor of an alternate mechanism to explain the differential nitrate reductase activity of certain PGG1 subspecies of the MTC. Overall, these findings may help to establish the precise evolutionary history of important genotype families such as W-Beijing and suggest that the –215T genotype may have contributed the virulence, spread, and evolutionary success of "modern" M. tuberculosis strains compared to the remaining MTC organisms.
INTRODUCTION
Together with niacin production, catalase/peroxidase activity, and urease production, the assay of nitrate reductase activity, through the accumulation of nitrite, remains a basic phenotypic criterion for differentiating Mycobacterium tuberculosis from Mycobacterium bovis in diagnostic mycobacteriology (6). Contrary to M. tuberculosis, which presents a strong nitrate reductase activity under aerobic conditions, M. bovis and the M. bovis-derived BCG vaccine strain fail to rapidly accumulate nitrite from nitrate. However, both M. tuberculosis and M. bovis possess the narGHJI operon, which is expressed under anaerobic conditions in both subspecies and the product of which is a membrane-bound anaerobic nitrate reductase complex (25). As an alternative to using oxygen as a terminal electron acceptor, the NarGHJI complex couples this process to the reduction of nitrate. It was shown previously that this enzyme is induced under anaerobic conditions in Bacillus subtilis (18), and the switch to anaerobic metabolism is believed to be critical in the establishment of latent infection by M. tuberculosis and possibly the other tubercle bacilli. Recently, a C-to-T transition at position –215 upstream of the GTG start codon was identified in the putative promoter region of the narGHJI operon (25). This transition was hypothesized to be responsible for the differential nitrate reductase activity between M. tuberculosis and M. bovis as a result of increased gene expression in M. tuberculosis, and –215T was presented as an M. tuberculosis-specific single-nucleotide polymorphism (SNP) (25, 26). However, this work did not cover the full range of M. tuberculosis complex (MTC) subspecies now known to exist, and it was unclear whether the evaluated M. tuberculosis clinical isolates represented both "modern" and "ancestral" M. tuberculosis strains.
Indeed, phylogenetic segregation of M. tuberculosis into "ancestral" versus "modern" lineages is based upon the presence or absence, respectively, of a chromosomal deletion dubbed TbD1 (1). Examples of "modern" M. tuberculosis families include W-Beijing, Haarlem, and Central-Asian1 (CAS1) as well as the M. tuberculosis laboratory strain H37Rv, whereas the East-African-Indian (EAI) family represents an "ancestral" M. tuberculosis lineage (1, 22). To date, all other MTC subspecies, including "Mycobacterium canettii," Mycobacterium africanum, Mycobacterium microti, Mycobacterium pinnipedii, and M. bovis, as well as the so-called dassie bacillus, have also been shown to retain TbD1 intact (reference 1 and Richard C. Huard, unpublished data). Notably, all tubercle bacilli that retain the TbD1 locus are also independently classified as principal genetic group 1 (PGG1) organisms by katG463-gyrA95 SNP analysis (24), while all "modern" M. tuberculosis strains fall into either PGG1, PGG2, or PGG3. As a result, the prevailing theory posits that the TbD1 deletion occurred in a PGG1 M. tuberculosis clone whose progeny then went on to establish the "modern" PGG1, PGG2, and PGG3 M. tuberculosis lineages, and that all current "ancestral" PGG1 M. tuberculosis strains are direct descendants of pre-TbD1 deletion M. tuberculosis organisms (1). It is also important to mention that, for reasons that remain to be determined, the known "modern" M. tuberculosis families are far more prevalent worldwide than their "ancestral" counterparts (9, 10).
In the present study, we aimed to further determine the distribution of the –215 C-to-T narGHJI SNP within a well-characterized collection of 68 DNAs of the MTC using a newly devised restriction fragment length polymorphism analysis of PCR products (PCR-RFLP) protocol. The results showed that all M. tuberculosis strains are not necessarily confined to the –215T narGHJI SNP. Consequently, this mechanism cannot solely explain the differential nitrite accumulation within the MTC given that "M. canettii" and some "ancestral" M. tuberculosis strains have the –215C genotype and yet reduce nitrate under aerobic conditions. These results are discussed both in terms of differential virulence and from an evolutionary genetics perspective.
