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Division of Infectious Diseases and Geographic Medicine, Stanford University Medical Center, Stanford, California 94305
Tuberculosis Center, Public Health Research Institute, 225 Warren St., Newark, New Jersey 07103-3535
Mycobacteria Reference Department, Diagnostic Laboratory for Infectious Diseases and Perinatal Screening, National Institute of Public Health and the Environment, Bilthoven, The Netherlands
ABSTRACT
Beijing/W strains of Mycobacterium tuberculosis are geographically widespread and hypervirulent. To enhance our understanding of their origin and evolution, we sought phylogenetically informative large sequence polymorphisms (LSPs) within the Beijing/W family. Comparative whole-genome hybridization of Beijing/W strains revealed 21 LSPs, 7 of which were previously unreported. We show that some of these LSPs are unique event polymorphisms that can be used to define and subdivide the Beijing/W family. One LSP (RD105) was seen in all Beijing/W strains and thus serves as a useful marker for the identification of this family of strains. Additional LSPs (RD142, RD150, and RD181) further divided this family into four monophyletic subgroups, demonstrating a deeper population structure than previously appreciated. All Beijing/W strains were also observed to have an intact pks15/1 gene that is involved in the biosynthesis of a phenolic glycolipid, a putative virulence factor. A simple PCR assay using these Beijing/W strain-defining deletions will facilitate molecular epidemiological studies and may assist in the identification of the molecular basis of phenotypes associated with this important lineage of M. tuberculosis.
INTRODUCTION
Molecular epidemiological studies have identified a genetically related group of Mycobacterium tuberculosis strains called Beijing/W that are widespread in many regions of the world (3, 10). Strains from this group have been associated with drug resistance, most notably in an outbreak of multidrug-resistant tuberculosis in New York (1, 4, 5, 35) and more recently in Russian prisons (31, 32). This association with drug resistance led to the hypothesis that strains from the Beijing/W group might be hypermutable (25), but this has recently been refuted (37). In Vietnam, Beijing/W strains were also associated with relapses and treatment failures (17). In the laboratory, these strains have a hypervirulent phenotype in mice and elicit a distinct immune response with a reduced level of cytokines, resulting in a failure to induce Th1 type immunity (19, 20) of which a polyketide synthase-derived phenolic glycolipid (PGL) is thought to play a principal role (26). Beijing/W strains also have a faster growth rate within human monocytes than do control strains (18). The bacterial genetic factors underlying this virulence are unknown, but the genetic relatedness of these Beijing/W strains suggests clonal expansion (19, 25, 29). It has even been hypothesized that vaccination with Mycobacterium bovis BCG may protect less well against Beijing/W strains, suggesting that they are escape variants (11, 19, 36).
Beijing/W strains share genetic markers, such as similar IS6110 restriction fragment length polymorphism patterns and spoligotypes (3, 15). Currently, Beijing/W strains are principally identified by the number of spacers in the direct repeat (DR) region of the M. tuberculosis genome, namely, spoligotype S00034, or ST1, which is characterized by the deletion of spacers 1 to 34 (36) and, in a global survey of spoligotypes, type S00034 was found to be the most common (9). These genetic markers have been powerful and widely used in identifying strains for molecular epidemiology studies of tuberculosis outbreaks. Large sequence polymorphisms (LSPs) have been described in numerous studies of organisms from the M. tuberculosis complex, giving important insights into their evolution and biology (6, 12, 21, 23, 33). An improved understanding of the degree of LSPs in the Beijing/W family will provide markers that will enable a more refined placing of these strains within the broader genetic population structure of the M. tuberculosis species.
In the present study, we seek to classify the Beijing/W strains from a phylogenetic perspective by identifying new LSPs that are unique-event polymorphisms (UEPs). Our data show that the Beijing/W strains are monophyletic within the M. tuberculosis species and also reveal further subpopulations within this family. PCR screens with LSPs can be used to facilitate further epidemiological studies to elucidate the prevalence of these strains in the worldwide epidemic of tuberculosis. Furthermore, this new robust classification provides a rational sampling framework for the selection of strains for exploring fundamental biological questions, such as whether there are specific bacterial factors that promote the geographic dispersion of the Beijing/W family.
MATERIALS AND METHODS
Strain selection and study design. A total of 179 M. tuberculosis strains were used in the present study. In order to identify the genomic deletions in the Beijing/W family of M. tuberculosis, reference strains that represent this family were obtained from the groups that originally described them. Nine strains of M. tuberculosis were provided by D. Van Soolingen (Bilthoven, The Netherlands) representing "Beijing" (16, 36) and five strains were provided by B. Kreiswirth (Newark, NJ) representing "W" (4). LSPs identified in these prototype strains were compared to deletions identified in a separate study of 100 randomly selected clinical isolates from San Francisco (33). For verification, genomic deletions that were found to be common to all Beijing/W strains of M. tuberculosis were used to screen a further 65 clinical strains in San Francisco that had spoligotype S00034, using real-time PCR.
