Proteomic profile of culture filtrate from the Brazilian vaccine strain Mycobacterium bovis BCG Moreau compared to M. bovis BCG Pasteur

Abstract

Background: Bacille Calmette-Guerin (BCG) is currently the only available vaccine against tuberculosis (TB) and comprises a heterogeneous family of sub-strains with genotypic and phenotypic differences. The World Health Organization (WHO) affirms that the characterization of BCG sub-strains, both on genomic and proteomic levels, is crucial for a better comprehension of the vaccine. In addition, these studies can contribute in the development of a more efficient vaccine against TB. Here, we combine two-dimensional electrophoresis (2DE) and mass spectrometry to analyse the proteomic profile of culture filtrate proteins (CFPs) from M. bovis BCG Moreau, the Brazilian vaccine strain, comparing it to that of BCG Pasteur. CFPs are considered of great importance given their dominant immunogenicity and role in pathogenesis, being available for interaction with host cells since early infection.

Results: The 2DE proteomic map of M. bovis BCG Moreau CFPs in the pH range 3-8 allowed the identification of 158 spots corresponding to 101 different proteins, identified by MS/MS. Comparison to BCG Pasteur highlights the great similarity between these BCG strains. However, quantitative analysis shows a higher expression of immunogenic proteins such as Rv1860 (BCG1896, Apa), Rv1926c (BCG1965c, Mpb63) and Rv1886c (BCG1923c, Ag85B) in BCG Moreau when compared to BCG Pasteur, while some heat shock proteins, such as Rv0440 (BCG0479, GroEL2) and Rv0350 (BCG0389, DnaK), show the opposite pattern.

Conclusions: Here we report the detailed 2DE profile of CFPs from M. bovis BCG Moreau and its comparison to BCG Pasteur, identifying differences that may provide relevant information on vaccine efficacy. These findings contribute to the detailed characterization of the Brazilian vaccine strain against TB, revealing aspects that may lead to a better understanding of the factors leading to BCG's variable protective efficacy against TB.

Figure 1

2DE proteomic profile of CFPs from M. bovis BCG Moreau. Proteins (500 ug) were applied to 17 cm IPG strips in the pH intervals of 3 - 6 (panels A and C) and 5 - 8 (panels B and D) and separated in the second dimension across 12% (panels A and B) and 15% (panels C and D) SDS-PAGE. The images were merged to obtain a composite map in the pH range 3 - 8 (panel E). Protein spots were visualized by colloidal CBB-G250 staining. Identified proteins are numbered in panel E and detailed in Additional file 2, Table S1. Molecular weight standards indicated in kDa.

Figure 2

Correlation between experimentally determined and theoretical pI and M_r and distribution of predicted cellular localization of the identified proteins. The experimental and theoretical pI (panel A) and M_r (panel B) values for the identified protein were compared. Overall, a positive Spearman correlation coefficient r = 0.75, p < 0.0001 and r = 0.95, p < 0.0001, is observed for the data in the pI range between 3 and 8 and M_r range of 9 to 120 kDa, respectively.

Figure 3

Functional classification of the identified M. bovis BCG Moreau CFPs. Identified proteins were classified into functional categories according to BCGList (http://genolist.pasteur.fr/BCGList/).

Figure 4

Comparative 2DE profiles of CFPs from M. bovis BCG strains Moreau and Pasteur. Proteins (500 ug) were applied to IPG strips in the pH intervals of 3 - 6 (panels A and B) and 4 - 7 (panels C and D) and separated in the second dimension in 12% (panels A and B) and 15% (panels C and D) SDS-PAGE. Protein spots were visualized by colloidal CBB-G250 staining and the gels images compared with PDQuest (Bio-Rad). Molecular weight standards indicated in kDa. The sectors shown in more detail in Additional files 5 and 6, Figures S2 and S3, are indicated in the figure (sectors A - G).

Figure 5

CFPs differentially expressed between BCG strains Moreau and Pasteur. Bars represent fold increase (mean ± SD of the pixel intensity ratios for each specified protein spot between strains). Protein spots more expressed in BCG Moreau compared to Pasteur are represented by blue bars while those more expressed in BCG Pasteur compared to Moreau are represented by red bars. Individual values are detailed in Table 1.