MATERIALS AND METHODS
Strains and DNAs. The studied collection comprised a set of 68 DNAs from many strains of the MTC that were well characterized within the international spoligotyping database project SpolDB4 (K. Brudey, unpublished data). A previous version of this database (SpolDB3) was published recently and provides the basis for most of the defined MTC clades (9, 10). The strains comprised 14 W-Beijing, 8 Central Asian1 (CAS1), and 14 "ancestral" East-African-Indian (EAI1, EAI3, EAI2/Manila) genotypes within the PGG1, 11 PGG2, or 3 strains of unknown genotype or belonging to the Haarlem, X, T1, Cameroon (CAM), or Latin American and Mediterranean (LAM) families. For a detailed comparison with the various members within the MTC, DNAs representing the following species were used: the M. tuberculosis reference strain H37Rv, two strains of M. africanum, three strains of M. bovis, one strain of M. bovis BCG, two strains of M. microti (3), two strains of M. pinnipedii (4), eight strains of "M. canettii" (8, 29), and two strains identified as the dassie bacillus (5).
Spoligotyping and katG463-gyrA95 polymorphism. Most spoligotyping results were obtained at the Institute Pasteur of Guadeloupe, using a previously published procedure and homemade membranes (14, 27). The katG463-gyrA95 results for M. tuberculosis complex (24) have been previously published elsewhere (references 1, 13, and 17 and Richard C. Huard, unpublished) or were implied according to the correlation established between PGG and spoligotyping (21).
Nitrate reduction and assay of the –215 narGHJI promoter polymorphism. Nitrate reduction data were obtained in house for available clinical isolates using a classical method (6) or recorded from providing laboratories when the assay had already been performed. A genome search into NCBI-PubMed (http://www.ncbi.nlm.nih.gov/entrez) retrieved the full sequence of both M. tuberculosis CDC1551 and M. bovis AF2122/97 (respective accession numbers NC002755 and NC002945). The narGHJI sequences were located from these sequences, and a 155-bp DNA fragment of the 5' untranslated region prior to narG, which contained the SNP at position –215, was amplified using LC66 (AACCGACGGTGTGGTTGAC) as a forward primer and LC67 (ATCTCGATGGATGGGCGTC) as a reverse primer (26). The single difference between M. bovis and M. tuberculosis corresponded to a C-to-T transition at position 215 in M. tuberculosis. PCR conditions were as follows: a 7-min initial denaturation phase at 94°C, followed by a 1-min annealing at 60°C and a 1-min synthesis at 72°C. A total of 45 cycles was used to increase product yields. Each reaction tube (50 μl) contained 33 μl of ultrapure water, 5 μl of 10x PCR buffer, 2 μl of MgCl2 (25 mM), 0.5 μl of a mixture of A, C, G, and T deoxynucleotides (25 mM), 2 μl of each of the LC66 and LC67 primers (10 pmol/μl), 0.2 μl of rTaq DNA polymerase (Amersham, Buckinghamshire, England), and 5 μl of DNA that was microbead extracted (7) or using a dilution of cetyl-trimethyl-ammonium-bromide-extracted DNA (28). A single PCR product was observed for each DNA isolate. A 10-μl aliquot of the amplified fragments (roughly 155 to 160 bp on gel) was further digested using either Sau3AI or DpnII according to the manufacturer's instructions (New England Biolabs, Beverly, MA, for DpnII and Roche-Biomedicals, Meylan, France for Sau3AI). These enzymes specifically cut at the GATC sequence overlapping the M. bovis-like –215C narGHJI promoter site, producing two bands (of approximately 90 and of 70 bp) in PCR-RFLP of the LC66-LC67 PCR fragment. In the case of the M. tuberculosis-like –215T sequence, the restriction site is absent and as a result PCR-RFLP of the narGHJI amplicon yields a single uncut band. The restriction digest was visualized by agarose gel electrophoresis on 3% Metaphor (FMC Bioproducts, Rockland, Maine) and run in Tris-borate-EDTA (1x) buffer, followed by ethidium-bromide staining. Pictures were taken electronically and were analyzed using the Taxotron software (PAD Grimont, Taxolab; Institut Pasteur) as previously reported (11).