Identification of genomic deletions. Whole genomic hybridization by using the Affymetrix M. tuberculosis GeneChip was used to identify genomic deletions that were absent from the fully sequenced strain H37Rv. The precise location of each deletion was confirmed by PCR and DNA sequencing. A detailed explanation for the methodology can be found in our recent article (33).
Spoligotyping. All strains in the present study were analyzed by spoligotyping using a method that determines the position and number of spacers and DR sequences of the DR region of M. tuberculosis as previously described (14).
Rapid identification of Genomic LSPs and SNPs using Real Time-PCR. Rapid identification of genomic deletions in M. tuberculosis strains were performed by real-time PCR in 96-well plates with the following TaqMan primers and probes: for rrs (ribosomal 16S rRNA, internal control), L_primer CGT TCC CGG GCC TTG TAC, R_primer CGG GTG TTA CCG ACT TTC ATG, and VIC probe CAC CGC CCG TCA CG; for RD105, L_primer AAC GAA CTG CGC ACT GAA CTC, R_primer TCC CGC ACC GGT TGA G, and FAM probe AGA GTG GAC AGT TTC G; for RD207, L_primer TCC GGC TCA CAA GAG GAA GT, R_primer TGT GCC CCA TCG GTG TTC, and FAM probe TTT CGA AGC GTT CGT CA; for RD142, L_primer CGG CAA GAT CAC AAA AAT CCA, R_primer CCC GTA ACG GGT CCA ATG T, and FAM probe TCT TAG TGC AGC CTT C; and for RD150, L_primer TGC TCG GTG GGC AAC TG, R_primer CGG CAC AGC GAG ACG AA, and FAM probe AAC GAC ATC CAC GCA TT. Real-time PCR was also used to detect the single nucleotide polymorphism at katG463 (CTG codon) and gyrA95 (ACC codon) (30), with the following TaqMan primers: for KatG463_SNP, L_primer CCG AGA TTGCCA GCC TTA AG, R_primer GAA ACT AGC TGT GAG ACA GTC AAT CC, KatG463_cgg_FAM probe CAG ATC CGG GCA TC, and KATg463_ctg_VIV probe CCA GAT CCT GGC ATC; and for GyrA95_SNP, L_primer CCG AGA CCA TGG GCA ACT AC, R_primer GGG CTT CGG TGT ACC TCA TC, GyrA95_agc_FAM probe TAC GAC AGC CTG GTG CG, and GyrA95_acc_VIC probe TAC GAC ACC CTG GTG CG. Allelic discrimination was performed to detect SNPs and deletions comparing amplification signal with the 16S rRNA internal control by using an ABI 7000 Real Time Thermocycler with standard conditions.
Analysis of pks15/1 polymorphism. The pks15/1 locus is described to be polymorphic among members of the M. tuberculosis complex (7). This region was analyzed for variability by PCR and sequencing with the oligonucleotide primers pks1L CTG GGT TGG CCT GCA CGT GGG CCA TAA and pks15R GCC CCC GCA GAG GCG CCG GTT with the following conditions of 40 cycles of amplification: 94°C for 0.40 min, 62°C for 0.40 min, and 72°C for 2.00 min. The PCR products obtained were sequenced by using an ABI 377 autosequencer and standard conditions and sequences analyzed by using SeqMan (DNAStar, Inc., Madison, WI).
RESULTS AND DISCUSSION
Characteristics of Beijing/W genomic deletions. In order to identify LSPs that will identify and further classify the Beijing/W family, we analyzed reference Beijing/W strains and a collection of Beijing/W strains from San Francisco at the whole-genome level. Using the Affymetrix GeneChip microarray, a total of 21 distinct LSPs were identified that were present in the sequenced strain H37Rv but absent from the Beijing/W strains. Of these, 13 deletions were found in the 14 reference strains and a further 8 deletions were found in the Beijing/W strains from San Francisco (12, 33). The LSPs ranged in size from 224 to 11,985 bp and partially or completely deleted 91 M. tuberculosis genes. The genomic address, genes deleted, and PCR primers used to amplify and sequence the LSPs boundaries are given in Table 1. The sequences of the LSPs boundaries were found to be conserved. These LSPs have been designated regions of difference (RD) by using the established convention (6). Fourteen LSPs have been described previously (33), while seven are new (RD139BW, RD144BW, RD168BW, RD200BW, RD213BW, RD220BW, and RD224c). Based on frequency, three classes of LSPs were identified: four common LSPs (RD105, RD149, RD152, and RD207) were found in all Beijing/W strains. Three (RD181, RD142, and RD150) were found variably deleted, and the remaining 14 LSPs were unique to a single strain.
