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Comparison of the membrane proteome of virulent Mycobacterium tuberculosis and the attenuated Mycobacterium bovis BCG vaccine strain by label-free quantitative proteomics.

ABSTRACT: The Mycobacterium tuberculosis membrane is rich in antigens that are potential targets for diagnostics and the development of new vaccines. To better understand the mechanisms underlying MTB virulence and identify new targets for therapeutic intervention, we investigated the differential composition of membrane proteomes between virulent M. tuberculosis H37Rv (MTB) and the Mycobacterium bovis BCG vaccine strain. To compare the membrane proteomes, we used LC-MS/MS analysis in combination with label-free quantitative proteomics, utilizing the area under the curve of the extracted ion chromatograms of peptides obtained from m/z and retention time alignment of MS1 features. With this approach, we obtained relative abundance ratios for 2203 identified membrane-associated proteins in high confidence. Of these proteins, 294 showed statistically significant differences of at least two fold in relative abundance between MTB and BCG membrane fractions. Our comparative analysis detected several proteins associated with known genomic regions of difference between MTB and BCG as being absent, which validated the accuracy of our approach. In further support of our label-free quantitative data, we verified select protein differences by immunoblotting. To our knowledge, we have generated the first comprehensive and high-coverage profile of comparative membrane proteome changes between virulent MTB and its attenuated relative BCG, which helps elucidate the proteomic basis of the intrinsic virulence of the MTB pathogen.

SUBMITTER: Gunawardena HP

PROVIDER: S-EPMC4360911 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Comparison of the membrane proteome of virulent Mycobacterium tuberculosis and the attenuated Mycobacterium bovis BCG vaccine strain by label-free quantitative proteomics.

Gunawardena Harsha P HP Feltcher Meghan E ME Wrobel John A JA Gu Sheng S Braunstein Miriam M Chen Xian X

Journal of proteome research 20131028 12

The Mycobacterium tuberculosis membrane is rich in antigens that are potential targets for diagnostics and the development of new vaccines. To better understand the mechanisms underlying MTB virulence and identify new targets for therapeutic intervention, we investigated the differential composition of membrane proteomes between virulent M. tuberculosis H37Rv (MTB) and the Mycobacterium bovis BCG vaccine strain. To compare the membrane proteomes, we used LC-MS/MS analysis in combination with lab ...[more]

PMID: 24093440

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Project description:The live attenuated bacillus Calmette-Guérin (BCG) vaccine for the prevention of disease associated with Mycobacterium tuberculosis was derived from the closely related virulent tubercle bacillus, Mycobacterium bovis. Although the BCG vaccine has been one of the most widely used vaccines in the world for over 40 years, the genetic basis of BCG's attenuation has never been elucidated. We employed subtractive genomic hybridization to identify genetic differences between virulent M. bovis and M. tuberculosis and avirulent BCG. Three distinct genomic regions of difference (designated RD1 to RD3) were found to be deleted from BCG, and the precise junctions and DNA sequence of each deletion were determined. RD3, a 9.3-kb genomic segment present in virulent laboratory strains of M. bovis and M. tuberculosis, was absent from BCG and 84% of virulent clinical isolates. RD2, a 10.7-kb DNA segment containing a novel repetitive element and the previously identified mpt-64 gene, was conserved in all virulent laboratory and clinical tubercle bacilli tested and was deleted only from substrains derived from the original BCG Pasteur strain after 1925. Thus, the RD2 deletion occurred after the original derivation of BCG. RD1, a 9.5-kb DNA segment found to be deleted from all BCG substrains, was conserved in all virulent laboratory and clinical isolates of M. bovis and M. tuberculosis tested. The reintroduction of RD1 into BCG repressed the expression of at least 10 proteins and resulted in a protein expression profile almost identical to that of virulent M. bovis and M. tuberculosis, as determined by two-dimensional gel electrophoresis. These data indicate a role for RD1 in the regulation of multiple genetic loci, suggesting that the loss of virulence by BCG is due to a regulatory mutation. These findings may be applicable to the rational design of a new attenuated tuberculosis vaccine and the development of new diagnostic tests to distinguish BCG vaccination from tuberculosis infection.

Dataset Information

Comparison of the membrane proteome of virulent Mycobacterium tuberculosis and the attenuated Mycobacterium bovis BCG vaccine strain by label-free quantitative proteomics.

Publications

Comparison of the membrane proteome of virulent Mycobacterium tuberculosis and the attenuated Mycobacterium bovis BCG vaccine strain by label-free quantitative proteomics.

Similar Datasets

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