RESULTS AND DISCUSSION
Results of narGHJI PCR-RFLP analysis, nitrate reduction testing, spoligotyping, and PGG groupings based on katG463-gyrA95 polymorphism are summarized in Fig. 1 and detailed in Tables 1 and 2. With respect to MTC organisms other than M. tuberculosis, all test isolates were PGG1 and bore the –215C narGHJI promoter SNP as determined by the two-band pattern observed in PCR-RFLP of the LC66-LC67 PCR fragment (Table 1). With the exception of the "M. canettii" strains, each of these isolates failed to reduce nitrate. The results also showed that all "modern" M. tuberculosis strains produced a single band in narGHJI PCR-RFLP and accumulated nitrite independently of PGG genotype (Table 2). However, even though each "ancestral" M. tuberculosis tested was PGG1, in narGHJI PCR-RFLP the isolates produced a two-band pattern indicative of a –215C genotype and nitrate reduction was variable.
Given that the –215T SNP has been shown to enhance gene expression of the nitrate reductase cluster (26), our data raise several points with interesting implications. First, since "M. canettii" and some "ancestral" M. tuberculosis strains accumulated nitrite despite having the –215C genotype, then this polymorphism cannot be solely accountable for differential nitrate reductase activity within the MTC. It remains possible that "M. canettii" and some "ancestral" M. tuberculosis strains have unidentified compensatory mechanisms that normalize narGHJI activity and represent differences that are absent from M. bovis and its nitrite-negative brethren. Alternatively, given the respective positions of each MTC organism in recently proposed phylogenetic maps for the evolution of the MTC (1, 17), it remains possible that "M. canettii" and some "ancestral" M. tuberculosis strains, which are theoretically among the oldest MTC lineages, possess additional unrecognized nitrate reductase genes that have been lost from phylogenetically younger lineages. Indeed, additional nitrate reductases have been described in conjunction with narGHJI for other bacterial species (25) and it cannot be presently excluded that this may also be the case for "M. canettii" and some "ancestral" M. tuberculosis strains. In this connection, it may be interesting to mention previous work demonstrating that the opportunistic pathogen Mycobacterium avium may be grown in media containing nitrate or nitrite as a single nitrogen source, with nitrite being removed more rapidly than nitrate; as M. avium is negative for nitrate reductase in routine biochemical identification, McCarthy suggested that growth was due to the rapid reduction of nitrite spontaneously produced from nitrate (16). Such observations should be kept in mind while looking for additional nitrate reductase genes among M. tuberculosis complex members.
Second, and contrary to previous thought (25), all M. tuberculosis strains are not restricted to the –215T genotype since every "ancestral" M. tuberculosis strain that was tested in this study was –215C. However, narGHJI PCR-RFLP detection of a two-banded pattern may be a helpful marker for the precise discrimination of "modern" M. tuberculosis isolates from "ancestral" M. tuberculosis strains and other MTC subspecies. In so being, this SNP and TbD1 represent valid markers to segregate M. tuberculosis strains along these lines.
Third, the fact that the W-Beijing and CAS1 clinical isolates did not present the –215C genotype observed both in "ancestral" EAI strains and non-M. tuberculosis subspecies of the MTC, although belonging to PGG1, strongly argues in favor of a relatively younger evolutionary age for the W-Beijing genotype than that of the EAI clade of M. tuberculosis (15). Moreover, since the most prevalent M. tuberculosis strains worldwide are from "modern" lineages (9, 10), it is reasonable to hypothesize that the elevated nitrate reductase activity associated with the –215T genotype may have contributed to the evolutionary success of "modern" M. tuberculosis, possibly making these lineages better human pathogens, enhancing their virulence, and facilitating their near-worldwide penetration. Indeed, SCID mouse infection studies, utilizing completely nitrate reductase deficient narG knockout M. bovis BCG, have shown that the mutant was significantly less virulent than the parental strain in terms of bacillary load, granuloma size, and mortality (30). As a result, these data indicated that the process of oxygen-independent respiration by narGHJI plays an important role in the adaptation of tubercle bacilli to the anaerobic environments that they face when intracellular and/or within the granuloma. As such, the elevated nitrate reductase activity of "modern" M. tuberculosis can certainly be envisioned to confer a heightened ability to flourish under anaerobic conditions or when oxygen is limited. These are important lines of inquiry that need to be further addressed in future studies.
