Elsevier

Tuberculosis

Volume 89, Issue 2, March 2009, Pages 126-135
Tuberculosis

General
Phenotypic differences between BCG vaccines at the proteome level

https://doi.org/10.1016/j.tube.2008.12.001Get rights and content

Summary

To contribute to Mycobacterium bovis BCG characterization, two substrains were analyzed using two-dimensional gel electrophoresis (2D-PAGE) and mass spectrometry (MS), based on their protective efficacy in a pulmonary-tuberculosis mouse model. Cell-fraction proteins of BCG Denmark and Phipps substrains were separated into ∼500 spots in 2D-PAGE. The proteomes were similar in protein number, and isoelectric point (pI) and molecular mass (MM) distribution. Statistical analysis, resulted in 72 spots with no change, and 168 and 90 unique for BCG Phipps or Denmark, respectively. Two hundred and fourteen spots showed changes in intensity of >1-fold, 138 of Denmark, and 76 of Phipps. Seventeen spots were selected for MS-based identification (13 from Phipps and 4 from Denmark), including unique, as well as proteins with changes in intensity. The proteins identified participate in virulence, detoxification, adaptation, lipid metabolism, information pathways, cell wall and cell processes, intermediary metabolism and respiration, or still hypotheticals. Our findings contribute to phenotype characterization of BCG substrains and provide new elements to consider for the design of diagnostic tools, drug targets and a new vaccine against tuberculosis based upon protein expression through quantitative statistical analysis.

Introduction

Mycobacterium bovis Bacille Calmette-Guèrin (BCG) remains the only vaccine to protect against miliary and meningeal tuberculosis (TB) in humans.1 Since its development in the 1920s, many substrains were derived from the original BCG due to worldwide distribution and different local conditions for vaccine production and preservation.2 Not all BCG substrains remain available, and many studies have described differences among original BCG vaccine-derived substrains at several levels, including protective efficacy,1 culture filtrate analysis,3, 4 genetic coding,5 or protein comparison.6, 7, 8

According to the World Health Organization (WHO), it is mandatory to complete characterization of the BCG substrains to reduce TB threat.9 High throughput gene sequencing has generated increased databases useful for predicting coding genes and protein function. By these means, the BCG Pasteur 1173P2 genome was sequenced in its 4,374,522-bp (3954 protein-coding genes).10 Proteomic approaches (mainly two-dimensional polyacrylamide gel electrophoresis [2D-PAGE] and mass spectrometry [MS]-based analysis) have identified proteins involved in stress response, low-oxygen tension,11 intracellular response, or nutrient starvation in Mycobacterium tuberculosis.4 BCG substrains, including Chicago, Copenhagen, and Pasteur, have also been studied using proteomic approaches,6 and several similarities and differences under standard culture conditions in the cell-fraction,7 culture filtrate,4 and more recently the membrane subproteome12 have been described.

Recently we reported the protection level of 10 different BCG substrains in a pulmonary-tuberculosis mouse model.13 On evaluating colony forming units (CFU), Delayed Type Hypersensitivity (DTH) response, and serological cytokine profile, we showed that BCG Phipps and Denmark induced similar protection against the challenge with M. tuberculosis H37Rv, nevertheless, animals vaccinated with BCG Denmark showed the highest pneumonic area in lungs – one of the most important components of the disease. Studying differences between BCG substrains at proteome level and their role in protective immunity will help unravel causes of success or failure of BCG substrains in protecting against TB. To analyze such differences, in this work we studied the cell-fraction proteome of these BCG substrains under standard culture conditions – equivalent to that when were administrated into mice (mid-log, enriched media culture). Thirteen proteins with increasing intensity in BCG Phipps and four in BCG Denmark were identified, including virulence-related and hypothetical proteins. This knowledge, together with in vivo data for each BCG substrain, will contribute to explaining host–pathogen interaction and aid us in designing a new vaccine better than the current BCG.

Section snippets

Strains

M. bovis BCG Phipps (kindly provided by Marcel Behr [General Hospital, McGill University]), and Denmark (kindly provided by Raúl Mancilla [Instituto de Investigaciones Biomédicas, UNAM]) substrains were grown in Middlebrook 7H9 broth supplemented with ADC (Albumin Dextrose Catalase) 10% (v/v) and 0.2% Tween-80 (v/v) for 8 days at 37 °C with shaking, harvested by centrifugation, washed three times, and suspended in sterile deionised water for lysis.

Sample preparation and 2D-PAGE

Cellular proteins were obtained by sonication of

2D-PAGE resolution and analysis of BCG cell-fraction proteins

A BCG substrains growth curve was done for 20 days; no differences in growth rates were found. The genetic profile of substrains was determined by multiplex PCR18 (Supplemental material, Figure S2). Bacteria from mid-logarithmic phase (day 8) were used for experiments. Up to 500 spots were well resolved in 2D-PAGE, with high reproducibility and low variation among triplicate experiments (Table 1). Quantitative comparison was made up of 544 spots (Figure 1). Spot-intensity-difference

Discussion

Spot number and distribution in our 2D-PAGE closely correlated with an equivalent previous report,8 and with that predicted in M. tuberculosis H37Rv genome20 (Supplemental material, Figure S1). Several proteins identified were previously described by genomic and proteomic approaches6, 7; nevertheless, to our knowledge the present is the first study in which the BCG substrains studied were selected based on the immunological response they induced against tuberculosis in standardized animal model.

Acknowledgments

The authors are grateful to Gabriel Martínez-Batallar and Sandra Contreras for expert advice on proteomic techniques. To Patricia Orduña, for BCG genotyping.

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