These LSPs have deleted and disrupted genes that may have an influence on gene expression and perhaps even be advantageous to Beijing/W. These genomic regions are intriguing for further study. Among the common LSPs, RD105 is 3,467 bp in size and completely deletes Rv0072 and Rv0073 and truncates Rv0071 and Rv0074. Rv0071 has a derived protein sequence similar to proteins of group II intron maturases (22). A variable repeat was also identified located 21 nucleotides downstream of the ATG start codon of Rv0071 and 25 nucleotides before the RD105 deletion. This is a 9-bp sequence that is repeated five times in H37Rv while in Beijing/W strains variability in the number of repeats was observed ranging from three to nine repeats, with a median of seven repeats (results not shown) and is in frame with the open reading frame of Rv0071. The significance of this repeat region is unknown, but it displays homology to five VDP repeats at the N terminus of hypothetical proteins found in two Streptococcus spp. and M. leprae and to internalin, a putative peptidoglycan bound protein from Listeria monocytogenes (8). The location of this repeat region is in close proximity to Rv0071 and may be phase variable. Phase variation is a common mechanism of transcriptional gene regulation in many other pathogenic bacteria (34). Among the other common LSPs, RD207 is 7,399 bp and is responsible for the partial deletion of the DR region and seven genes, including IS6110 (28). One of these genes, Rv2819c, is of unknown function and was found to be downregulated in the attenuated strain H37Ra (27). RD207 is responsible for the nine-spacer S00034 spoligotype characteristic of Beijing/W strains (36) and predictably occurs in all 14 reference strains. RD149 (or RD3) and RD152 are associated with mobile genetic elements, e.g., prophage and hot spots for IS6110 insertion, respectively (6, 13). Of the unique LSPs, RD139BW and RD213BW are of particular interest. RD139BW deletes three genes involved in the Kdp complex, and a gene (trcS) that is part of a two-component regulatory system. In the SCID mouse model, mice infected with deletion mutants of trcS, died more rapidly than those infected with wild-type bacteria, suggesting a role of trcS in virulence (24). RD213BW is 2,119 bp in size and deletes a section of the pks1 gene. Polymorphisms in the pks15/1 genes have previously been described, one of which curtails the production of PGL (7). Considering these parallel deletion events in the pks15/1 region, a positive selection pressure favoring the loss of PGL production might exist under certain circumstances (26). However, most deletions are thought to be slightly deleterious to the fitness of the M. tuberculosis (12). In summary, there is anecdotal evidence suggesting that deleted genes in the Beijing/W strains may contribute to virulence, butno firm associations were observed. However, the frequency of these deletions suggests that some are useful markers for defining the Beijing/W family of M. tuberculosis, which may signify a clonal expansion of these strains through selective pressures. It has been postulated that Beijing/W strains could have emerged as escape variants resulting from insufficient protection by M. bovis BCG vaccination (11, 19, 36). This is an intriguing hypothesis, and these new molecular markers will facilitate future studies on the spread the Beijing/W strains and their role in the tuberculosis epidemic.
Genomic deletions place Beijing/W strains in a monophyletic grouping. In our recent work, we described a phylogeny for the M. tuberculosis species constructed from the 68 LSPs identified in 100 clinical isolates of M. tuberculosis from San Francisco (12). We took this phylogeny and compared the LSPs from the 14 reference Beijing/W strains to observe where they may fit within the M. tuberculosis phylogenetic tree. All of the 14 reference strains were found to have RD105, RD207, RD152, and RD149 LSPs in common with those of the East Asia clade of this phylogeny (12). In order to verify these observations, we performed spoligotyping on all 100 isolates from San Francisco and also found that each of the 23 strains in the East Asia phylogenetic clade had the S00034 spoligotype characteristic of the Beijing/W strains (Fig. 1). Furthermore, all of these strains were also found to be principle genetic group 1, having the katG463/gyrA95 genotype (30), which is another characteristic of Beijing/W strains (3). RD150 was only observed among six strains from East Asia clade (12, 33) and not in the 14 reference strains. Moreover, an additional seven (unique) Beijing/W deletions (RD108, RD110a, RD127, RD129, RD165, RD166, and RD182a) were found only in the strains from East Asia clade, showing that our broader sampling from another data set, refined the population genetic structure within this family (12, 33).
