RD and DU Profile of 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 1). 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 1). 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, 26].

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 2). 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 1).

The genome of BCG Mexico 1931 contains fewer genes (3,904) than those of BCG Pasteur (3,954) and BCG Tokyo (4,033) [9, 27]. 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).

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 (fadD23), 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 BCG3926) and another gene encoding a putative transcriptional regulator [BCG3925c (whiB6)] belonging to the WhiB protein family (1-7). This family has been proposed to form part of a new redox system in M. tuberculosis[30]. 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, 31]. 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 3 and 4). 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 3).

SNP	Location1	Gene	Change	In comparison with BCG
1	39256	BCGMEX_0037c	NS	Pasteur
2	89198	BCGMEX_0084	NS	Tokyo
3	338587	PE_PGRS4	NS	Tokyo
4	535543	sigK	NS	Tokyo
5	562401	pcaA	NS	Tokyo
6	582595	regX3	NS	Tokyo
7	585522	BCGMEX_0506c	NS	Tokyo and Pasteur
8	644544	BCGMEX_0568	NS	Tokyo
9	739067	mmaA3	NS	Tokyo
10	1165878	BCGMEX_1072c	NS	Tokyo
11	1294737	narJ	NS	Pasteur
12	1465719	BCGMEX_1346c	NS	Tokyo
13	1908146	PPE22	NS	Pasteur
14	1987564	BCGMEX_1787c	NA	Pasteur
15	2171779	BCGMEX_1957	NS	Tokyo and Pasteur
16	2261942	pks12	NS	Pasteur
17	2268073	pks12	NS	Pasteur
18	2599245	hrcA	NS	Tokyo
19	2625029	BCGMEX_2390	NA	Tokyo and Pasteur
20	2637892	PE_PGRS41	NS	Tokyo
21	2839572	BCGMEX_2587c	NS	Pasteur
22	3294834	ilvN	NS	Pasteur
23	3433045	PPE50	NS	Tokyo
24	3433175	PPE50	NS	Tokyo
25	3537078	BCGMEX_3256	NS	Tokyo
26	3592845	BCGMEX_3305c	NS	Tokyo
27	3674969	sdhB	NS	Tokyo
28	3785919	BCGMEX_3469	NS	Tokyo
29	4015927	hpt	NS	Tokyo
30	4059882	BCGMEX_3724	NA	Pasteur
31	4069095	BCGMEX_3734	NS	Tokyo
32	4075693	ponA2	NS	Tokyo and Pasteur
33	4087843	BCGMEX_3752	NS	Tokyo

¹Locations and names of affected genes follow BCG Mexico 1931; bold text indicates previously reported SNPs; NS = non-synonymous; NA = not annotated

SNP	Location1	Gene	Change	In comparison with BCG
1	1671425	BCGMEX_1520	Insertion	Tokyo
2	2744951	PE_PGRS43b	Insertion	Tokyo
3	4060893	acs	Insertion	Tokyo
4	4252183	fadD23	Pseudogene	Pasteur

¹Locations and names of affected genes follow BCG Mexico 1931; bold text indicates previously reported SNPs.

We found SNPs within BCG0510c (pcaA), BCG0532 (regX3), BCG0692c (mma3), BCG0484c (sigK) and BCG3734 (Table 3). The SNPs in the last three genes have been described in previous studies [7, 31]. The SNP found in BCG0692c (mma3) 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 (sigK) 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 (regX3) and Rv0470c (pcaA) 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, 35]. Additionally, regX3 is part of a two-component system regulated by Pi (SenX3-RegX3) that is involved in the virulence of M. tuberculosis[36, 37].

Interestingly, a specific nsSNP from BCG Mexico 1931 causes an amino acid change in BCG3741 (ponA2). Mutations in this gene have been associated with increased sensitivity to heat shock (24 h at 45°C) and exposure to H₂O₂ compared to wild-type M. tuberculosis. Additionally, a ponA2 mutant was found to exhibit lower survival in mice compared to wild-type M. tuberculosis[38].

We also identified six SNPs in PE_PGRS4, PPE22, PE_PGRS41, PPE50 and PE_PGRS43b (Tables 3 and 4). 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[39, 40]. 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 4), 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 4).

Previous studies have shown that BCG strains (Connaught, Tice, Danish and Phipps) differ in their protein profiles [41–43]. 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 (crp) and BCG0484c (sigK) 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 5A 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 5B). 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 5C). 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 [44–47]. 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.