Finally, recent studies have found that some of the PGG1 MTC strains originally identified phenotypically as M. africanum subtype II were actually M. tuberculosis strains when speciated by genetic means (13; Richard C. Huard, unpublished). The results of the present study may explain why these isolates were misidentified: they probably exhibited one or more M. tuberculosis-like phenotypic features, with the exception of a lack of aerobic nitrite accumulation, which is an M. bovis characteristic. As a result, these isolates were probably given the default designation of M. africanum subtype II, a category that has been used to denote organisms that represent the phenotypic continuum between M. tuberculosis and M. bovis (12). Hence, our data support the call to do away with the unnecessary "subtype II/East African/M. tuberculosis-like" M. africanum designation (1, 23) since, in every case reported to date, strains identified as such were later found to really be either M. tuberculosis PGG1 or PGG2 isolates rather than true M. africanum when reevaluated by molecular means (1, 2, 13, 19, 20).
In conclusion, our study demonstrates that (i) the W-Beijing and CAS1 families do not share the –215C genotype of other PGG1 MTC organisms, a result that suggests a relatively younger evolutionary age; (ii) the EAI clade which expresses a phenotypically variable nitrate reductase phenotype is genotypically similar to other PGG1 organisms (M. africanum, M. bovis, M. microti, M. pinnipedii, dassie bacillus) but not "modern" M. tuberculosis on the basis of the –215 polymorphic nucleotide within the promoter of the narGHJI operon; (iii) for "M. canettii" and some "ancestral" M. tuberculosis strains, an alternate mechanism to the presence of –215C within the promoter of the narGHJI operon may be hypothesized to explain their nitrate reductase positive phenotype; and (iv) the simple narGHJI PCR-RFLP protocol described herein may be a helpful marker for the precise segregation of "modern" M. tuberculosis isolates from "ancestral" M. tuberculosis strains and other MTC subspecies. Taken together, our results underscore that the use of nitrate as an electron acceptor in anaerobic conditions (e.g., within the granulomatous lesion) appears to be a complex phenotypic-genotypic trait within the MTC and may be linked to virulence. Clearly, these findings deserve further study to be fully understood and appreciated.
ACKNOWLEDGMENTS
We thank many investigators for sharing their DNAs under the spolDB3 and spolDB4 open projects on construction of spoligotyping databases and in particular S. A. L. Al-Hajoj, J. Maugein, J. W. Pape, V. Rasolofo-Razanamparany, M. Ridell, and L. Sechi. We thank M. Fabre for providing the DNAs of some "M. canettii" and M. africanum strains, isolated at the Percy Hospital, Clamart, France. R.C.H. is grateful to D. Cousins, W. R. Butler, and S. Massarella for having shared various other M. tuberculosis complex isolates and to J. L. Ho and W. D. Johnson, Jr., for the support needed to complete this project.
This study was supported by funds obtained through the International network of the Pasteur Institutes-RIIP, Paris, France.
Present address: Clinical Microbiology Laboratory Service and the Department of Pathology in Medicine, New York Presbyterian Hospital, Columbia Medical Center, New York, NY.
