Phenotypic differences between BCG vaccines at the proteome level

Tuberculosis 89 (2009) 126–135

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GENERAL

Phenotypic differences between BCG vaccines at the proteome level Mauricio Rodrı´guez-Alvarez a, Guillermo Mendoza-Herna´ndez b, Sergio Encarnacio´n c, Juan Jose´ Calva d, Yolanda Lo´pez-Vidal a, * a ´noma de Me´xico (UNAM), Programa de Inmunologı´a Molecular Microbiana, Departamento de Microbiologı´a y Parasitologı´a, Facultad de Medicina, Universidad Nacional Auto ´ n, 04510 Me´xico, D.F., Mexico ´n, 4 piso, Av. Universidad #3000, Coyoaca Edificio de Investigacio b ´noma de Me´xico (UNAM), Edificio de Investigacio ´n, 2 piso, Av. Laboratorio de Pe´ptidos y Proteı´nas, Departamento de Bioquı´mica, Facultad de Medicina, Universidad Nacional Auto ´ n, 04510, Me´xico D.F., Mexico Universidad #3000, Coyoaca c ´ lica, Centro de Ciencias Geno ´micas, Universidad Nacional Auto ´noma de Me´xico (UNAM). Apdo. Postal 565-A, 62210 Cuernavaca, Morelos, Mexico Laboratorio de Ingenierı´a Metabo d ´ n (INCMNSZ), Vasco de Quiroga #15, Colonia Seccio ´n Salvador Zubira ´n XVI, Tlalpan, 14000 Unidad de Epidemiologı´a Clı´nica, Instituto Nacional de Ciencias Me´dicas y de la Nutricio Me´xico, D.F., Mexico

a r t i c l e i n f o

s u m m a r y

Article history: Received 10 June 2008 Received in revised form 28 October 2008 Accepted 14 December 2008

To contribute to Mycobacterium bovis BCG characterization, two substrains were analyzed using twodimensional 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 w500 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. Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: Proteomics BCG 2D-PAGE Tuberculosis Protein identification

1. Introduction Mycobacterium bovis Bacille Calmette-Gue`rin (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.6–8 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

* Corresponding author. Tel./fax: þ52 55 56 16 08 44. E-mail address: [email protected] (Y. Lo´pez-Vidal). 1472-9792/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tube.2008.12.001

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, lowoxygen 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

M. Rodrı´guez-Alvarez et al. / Tuberculosis 89 (2009) 126–135

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. 2. Materials and methods 2.1. Strains M. bovis BCG Phipps (kindly provided by Marcel Behr [General Hospital, McGill University]), and Denmark (kindly provided by Rau´l Mancilla [Instituto de Investigaciones Biome´dicas, UNAM]) substrains were grown in Middlebrook 7H9 broth supplemented with ADC (Albumin Dextrose Catalase) 10% (v/v) and 0.2% Tween80 (v/v) for 8 days at 37  C with shaking, harvested by centrifugation, washed three times, and suspended in sterile deionised water for lysis. 2.2. Sample preparation and 2D-PAGE Cellular proteins were obtained by sonication of bacteria in presence of a protease inhibitor (20 mM PMSF) (15 cycles: 1 min ON/1 min OFF) at 4  C. To solubilize, denature, and reduce proteins, sample was mixed with 4% CHAPS, 9 M urea, and 70 mM DTT, and centrifuged (30 min at 100,000  g).14 For isoelectric focusing (IEF), 80 or 400 mg of protein (for analytical or preparative gels, respectively) plus 0.02% Bio-Lyte 3-10 buffer (v/v) were used for rehydrating 11-cm immobilized pH gradient (IPG)-ready strips (Bio-Rad) pH 4–7 for 16 h at room temperature. IEF was carried out on a Multiphor II (Amersham Biosciences) at 17  C until 52,000 or 80,000 VH were completed for analytical and preparative gels, respectively. Prior to second dimension, IPG-strips were equilibrated in a solution containing 6 M urea, 30% (v/v) glycerol, 25 mM Tris–base, and 3.5 mM SDS; first, 15 min with 70 mM DTT, and then 15 min with 120 mM iodoacetamide; afterward, these were placed in contact with 12.5% polyacrylamide gels 1-mm thick, 16  18-cm gels, and sealed with 0.5% agarose (w/v) containing a trace of bromophenol blue. The second dimension was run in a Hoeffer SE600 (Amersham Biosciences) at constant voltage (100 V) at 10  C. 2.3. Gel analysis and spot selection All 2D-PAGE were performed three times with mycobacteria from different independent cultures to overcome technical variation. Gel analysis was performed on silver-stained,15 using PDQuest-Advanced 2D Analysis V8.0 (Bio-Rad). A Master Image Gel (MIG) was integrated with the three gels of each substrain and was utilized for comparison. Detailed comparison of BCG Denmark and Phipps MIG was conducted; up to 50 landmarks were placed in all gel images. All spots present in at least two of six gels were included. Statistical spot-intensity analysis was performed by normalizing spot values according to total image density of the gel to which spots belonged; median and interquartile range for each spot was calculated. Statistical spot-intensity comparison between strains was conducted by Mann–Whitney U-test, considering p < 0.05 (one-tailed) as statistically significant. XLSTAT V.2008.1.01 (Addinsoft) in Microsoft-Excel (Microsoft) was employed. For data management, analysis and presentation, spots

