Exploring the potential role of moonlighting function of the surface-associated proteins from Mycobacterium bovis BCG Moreau and Pasteur by comparative proteomic
Tuberculosis (TB) is one of major causes of death worldwide. Bacillus Calmette-Guerin (BCG) is the only licensed TB vaccine and its inability to protect against adult pulmonary TB can be due to genetic differences among strains described since the 1940s. In this work, we compared the proteomic profile of the surface-associated proteins from M. bovis BCG Moreau, the Brazilian vaccine strain, and the BCG Pasteur reference strain. The methodology used was 2D-gel electrophoresis combined with mass spectrometry techniques (MALDI-TOF/TOF). We identified 115 proteins. Of these, 24 proteins showed differential expression between the two BCG strains. Furthermore, 27 proteins previously described as displaying moonlighting function were identified, 8 of these proteins showed variation in abundance comparing BCG Moreau to Pasteur and 2 of them presented two different domain hits.
Sample Processing Protocol
Bi-dimensional electrophoresis: IPG strips and all 2DE reagents were purchased from Bio-Rad, (Hercules, CA, USA). For the first dimension, 500 μg of proteins were diluted to a final volume of 300 μL of rehydration solution (8Murea, 2% CHAPS), 4mM Tributyl phosphine (TBP), 0.4% ampholytes pH 3-10, trace of bromophenol blue).The samples were applied to IPG strips (17cm, pH interval of 4-7) by in-gel rehydration and incubated for 1 h at room temperature. All isoelectric focusing was performed on a Protean® IEF cell (Bio-Rad) with a temperature of 20 ºC and a maximum current of 50 µA/strip. Running conditions: active rehydration (50V) for 11 h; step 1- linear gradient from 1 to 250V over 20 min; step 2 - linear gradient from 250 to10000V over 2 h; step 3- constant 10,000V until complete 80,000 Vh. For IPG strips in the pH range 3-6, step 3 was constant 10,000V until 60,000Vh was achieved. After isoelectric focusing, proteins were reduced in 130 mM DTT and alkylated in 270 mM iodoacetamide, both in equilibration buffer (6M urea, 2% SDS, 375mM Tris-HCl pH 8.8, 20% glycerol). Second dimension separation was done in 17cm, 12% SDS-PAGE gel, 1.0mm thick, using a vertical system (Bio-Rad) in standard Tris/glycine/SDS buffer at 40 mA/gel, 10 ºC, until the tracking dye left the gel. Proteins were visualized with CBB following procedures described elsewhere. Image analysis: Gel images were documented using a GS-800TM calibrated imaging densitometer (Bio-Rad) and image analysis was performed using PDQuestTM software (Bio-Rad). During the alignment of the images, to compensate for subtle differences in sample loading, gel staining, and destaining, the volume of each spot was normalized in relation to the total density of valid spots present in the gel image. Comparison of 2DE maps derived from three independent protein preparations, each one obtained from three independent BCG cultures, was performed. To determine experimental pI and Mr coordinates for each single spot, 2DE gels were calibrated using a select set of reliable identification landmarks distributed throughout the entire gel. The theoretical pI and Mr of proteins identified by mass spectrometry were obtained using BCG Moreau RDJ genome reference. Protein digestion, peptide extraction and MALDI-TOF/TOF analysis: In-gel digestion of the 2D SDS-PAGE separated proteins was carried out using the procedure according to Shevchenko. Briefly, protein spots were excised and the gel pieces were washed three times with 50% (v/v) acetonitrile in 25 mM ammonium bicarbonate for 15 min each, dehydrated in acetonitrile, and dried in a vacuum centrifuge. Gel pieces were rehydrated in 15 µL of 50 mM ammonium bicarbonate containing 20 ng of sequencing grade modified trypsin (Promega). After 15 min, 20 µL of 50 mM ammonium bicarbonate were added to keep the gel pieces wet during tryptic digestion (37 °C, 16 h). To extract peptides, 20 µL of 0.5% (v/v) TFA in 50% (v/v) acetonitrile were added and samples were sonicated for 30 min. The separated liquid was concentrated under vacuum to an approximate volume of 10 µL. The resulting peptides were extracted, partially dried, and salts were removed using ZipTipC18 (Millipore, Bedford, MA) following the manufacturer’s instructions. The tryptic peptides were analyzed on a 4700-Proteomics Analyzer MALDI-TOF/TOF (Applied Biosystems, Foster City, CA). All mass spectra were acquired on positive ion reflector mode with 2,000 shots per spot and externally mass calibrated with a peptide mixture. The 10 most intense ion peaks from the peptide mass fingerprinting (or MS run) were further submitted to fragmentation using PSD mode with CID gas off and 1 keV collision energy.
Data Processing Protocol
Data Analyses and protein identification: Following MS/MS acquisition, the processed data files (ppw files) from the MALDI-TOF/TOF were analyzed on a Mascot Server license v. 2.2. The mass spectra were searched against the Mycobacterium bovis BCG str. Moreau RDJ protein database. The parameters used for the search were as follows: peptide and fragment ions mass tolerance was set at 0.5 Da; maximum of one miss cleavage site by trypsin; carbamidomethylation of cysteine residues as fixed modification, whereas oxidation of methionine/tryptophan, acetylation of the N-terminal, pyroglutamic acid, pyroglutamine and deamidation of asparagine / glutamine were considered as variable modifications. Positive protein hit identification was accepted with at least 1 matched unique peptide. False discovery rate was estimated less than 1%. The peptide ion score was considered greater than 15 with a significance threshold of p<0.05, whereas the protein score was above 20. Finally, a good correlation between the experimental and theoretical molecular mass and pI was also considered for positive identifications. Putative signal peptide for protein export were predicted using SignalP 4.1, LipoP 1.0, TatP 1.0 and SecretomeP 2.0 in order to predict protein localization. Potential transmembrane domains were predicted with TMHMM 2.0. Beta-barrel membrane proteins structural subclass from integral membrane proteins were discriminated using a Hidden Markov Model method with PRED-TMBB. In order to go further in the characterization of the proteins, functional classification using TubercuList was performed.
Pagani TD, Guimarães ACR, Waghabi MC, Corrêa PR, Kalume DE, Berrêdo-Pinho M, Degrave WM, Mendonça-Lima L. Exploring the Potential Role of Moonlighting Function of the Surface-Associated Proteins From Mycobacterium bovis BCG Moreau and Pasteur by Comparative Proteomic. Front Immunol. 2019 10:716 PubMed: 31080447