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Division of International Medicine and Infectious Diseases, Department of Medicine, Joan and Sanford I. Weill Medical College, Cornell University, New York, New York
ABSTRACT
A well-characterized collection of Mycobacterium tuberculosis complex (MTC) isolates, representing all known subspecies as well as some relevant genotypic families of M. tuberculosis, was analyzed for the newly discovered narGHJI –215 C-to-T promoter single-nucleotide polymorphism (SNP). This point mutation has been shown in earlier studies to be responsible for the differential nitrate reductase activity of M. tuberculosis versus M. bovis. As previously defined by the presence or the absence of the TbD1 genetic locus, the group included both the "modern" W-Beijing, Haarlem, and Central-Asian1 (CAS1) families as well as the "ancestral" East-African-Indian (EAI) clade. Interestingly, among "modern" M. tuberculosis isolates, those previously classified as Principal Genetic Group 1 (PGG1) organisms by katG463-gyrA95 polymorphism analysis did not present the two-banded narGHJI restriction fragment length polymorphism analysis of PCR products pattern common to the other PGG1 MTC members, including the "ancestral" M. tuberculosis isolates. Instead, they showed a one-banded pattern, aligning them with other evolutionarily recent M. tuberculosis isolates of the PGG2 and PGG3 groups, such as Haarlem, Latin-American and Mediterranean (LAM), and X families. The presence of a nitrate reductase producer phenotype in "Mycobacterium canettii" and some "ancestral" M. tuberculosis isolates, despite a two-band –215C genotype, argues in favor of an alternate mechanism to explain the differential nitrate reductase activity of certain PGG1 subspecies of the MTC. Overall, these findings may help to establish the precise evolutionary history of important genotype families such as W-Beijing and suggest that the –215T genotype may have contributed the virulence, spread, and evolutionary success of "modern" M. tuberculosis strains compared to the remaining MTC organisms.
INTRODUCTION
Together with niacin production, catalase/peroxidase activity, and urease production, the assay of nitrate reductase activity, through the accumulation of nitrite, remains a basic phenotypic criterion for differentiating Mycobacterium tuberculosis from Mycobacterium bovis in diagnostic mycobacteriology (6). Contrary to M. tuberculosis, which presents a strong nitrate reductase activity under aerobic conditions, M. bovis and the M. bovis-derived BCG vaccine strain fail to rapidly accumulate nitrite from nitrate. However, both M. tuberculosis and M. bovis possess the narGHJI operon, which is expressed under anaerobic conditions in both subspecies and the product of which is a membrane-bound anaerobic nitrate reductase complex (25). As an alternative to using oxygen as a terminal electron acceptor, the NarGHJI complex couples this process to the reduction of nitrate. It was shown previously that this enzyme is induced under anaerobic conditions in Bacillus subtilis (18), and the switch to anaerobic metabolism is believed to be critical in the establishment of latent infection by M. tuberculosis and possibly the other tubercle bacilli. Recently, a C-to-T transition at position –215 upstream of the GTG start codon was identified in the putative promoter region of the narGHJI operon (25). This transition was hypothesized to be responsible for the differential nitrate reductase activity between M. tuberculosis and M. bovis as a result of increased gene expression in M. tuberculosis, and –215T was presented as an M. tuberculosis-specific single-nucleotide polymorphism (SNP) (25, 26). However, this work did not cover the full range of M. tuberculosis complex (MTC) subspecies now known to exist, and it was unclear whether the evaluated M. tuberculosis clinical isolates represented both "modern" and "ancestral" M. tuberculosis strains.
Indeed, phylogenetic segregation of M. tuberculosis into "ancestral" versus "modern" lineages is based upon the presence or absence, respectively, of a chromosomal deletion dubbed TbD1 (1). Examples of "modern" M. tuberculosis families include W-Beijing, Haarlem, and Central-Asian1 (CAS1) as well as the M. tuberculosis laboratory strain H37Rv, whereas the East-African-Indian (EAI) family represents an "ancestral" M. tuberculosis lineage (1, 22). To date, all other MTC subspecies, including "Mycobacterium canettii," Mycobacterium africanum, Mycobacterium microti, Mycobacterium pinnipedii, and M. bovis, as well as the so-called dassie bacillus, have also been shown to retain TbD1 intact (reference 1 and Richard C. Huard, unpublished data). Notably, all tubercle bacilli that retain the TbD1 locus are also independently classified as principal genetic group 1 (PGG1) organisms by katG463-gyrA95 SNP analysis (24), while all "modern" M. tuberculosis strains fall into either PGG1, PGG2, or PGG3. As a result, the prevailing theory posits that the TbD1 deletion occurred in a PGG1 M. tuberculosis clone whose progeny then went on to establish the "modern" PGG1, PGG2, and PGG3 M. tuberculosis lineages, and that all current "ancestral" PGG1 M. tuberculosis strains are direct descendants of pre-TbD1 deletion M. tuberculosis organisms (1). It is also important to mention that, for reasons that remain to be determined, the known "modern" M. tuberculosis families are far more prevalent worldwide than their "ancestral" counterparts (9, 10).