In agreement with previous studies (6, 12), three types of LSPs were identified from this analysis. The first two types include repetitive DNA, mobile genetic elements (e.g., RD149), and LSPs resulting from homologous recombination between IS6110 (e.g., RD152 and RD207). These are likely to occur independently in different strains. In contrast, a third type of LSPs has been identified that are not associated with either repetitive DNA or mobile genetic elements. This latter type (e.g., RD105, RD142, RD150, and RD181) truncate genes at specific positions and are therefore likely to represent UEPs (6, 12). Based on this definition, the Beijing/W lineage is principally defined by deletion RD105, which occurs in all of the Beijing/W strains. RD105 was not found deleted in any non-Beijing/W strains (12). RD149, RD152, and RD207 also occur in all of the Beijing/W strains but are associated with mobile genetic elements and therefore may not be UEPs (Fig. 1). RD142, RD150, and RD181 are variably deleted in Beijing/W strains but are monophyletic for subdivisions of the Beijing/W group. RD142 and RD150 were only observed in strains with RD181 and RD105. These subdivisions are useful for further refining the phylogenetic structure of this lineage and may identify strains within the Beijing/W group with differing phenotypes.
Taken together, these data show that the Beijing/W family of strains is monophyletic and therefore a true clonal lineage within M. tuberculosis. The phylogeny of the Beijing/W lineage of M. tuberculosis (Fig. 2) is a robust classification, providing specific markers that allow rapid and simple identification. It not only defines the Beijing/W lineage but also subdivides it further, demonstrating a refined population structure within this family. This new classification also provides a robust framework for the selection of representative isolates for further study in the laboratory and in molecular epidemiological studies. To illustrate this and to further validate this new typing tool, we screened by using real-time PCR an additional 65 strains from San Francisco that had spoligotype S00034 for these lineage-defining deletions. All 65 strains had RD105 deleted, thereby confirming its utility in identifying Beijing/W strains. These strains were further examined for the presence of RD181, RD150, and RD142 and were found to be variably deleted 59, 12, and 1 time, respectively (i.e., these sequences were not deleted from all strains). This new classification will be a valuable tool for understanding the contribution of Beijing/W strains in the worldwide pandemic of tuberculosis and the evolution and pathogenesis of the tubercle bacterium. Selection of strains for further study using these Beijing/W-defining LSPs could have important implications for the evaluation of new diagnostics and the efficacy of vaccines in different geographic regions. Beijing/W is one of a number of genotypes of M. tuberculosis influencing the current worldwide epidemic of tuberculosis. It has been proposed that a prevalent genotype in Delhi may be a close relative or progenitor of Beijing/W (2). Deletion analysis of this Delhi group, as well as other genotype families, will refine further their origin and evolution.
pks15/1 polymorphisms are absent in all groups of the Beijing/W lineage. A recent study described polymorphisms in the pks15/1 region of mycobacteria that has given insights into the hypervirulence of Beijing/W strains (7, 36). In M. tuberculosis 210 (a Beijing/W strain) and in Mycobacterium canettii the pks15/1 region is an intact open reading frame, whereas in Mycobacterium bovis there is a 6-bp deletion and in the H37Rv reference strain there is a 7-bp deletion (7). The pks15/1 region is involved in the production of a PGL, which is immunomodulatory and associated with a hypervirulent phenotype in the mouse model, including the inhibition of the proinflammatory immune response (26). Loss of this gene could therefore account for the various phenotypes attributed to the Beijing/W strain family.
We investigated the pks15/1 region in the Beijing/W strains from the present study to determine whether all four subgroups could potentially produce PGL. PCR amplification and sequencing of this pks15/1 region was performed on the 102 Beijing/W strains (14 reference strains, 23 strains from the East Asia clade [12], and the 65 strains from San Francisco). The results revealed that all four subgroups of Beijing/W strains had an intact pks15/1 open reading frame (Fig. 1) and potentially could produce PGL. This confirms and expands the findings of a study which used this polymorphism to refine the phylogeny of the M. tuberculosis complex (21).
Concluding remarks. Comparative whole-genome hybridization was previously used to provide insights into the evolution of M. tuberculosis (6, 23, 33). We applied the same approach to investigate genetic diversity in the Beijing/W family, a set of strains that are geographically widely disseminated. LSPs were found that were associated with this family of strains and that define it as a lineage within M. tuberculosis. Interestingly, a group of LSPs were also identified that subdivide the Beijing/W family into at least four exclusive subgroups, raising the possibility that there are phenotypic differences even within the Beijing/W family. Future studies attributing phenotypes to the Beijing/W family should ensure all genetic subgroups are represented. We thus have characterized the Beijing/W family within the broader phylogeny of M. tuberculosis and have identified robust markers that will help to elucidate the origin and role of this lineage in the global tuberculosis pandemic.
ACKNOWLEDGMENTS
We gratefully acknowledge Pablo J. Bifani for help in the selection of strains and Natalia Kurepina, Ruth Griffin, Brian Robertson, and Saurabh Johri for helpful comments on the manuscript.
This study was funded by National Institute of Allergy and Infectious Diseases grant AI34238 (to P.M.S.).
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