RD and DU Profile of BCG Mexico strains
Our RD and DU profile analysis of BCG Mexico 1931 demonstrated the presence of the RD8, RD14, RD16 and RD Danish/Glaxo regions and the absence of the RD1, RD2 and N-RD18 regions, as well as a single copy of the insertion sequence IS6110. These properties are similar to those observed for BCG Phipps and BCG Tice (Table ). In contrast, BCG Mexico 1988 and BCG Mexico 1997 exhibited identical RD and DU profiles, with the RD1, RD2 and RD Danish/Glaxo regions and one copy of IS6110 missing (Table ). This profile is identical to that of BCG Danish. The absence of the RD Danish/Glaxo region, which is specific to BCG Danish, in BCG Mexico 1988 and 1997 confirms this result and is consistent with historical records indicating that BCG vaccine production in Mexico utilised the BCG Danish 1331 strain beginning in 1970.
Region of difference (RD) profiles in BCG Mexico strains.
The amplification pattern of DU regions in BCG Mexico 1931 indicated duplication of DU2-IV, in contrast to those of BCG Mexico 1988 and BCG Mexico 1997, which showed duplication of DU2-III (Table ). These differences in RDs and DUs confirm that BCG Mexico 1931 is a different strain from BCG Mexico 1988 and 1997, which are related to BCG Danish.
The above results and subsequent sequencing of the BCG Mexico 1931 genome place this strain in DU2 group IV within the genealogy of BCG strains (Figure ). These results differ from findings of previous studies in which BCG Mexico was placed in DU2 group III as a strain derived from BCG Danish, which is an erroneous result that can be attributed to the different BCG vaccine production periods in Mexico [17
Figure 1 Genealogy of BCG vaccines, adapted from Brosch et al. . The BCG Mexico 1931 strain was included in this study.
Genome Sequence of BCG Mexico 1931
Mycobacterium bovis BCG Mexico 1931 has a circular chromosome of 4,350,386 bp with an overall G+C content of 65.7% [GenBank: CP002095]. The genome contains 3,904 genes that encode proteins (CDS), three genes that encode rRNAs and 45 genes that encode tRNAs. Additionally, 29 possible pseudogenes have been identified (Figure ). The BCG Mexico 1931 genome is 20 Kb smaller than those of BCG Pasteur 1173P2 (4,374,522 bp) and BCG Tokyo 172 (4,371,711 bp). The differences between BCG Mexico 1931 and BCG Pasteur are due to lack of DU1, the presence of specific deletions and the presence of RD14 in BCG Mexico 1931. With respect to BCG Tokyo 172, the difference in genome size can be explained by the loss of RD2, N-RD18 and one copy of insertion sequence IS6110 as well as by differences in the size of DU2 (Figure ).
Figure 2 Circular map of the M. bovis BCG Mexico 1931 chromosome. The scale is in bases and is shown in the black outer circle. The dark blue circle shows forward-strand CDS, and the light blue circle shows reverse-strand CDS. The next two circles moving inward (more ...)
The genome of BCG Mexico 1931 contains fewer genes (3,904) than those of BCG Pasteur (3,954) and BCG Tokyo (4,033) [9
]. This variation is due to the presence of different RDs in each strain and to differences in the criteria used for annotation of hypothetical proteins not previously described in other BCG strains but annotated in M. tuberculosis
strains (GenBank database).
A comparison of the BCG Mexico 1931 genome sequence with those of BCG Pasteur and BCG Tokyo revealed 95 SNPs and three new RD regions, which were designated RDMex01 (53 bp), RDMex02 (655 bp) and RDMex03 (2,847 bp). These regions were deleted in the BCG Mexico 1931 sequence based on alignment with the BCG Pasteur and BCG Tokyo sequences. The deletions were confirmed by alignment with the M. tuberculosis and M. bovis sequences. These new RD regions led to the loss of three genes [BCG3923, BCG3924 and BCG3925c (whiB6)] and the partial deletion of two genes [(BCG3926 and BCG3889 (fadD23)] in BCG Mexico 1931 (Table ). Interestingly, a PCR screen of these regions in nine BCG strains (Birkhaug, Connaught, Danish, Frappier, Moreau, Phipps, Tice, Tokyo and Sweden) showed that the RDMex02 and RDMex03 regions have been lost only in BCG Mexico 1931 and can therefore be used as molecular markers for this strain.