In the present study, we aimed to further determine the distribution of the –215 C-to-T narGHJI SNP within a well-characterized collection of 68 DNAs of the MTC using a newly devised restriction fragment length polymorphism analysis of PCR products (PCR-RFLP) protocol. The results showed that all M. tuberculosis strains are not necessarily confined to the –215T narGHJI SNP. Consequently, this mechanism cannot solely explain the differential nitrite accumulation within the MTC given that "M. canettii" and some "ancestral" M. tuberculosis strains have the –215C genotype and yet reduce nitrate under aerobic conditions. These results are discussed both in terms of differential virulence and from an evolutionary genetics perspective.
MATERIALS AND METHODS
Strains and DNAs. The studied collection comprised a set of 68 DNAs from many strains of the MTC that were well characterized within the international spoligotyping database project SpolDB4 (K. Brudey, unpublished data). A previous version of this database (SpolDB3) was published recently and provides the basis for most of the defined MTC clades (9, 10). The strains comprised 14 W-Beijing, 8 Central Asian1 (CAS1), and 14 "ancestral" East-African-Indian (EAI1, EAI3, EAI2/Manila) genotypes within the PGG1, 11 PGG2, or 3 strains of unknown genotype or belonging to the Haarlem, X, T1, Cameroon (CAM), or Latin American and Mediterranean (LAM) families. For a detailed comparison with the various members within the MTC, DNAs representing the following species were used: the M. tuberculosis reference strain H37Rv, two strains of M. africanum, three strains of M. bovis, one strain of M. bovis BCG, two strains of M. microti (3), two strains of M. pinnipedii (4), eight strains of "M. canettii" (8, 29), and two strains identified as the dassie bacillus (5).
Spoligotyping and katG463-gyrA95 polymorphism. Most spoligotyping results were obtained at the Institute Pasteur of Guadeloupe, using a previously published procedure and homemade membranes (14, 27). The katG463-gyrA95 results for M. tuberculosis complex (24) have been previously published elsewhere (references 1, 13, and 17 and Richard C. Huard, unpublished) or were implied according to the correlation established between PGG and spoligotyping (21).
Nitrate reduction and assay of the –215 narGHJI promoter polymorphism. Nitrate reduction data were obtained in house for available clinical isolates using a classical method (6) or recorded from providing laboratories when the assay had already been performed. A genome search into NCBI-PubMed (http://www.ncbi.nlm.nih.gov/entrez) retrieved the full sequence of both M. tuberculosis CDC1551 and M. bovis AF2122/97 (respective accession numbers NC002755 and NC002945). The narGHJI sequences were located from these sequences, and a 155-bp DNA fragment of the 5' untranslated region prior to narG, which contained the SNP at position –215, was amplified using LC66 (AACCGACGGTGTGGTTGAC) as a forward primer and LC67 (ATCTCGATGGATGGGCGTC) as a reverse primer (26). The single difference between M. bovis and M. tuberculosis corresponded to a C-to-T transition at position 215 in M. tuberculosis. PCR conditions were as follows: a 7-min initial denaturation phase at 94°C, followed by a 1-min annealing at 60°C and a 1-min synthesis at 72°C. A total of 45 cycles was used to increase product yields. Each reaction tube (50 μl) contained 33 μl of ultrapure water, 5 μl of 10x PCR buffer, 2 μl of MgCl2 (25 mM), 0.5 μl of a mixture of A, C, G, and T deoxynucleotides (25 mM), 2 μl of each of the LC66 and LC67 primers (10 pmol/μl), 0.2 μl of rTaq DNA polymerase (Amersham, Buckinghamshire, England), and 5 μl of DNA that was microbead extracted (7) or using a dilution of cetyl-trimethyl-ammonium-bromide-extracted DNA (28). A single PCR product was observed for each DNA isolate. A 10-μl aliquot of the amplified fragments (roughly 155 to 160 bp on gel) was further digested using either Sau3AI or DpnII according to the manufacturer's instructions (New England Biolabs, Beverly, MA, for DpnII and Roche-Biomedicals, Meylan, France for Sau3AI). These enzymes specifically cut at the GATC sequence overlapping the M. bovis-like –215C narGHJI promoter site, producing two bands (of approximately 90 and of 70 bp) in PCR-RFLP of the LC66-LC67 PCR fragment. In the case of the M. tuberculosis-like –215T sequence, the restriction site is absent and as a result PCR-RFLP of the narGHJI amplicon yields a single uncut band. The restriction digest was visualized by agarose gel electrophoresis on 3% Metaphor (FMC Bioproducts, Rockland, Maine) and run in Tris-borate-EDTA (1x) buffer, followed by ethidium-bromide staining. Pictures were taken electronically and were analyzed using the Taxotron software (PAD Grimont, Taxolab; Institut Pasteur) as previously reported (11).