Bacterial strains and DNA isolation

Mycobacterium bovis BCG substrains were grown in Sauton medium for 15 days at 37°C, harvested by centrifugation and stored at -70°C until use. The Birkhaug, Connaught, Danish 1331, Frappier, Moreau, Phipps, Tice and Sweden BCG strains were kindly provided by M. Behr (McGill University Health Centre, Canada) and BCG Tokyo 172 was provided by M. Macías (Instituto Nacional de Pediatría, México). The Mexican Instituto Nacional de Higiene provided the BCG Mexico 1931, 1988 and 1997 substrains. Genomic DNA was extracted by the phenol/chloroform method following established protocols [49].

Identification of regions of difference (RD) and tandem duplications (DU) by PCR

The RD regions of the BCG Mexico substrains were profiled by multiplex PCR using primer sequences and conditions reported by the WHO (personal communication) and by Bedwell et al. [50]. These primers amplify the RD1 (ET1, ET2, ET3), RD2 (RD2l, RD2r), RD8 (RD8l, RD8r), RD14 (RD14l, RD14r), RD16 (RD16l, RD16r), N-RD18 (RD18L, RD18R, RD18wtR) and RD Danish/Glaxo (RD-D/G L, RD-D/G R) regions and an IS6110 insertion sequence (IS6110L, IS6110R, RD2 wtR). Similarly, a DU profile was obtained by PCR using primer sequences reported by Brosch et al. [9]. PCR amplification of the DU regions was performed in a volume of 30 μL with 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl₂, 0.1 mM dNTPs, 20 pmol of each primer, 1 U of Platinum Taq DNA polymerase (Invitrogen, USA) and 50 ng of template DNA. The thermal profile consisted of one cycle at 94°C (5 min); 35 cycles at 94°C (1 min), 55°C (1 min) and 72°C (1 min); and one cycle at 72°C (5 min). Amplification products were analysed by horizontal electrophoresis on 3% (w/v) agarose gels stained with ethidium bromide.

BCG Mexico 1931 genome sequencing

The genome of M. bovis BCG Mexico 1931 was sequenced using 454 pyrosequencing, which was performed by the Sequencing Unit of CINVESTAV, Irapuato, Mexico. Additionally, a fosmid library with inserts of approximately 40 kb was constructed using the CopyControl™ pCCFOS™ system (Epicentre Technologies, USA), and the fos-end sequences of 250 clones were determined using the Sanger method (3730xl DNA Analyzer, Applied Biosystems, USA). Draft assemblies were based on 623,000 reads (36× coverage), and the Phred/Phrap/Consed software package was employed for sequence assembly and quality assessment [51] using the BCG Pasteur 1173P2 sequence as a reference [GenBank: AM408590]. To close gaps and resolve duplicated regions, the complete sequences of three fosmids (approximately 40 kb) and 110 PCR end reads were obtained. Annotation was performed using the RAST Server [52, 53], Artemis [54, 55] and BCGList [56].

BCG Mexico 1931 genome sequence analysis

The BCG Pasteur 1173P2, BCG Tokyo 172 [GenBank: AP010918] and BCG Mexico sequences were compared using Consed software [51] and the Basic Local Alignment and Search Tool (BLAST) [57]. The presence of the new regions of difference found in BCG Mexico 1931 (RDMex01, RDMex02 and RDMex03) in the Birkhaug, Connaught, Danish 1331, Frappier, Moreau, Phipps, Tice, Tokyo 172 and Sweden BCG strains was determined by PCR screening. The primer pairs used were 5'-GCCCAAACAGTTCGACGG-3' and 5'-CCAGACATATGCGAGGAC-3' for RDMex01; 5'-GCGATAATGGGCGATGTCG-3' and 5'-CCGCGGTTGTTGAGTTCG-3' for RDMex02; and 5'-GCAGCAGTGAACGCTTGG-3', 5'-CAGTGGAGCTGAAGGCAG-3' and 5'-GTTGCTTGGACGGCAATCG-3' for RDMex03. PCR amplifications were performed in a volume of 30 μL with 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl₂, 0.1 mM dNTPs, 20 pmol of each primer, 0.4 U Platinum Taq DNA polymerase (Invitrogen, USA) and 20 ng of template DNA. The thermal profile consisted of one cycle at 94°C (5 min); 35 cycles at 94°C (1 min), 55°C (1 min) and 72°C (1 min); and one cycle at 72°C (5 min). Amplification products were analysed by horizontal electrophoresis on 3% (w/v) agarose gels stained with ethidium bromide.