New RD regions identified in BCG Mexico 1931
RDMex01 is an intergenic deletion located between the BCG0767 (rpsN1) and BCG0768 (rpsH) genes, which encode two subunits of the 30S ribosomal protein. The biological effect of this deletion is unknown.
RDMex02 is associated with deletion of 218 aa from BCG3889
), affecting a conserved region of the protein that includes two transmembrane domains. This gene encodes a probable fatty-acyl CoA ligase involved in lipid degradation. Lynett et al
. have reported that this protein is involved in sulpholipid production and that disruption of the gene results in increased association between bacteria and macrophages [28
]. Molina et al
. found that BCG Mexico 1931 associates more strongly with macrophages (THP-1) compared to BCG Danish, BCG Moreau, BCG Phipps and BCG Tokyo172 [29
Finally, RDMex03 was the largest deletion found in the BCG Mexico 1931 genome. It affected four genes: three genes encoding hypothetical proteins (BCG3923, BCG3924
) and another gene encoding a putative transcriptional regulator [BCG3925c
)] belonging to the WhiB protein family (1-7). This family has been proposed to form part of a new redox system in M. tuberculosis
]. Interestingly, this deletion is situated in the extended RD1 region.
The new RDs described in BCG Mexico 1931 may contribute to understanding of the phenotypic differences between BCG Mexico 1931 and other BCG strains.
Our SNP analysis indicated the presence of 33 SNPs in BCG Mexico 1931 compared to BCG Pasteur and 77 SNPs in BCG Mexico 1931 compared to BCG Tokyo. Among these SNPs, at least 23 have been reported in two previous studies [27
]. Additionally, in agreement with the SNP-based phylogeny constructed by García Pelayo et al
., BCG Mexico 1931 was grouped with BCG Tice in our analysis [31
We found a total of 37 SNPs representing nonsynonymous mutations (nsSNPs), leading to amino acid substitutions (Tables and ). Four SNPs in this group were specific to BCG Mexico 1931 [BCGMEX_0506c, BCGMEX_1957, BCGMEX_2390 and BCG3741 (ponA2)]. On the basis of functional classes, the genes containing nsSNPs encoded hypothetical proteins (19%), proteins involved in intermediary metabolism and respiration (16%), proteins related to lipid metabolism (16%) and PE/PPE family proteins (16%) (Figure ).
Non-synonymous SNPs identified in BCG Mexico compared with BCG Pasteur 1173P2 and BCG Tokyo 172.
Insertions and pseudogenes identified in BCG Mexico 1931 compared with BCG Pasteur 1173P2 and BCG Tokyo 172
Functional classification of the genes affected by SNPs described in Table 3. Numbers in parentheses indicate the number of genes affected in each functional category.
We found SNPs within BCG0510c
) and BCG3734
(Table ). The SNPs in the last three genes have been described in previous studies [7
]. The SNP found in BCG0692c
) causes an amino acid change with a concomitant loss of methoxymycolates in BCG strains obtained from the Pasteur Institute after 1927 [18
]. This result is consistent with the findings of Hayashi et al
., who described the absence of these acids in BCG Mexico 1931 [26
]. An SNP in the start codon of BCG0484c
) is responsible for low expression of MPB70 and MPB83 in late BCG strains, including BCG Mexico 1931 [32
]. Moreover, mutations in BCG3734
, a CRP homologue global regulator, have been described as specific to BCG and are responsible for increased binding of CRP to its target DNA [33
]. Mutations in Rv0491
) and Rv0470c
) have been implicated in the virulence of M. tuberculosis
. The pcaA
gene encodes a mycolic acid cyclopropane synthetase and is important for growth, persistence in macrophages and proinflammatory activity [34
]. Additionally, regX3
is part of a two-component system regulated by Pi (SenX3-RegX3) that is involved in the virulence of M. tuberculosis
Interestingly, a specific nsSNP from BCG Mexico 1931 causes an amino acid change in BCG3741
). Mutations in this gene have been associated with increased sensitivity to heat shock (24 h at 45°C) and exposure to H2
compared to wild-type M. tuberculosis
. Additionally, a ponA2
mutant was found to exhibit lower survival in mice compared to wild-type M. tuberculosis
We also identified six SNPs in PE_PGRS4, PPE22, PE_PGRS41, PPE50
(Tables and ). These genes encode PE/PPE family proteins, which may play a role in the evasion of host immune responses, possibly via antigenic variation of mycobacteria [39
]. In previous studies, it has been shown that the PPE22 protein elicits B cell responses, while PPE50 is required for mycobacterial growth in vitro
]. Furthermore, we determined that PE_PGRS54 (6,285 bp) and PPE_PGRS55 (5,433 bp) correspond to a longer product in BCG Mexico compared with homologous sequences only for BCG Tokyo (6,153 and 5,088 bp, respectively). These results are consistent with data previously described [27
]. Importantly, the functional implications of these size variations remain unknown.