RESULTS AND DISCUSSION
Results of narGHJI PCR-RFLP analysis, nitrate reduction testing, spoligotyping, and PGG groupings based on katG463-gyrA95 polymorphism are summarized in Fig. 1 and detailed in Tables 1 and 2. With respect to MTC organisms other than M. tuberculosis, all test isolates were PGG1 and bore the –215C narGHJI promoter SNP as determined by the two-band pattern observed in PCR-RFLP of the LC66-LC67 PCR fragment (Table 1). With the exception of the "M. canettii" strains, each of these isolates failed to reduce nitrate. The results also showed that all "modern" M. tuberculosis strains produced a single band in narGHJI PCR-RFLP and accumulated nitrite independently of PGG genotype (Table 2). However, even though each "ancestral" M. tuberculosis tested was PGG1, in narGHJI PCR-RFLP the isolates produced a two-band pattern indicative of a –215C genotype and nitrate reduction was variable.
Given that the –215T SNP has been shown to enhance gene expression of the nitrate reductase cluster (26), our data raise several points with interesting implications. First, since "M. canettii" and some "ancestral" M. tuberculosis strains accumulated nitrite despite having the –215C genotype, then this polymorphism cannot be solely accountable for differential nitrate reductase activity within the MTC. It remains possible that "M. canettii" and some "ancestral" M. tuberculosis strains have unidentified compensatory mechanisms that normalize narGHJI activity and represent differences that are absent from M. bovis and its nitrite-negative brethren. Alternatively, given the respective positions of each MTC organism in recently proposed phylogenetic maps for the evolution of the MTC (1, 17), it remains possible that "M. canettii" and some "ancestral" M. tuberculosis strains, which are theoretically among the oldest MTC lineages, possess additional unrecognized nitrate reductase genes that have been lost from phylogenetically younger lineages. Indeed, additional nitrate reductases have been described in conjunction with narGHJI for other bacterial species (25) and it cannot be presently excluded that this may also be the case for "M. canettii" and some "ancestral" M. tuberculosis strains. In this connection, it may be interesting to mention previous work demonstrating that the opportunistic pathogen Mycobacterium avium may be grown in media containing nitrate or nitrite as a single nitrogen source, with nitrite being removed more rapidly than nitrate; as M. avium is negative for nitrate reductase in routine biochemical identification, McCarthy suggested that growth was due to the rapid reduction of nitrite spontaneously produced from nitrate (16). Such observations should be kept in mind while looking for additional nitrate reductase genes among M. tuberculosis complex members.
Second, and contrary to previous thought (25), all M. tuberculosis strains are not restricted to the –215T genotype since every "ancestral" M. tuberculosis strain that was tested in this study was –215C. However, narGHJI PCR-RFLP detection of a two-banded pattern may be a helpful marker for the precise discrimination of "modern" M. tuberculosis isolates from "ancestral" M. tuberculosis strains and other MTC subspecies. In so being, this SNP and TbD1 represent valid markers to segregate M. tuberculosis strains along these lines.