BCG Mexico 1931 proteome

M. bovis BCG Danish, BCG Mexico 1931, BCG Phipps and BCG Tokyo strains were grown in Middlebrook 7H9 medium (pH 7.2) for eight days at 37°C with shaking, harvested by centrifugation, washed and suspended in sterile water for lysis. Cellular proteins were obtained by sonication of mycobacteria (Ultrasonic Processor, Cole Parmer Corporation, USA) in the presence of a protease inhibitor (PMSF, 20 mM) at 4°C. The extracted proteins were quantified using a Bradford assay. For 2D-PAGE, approximately 100 mg of protein was solubilised, denatured, reduced in sample buffer [4% CHAPS, 2 M urea, 70 mM l-dithiothreitol (DTT), 0.001% bromophenol blue, and 0.1% 3-10 ampholyte] and used to rehydrate 11-cm pH 4-7 IPG strips (ReadyStrip™, IPG strips, Bio-Rad). Isoelectric focusing (IEF) was performed on a Multiphor II system (Amersham Biosciences, England) until reaching 52,000 VH at 17°C. The strips were equilibrated twice in a solution containing 4% urea, 30% glycerol (v/v), 50 mM Tris-HCl (pH 8.8), 2% SDS (w/v), and 0.002% bromophenol blue supplemented with 90 mM DTT for the first incubation (15 minutes) and 250 mM iodoacetamide (IAA) for the second incubation (15 minutes). Second-dimension electrophoresis was performed on a 12% polyacrylamide gel (Hoeffer SE-600, Amersham Biosciences, England) for approximately five hours with a voltage gradient of 50-200 V. Once fixed, the proteins were silver-stained, and gel images were captured in a digital format (Molecular Imager GS-800TM Calibrated Densitometer, Bio-Rad, USA). Gel analysis was performed using the program 2-D PDQuest Advance V.8.0 (Bio-Rad, USA). Duplicate gels with proteins obtained from independent cultures were included in the analysis. A master image gel was created using three replicates of each experiment and was used for comparison [42].

Immune blotting

To identify antigenic proteins in BCG Mexico 1931, we conducted an immune blot analysis from the 2D-PAGE gels. Proteins were transferred onto Hybond P polyvinylidene difluoride (PVDF) membranes (GE Healthcare, England) using a Trans-Blot SD Semi-Dry Transfer system (Bio-Rad, USA) for 1 h at 10 V. The membranes were blocked with Tris-buffered saline (TBS) containing 0.05% Tween-20 and 5% skim milk at 4°C overnight. The membranes were then incubated for 1 h at room temperature with sera from subjects with PPD+, PPD-, pulmonary TB or mycobacterioses caused by NTMs. The membranes were incubated with selected sera from each group having the highest IgG2 titres against each of the groups described above (data not shown). Then membranes were subsequently incubated with an alkaline phosphatase-conjugated mouse anti-human IgG2 antibody (1:5000; Zymed, Invitrogen, USA) for 1 h at room temperature. Immune detection was accomplished using the Inmobilon Western System (Millipore Co, USA). Chemiluminescence signals were measured using the Genius Plus system (TECAN, Switzerland).

Protein sequencing

Reactive proteins were sequenced using a 3200 QTRAP hybrid tandem mass spectrometer (3200 QTRAP, Applied Biosystems, USA) equipped with a nano-electrospray ion source (NanoSpray II) and a MicroIonSpray II head. Proteins were identified based on their MS/MS spectra datasets using the MASCOT search algorithm (Version 1.6b9, Matrix Science, London, UK). A BLAST search was conducted using the M. tuberculosis complex subset of the National Centre for Biotechnology Information (NCBI) non-redundant database (NCBI nr20070623).

Nucleotide and protein sequence accession numbers

The complete genome sequence of Mycobacterium bovis BCG Mexico 1931 has been deposited in the NCBI GenBank database under accession number CP002095. The GenBank accession numbers for the identified protein sequences are: JN034599 (pckA), JN034600 (tig), JN034601 (sahH), JN034602 (atpD), JN034603 (icl), JN034604 (fumC), JN034605 (lpd), JN034606 (tuf), JN034607 (kasA), JN034608 (fadA), JN034609 (kasB), JN034610 (BCGMEX_2464c), JN034611 (thrC), JN034612 (groEL2), JN034613 (BCGMEX_2988), JN034614 (BCGMEX_3456c), JN034615 (TB31.7), JN034616 (tsf), JN034617 (nusG), JN034618 (fixB), JN034619 (BCGMEX_0855c), JN034620 (cysA2), JN034621 (sucD), JN034622 (cfp30B), JN034623 (echA8), JN034624 (fbpC), JN034625 (fbpA), JN034626 (nuoC), JN034627 (ahpC), JN034628 (clpP), JN034629 (ssb), JN034630 (rplL), JN034631 (hspX) and JN034632 (ndkA).