Comparison of BCG proteomes
The protein contents of the cell fractions from four BCG substrains were analysed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) using bacteria in mid-logarithmic phase. A total of 812, 794, 791 and 701 spots were visualised for BCG Mexico 1931, BCG Danish, BCG Phipps and BCG Tokyo, respectively (Figure ), with high reproducibility and low variation between duplicate experiments (0.4%). A comparative analysis of the proteomes of these BCG strains revealed 185 spots common to all strains and 136 that were unique to BCG Mexico 1931. Additionally, this analysis showed higher percentages of spots in common between BCG Mexico 1931 and BCG Danish (62%) or BCG Phipps (61%) than between BCG Mexico 1931 and BCG Tokyo (48%) (Figure ).
Figure 4 Representative 2D-PAGE image for BCG Mexico and comparison with the proteomes of BCG Danish, Phipps and Tokyo. One hundred micrograms of cellular protein were loaded onto IPG strips (pH 4-7) for isoelectrofocusing at 52,000 VH and run on sodium dodecyl (more ...)
Previous studies have shown that BCG strains (Connaught, Tice, Danish and Phipps) differ in their protein profiles [41
]. Here, we observed that late strains (BCG Mexico 1931, Danish and Phipps) had a greater number of proteins in common compared to the early strain we studied (BCG Tokyo). This difference can be explained by mutations in transcriptional regulators such as BCG3734
) and BCG0484c
) in late BCGs. Furthermore, the proteins unique to BCG Mexico 1931 may be useful for characterising this strain and explaining the causes of the observed phenotypic differences compared to other BCG strains.
Characterisation of the immune response by immune blotting
To identify immunogenic proteins in BCG Mexico 1931, we performed an immune blotting analysis. We detected 39 reactive spots in the immune proteome (Figure and Additional file 1
: Table S1). The largest numbers of reactive spots were obtained when using serum from subjects with active TB (16) or positive tuberculin skin test results (PPD+) (14); 12 of these spots were unique to each serum type (Figure ). This result indicates high variability in the proteins recognised by each type of serum. We identified 37 proteins by sequencing (Additional file 1
: Table S1), the majority of which (17; 47%) corresponded to intermediary metabolism and respiration proteins (Figure ). Among the identified proteins, some have been previously described as virulence proteins in different strains of M. tuberculosis
: phosphoenolpyruvate carboxykinase (pckA
), isocitrate lyase (icl
), 3-oxoacyl synthase II (kasA
), groEL, TB27.3, the 85A and 85C antigens, alkyl-hydroperoxide reductase (ahpC
) and heat shock protein HspX [44
]. Rodriguez-Alvarez et al
. have reported that GroEL and AhpC are over-expressed in BCG Phipps [42
]. To our knowledge, phosphoenolpyruvate carboxykinase, isocitrate lyase, 3-oxoacyl synthase II and AhpC are described as antigenic proteins for the first time in this report.
Figure 5 Immunogenic proteins identified in BCG Mexico. A total of 37 immunogenic proteins were identified. Spots circled in red, green, dark blue and light blue represent proteins reactive to sera from subjects with active pulmonary tuberculosis, NTM mycobacterioses, (more ...)