Third, the fact that the W-Beijing and CAS1 clinical isolates did not present the –215C genotype observed both in "ancestral" EAI strains and non-M. tuberculosis subspecies of the MTC, although belonging to PGG1, strongly argues in favor of a relatively younger evolutionary age for the W-Beijing genotype than that of the EAI clade of M. tuberculosis (15). Moreover, since the most prevalent M. tuberculosis strains worldwide are from "modern" lineages (9, 10), it is reasonable to hypothesize that the elevated nitrate reductase activity associated with the –215T genotype may have contributed to the evolutionary success of "modern" M. tuberculosis, possibly making these lineages better human pathogens, enhancing their virulence, and facilitating their near-worldwide penetration. Indeed, SCID mouse infection studies, utilizing completely nitrate reductase deficient narG knockout M. bovis BCG, have shown that the mutant was significantly less virulent than the parental strain in terms of bacillary load, granuloma size, and mortality (30). As a result, these data indicated that the process of oxygen-independent respiration by narGHJI plays an important role in the adaptation of tubercle bacilli to the anaerobic environments that they face when intracellular and/or within the granuloma. As such, the elevated nitrate reductase activity of "modern" M. tuberculosis can certainly be envisioned to confer a heightened ability to flourish under anaerobic conditions or when oxygen is limited. These are important lines of inquiry that need to be further addressed in future studies.
Finally, recent studies have found that some of the PGG1 MTC strains originally identified phenotypically as M. africanum subtype II were actually M. tuberculosis strains when speciated by genetic means (13; Richard C. Huard, unpublished). The results of the present study may explain why these isolates were misidentified: they probably exhibited one or more M. tuberculosis-like phenotypic features, with the exception of a lack of aerobic nitrite accumulation, which is an M. bovis characteristic. As a result, these isolates were probably given the default designation of M. africanum subtype II, a category that has been used to denote organisms that represent the phenotypic continuum between M. tuberculosis and M. bovis (12). Hence, our data support the call to do away with the unnecessary "subtype II/East African/M. tuberculosis-like" M. africanum designation (1, 23) since, in every case reported to date, strains identified as such were later found to really be either M. tuberculosis PGG1 or PGG2 isolates rather than true M. africanum when reevaluated by molecular means (1, 2, 13, 19, 20).
In conclusion, our study demonstrates that (i) the W-Beijing and CAS1 families do not share the –215C genotype of other PGG1 MTC organisms, a result that suggests a relatively younger evolutionary age; (ii) the EAI clade which expresses a phenotypically variable nitrate reductase phenotype is genotypically similar to other PGG1 organisms (M. africanum, M. bovis, M. microti, M. pinnipedii, dassie bacillus) but not "modern" M. tuberculosis on the basis of the –215 polymorphic nucleotide within the promoter of the narGHJI operon; (iii) for "M. canettii" and some "ancestral" M. tuberculosis strains, an alternate mechanism to the presence of –215C within the promoter of the narGHJI operon may be hypothesized to explain their nitrate reductase positive phenotype; and (iv) the simple narGHJI PCR-RFLP protocol described herein may be a helpful marker for the precise segregation of "modern" M. tuberculosis isolates from "ancestral" M. tuberculosis strains and other MTC subspecies. Taken together, our results underscore that the use of nitrate as an electron acceptor in anaerobic conditions (e.g., within the granulomatous lesion) appears to be a complex phenotypic-genotypic trait within the MTC and may be linked to virulence. Clearly, these findings deserve further study to be fully understood and appreciated.
ACKNOWLEDGMENTS
We thank many investigators for sharing their DNAs under the spolDB3 and spolDB4 open projects on construction of spoligotyping databases and in particular S. A. L. Al-Hajoj, J. Maugein, J. W. Pape, V. Rasolofo-Razanamparany, M. Ridell, and L. Sechi. We thank M. Fabre for providing the DNAs of some "M. canettii" and M. africanum strains, isolated at the Percy Hospital, Clamart, France. R.C.H. is grateful to D. Cousins, W. R. Butler, and S. Massarella for having shared various other M. tuberculosis complex isolates and to J. L. Ho and W. D. Johnson, Jr., for the support needed to complete this project.
This study was supported by funds obtained through the International network of the Pasteur Institutes-RIIP, Paris, France.
Present address: Clinical Microbiology Laboratory Service and the Department of Pathology in Medicine, New York Presbyterian Hospital, Columbia Medical Center, New York, NY.
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