127

selected received the letter ‘‘H’’ followed by a number (i.e., H1, H2, etc.). 2.4. In-gel digestion and MS-based identification Selected spots from colloidal-Coomassie-stained gels15 were excised manually and frozen at 70  C until use. For MALDI-TOF– MS analysis, protein spots were destained, reduced, alkylated, and digested with trypsin, and desalted utilizing C18 ZipTip (Millipore).16 Mass spectra were performed using an Autoflex (Bruker Daltonics) operated in delayed extraction and reflectron mode. Spectra were externally calibrated using peptide calibration standards, while peptide mixtures were analyzed using saturated solution of alpha-cyano-4-hydroxycinnamic acid (HCCA) in 50% acetonitrile/0.1% trifluoroacetic acid. Peak lists of tryptic peptide masses were generated and searched against NCBInr databases utilizing Mascot search engine within MSDB 20050929 database and limited to M. tuberculosis complex (Matrix Science, http:// www.matrixscience.com); search parameters included unrestricted protein molecular mass range, trypsin digestion, peptide mass tolerance þ/0.2 Da, and monoisotopic mass value. Proteins not identified by MALDI-TOF–MS were excised again from gels (both silver- and Coomassie-stained) and analyzed by LC/ ESI-MS/MS using nanoflow liquid chromatography coupled with 3200 Q-TRAP system (Applied Biosystems) equipped with a nanoelectrospray source in automated mode employing Information Dependent Acquisition (IDA).17 Precursor ion determination was done using an Enhanced MS scan over a mass range of 400–1500 m/z at 4000 amu/s (with no trapping in Q0 and LIT fill time of 20.00 ms), with ion spray voltage of 2.4 kV applied to a Picotip FS360-75-15-N with ion spray gas (nitrogen). Precursors ions were collided in Q2 using rolling collision energy (maximum allowed, CE ¼ 80). Enhanced product ion scans (MS/MS) were performed over a mass range of 50–1700 m/z at 4000 amu/s; collision voltages were determined dynamically. All precursor ion mass/charge ratios were confirmed with Enhance Resolution scan. Protein identification was performed with Mascot search engine within MSDB 20050929 database and limited to M. tuberculosis complex; search parameters comprised trypsin digestion, MS/MS ion search, monoisotopic mass values, unrestricted protein mass range, þ/0.5 Da peptide mass tolerance, and þ/0.3 Da fragment mass tolerance. 3. Results 3.1. 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 quantification demonstrated that 72 (13.2%) spots had no changes between

Table 1 Spot quantification of Bacille Calmette-Gue`rin (BCG) vaccines’ proteomes analyzed on PDQuest-Advanced 2D Analysis V8.0 software.

BCG strain

Denmark Phipps

Total spots in Master Image Gel

SD

VC (%)

742 791

16.9 87.3

3.3 19.2

SD, standard deviation among triplicates. VC, variation coefficient ¼ (SD/mean)  100.

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substrains, while 168 (30.9%) and 90 (16.5%) spots were unique (defining unique spot as >140.4-fold increase in pixel intensity) for BCG Phipps and Denmark, respectively. Two hundred and fourteen (39.3%) spots showed change in intensity of 1-fold or more, 138 (25.4%) from BCG Denmark and 76 (13.9%) from Phipps (Table 2) (descriptive statistics in Supplemental material, Table S1). 3.2. Identification of proteins using MALDI-TOF–MS and LC/ESI-MS/MS Seventeen spots with increased pixel intensity (13 of BCG Phipps and 4 of Denmark) were identified by MS-based technologies. Table 3 depicts electrophoretic characteristics and changes in pixel intensity. Protein identification details are presented in Table 4. As technical-procedures control, we took one spot equally intense in both substrains (C1 in Figure 1); both spots were identified as a probable electron transferase sub-unit-beta flavo protein, FixA (BCG3052c or Rv3029c) (data not shown). H2, more intense in BCG Phipps, was identified as alkyl-hydroperoxide-reductase-C (AhpC) (BCG2447 or Rv2428), a protein involved in resistance to both reactive nitrogen intermediates (RNI) and isoniazide. This protein belongs to mycobacterial functional category (FC) 0: virulence, detoxification, and adaptation.19 H5, H13, H21, and H26 were identified as hypothetical proteins (FC 10) BCG1826 (Rv1794), TB 39.8 [BCG0050c (Rv0020c)], CFP17 [BCG1862 (Rv1827)], and conserved 35 kDa alanine-rich protein [BCG2760c (Rv2744c)], respectively; their functions are not yet described. H9, more intense in BCG Phipps, was a mixture of two proteins: a probable S-adenosylmethionine synthase (MetK) (BCG1453, Rv1392) involved in amino acid biosynthesis (FC 7: intermediary metabolism and respiration), and a probable glutamyl-tRNA-amidotransferase sub-unit A (GatA) (BCG3033c, Rv3011c), also involved in amino acid biosynthesis (FC 2: information pathways). Despite different electrophoretic mobility, two spots, H7 and H8, were identified as the same protein, namely the 65 kDa antigen (Cpn60-2) GroEL2 (BCG0479, Rv0440) (FC 0). H7 was highly intense in BCG Phipps, while H8 was in Denmark. H4, was identified as a probable serine proteinase A (PepA) (BCG0159, Rv0125), also called Mtb32A (FC 7); H6 was identified as Mpt32 antigen (Glycoprotein 45 kDa) (BCG1896, Rv1860), a protein belonging to FC 0. H10 was identified as GroEL1 (BCG3487c, Rv3417c), a chaperonin that participates in peptide-folding processes and during heat-shock response (FC 0). H24 was identified as enoyl-CoA-hydratase (BCG0679c, Rv0632c); H25, H28, and H30 were identified as a probable hydrolase (BCG3470, Rv3400), a possible thioredoxin (BCG1386, Rv1324), and glyceraldehyde-3phosphate dehydrogenase (BCG1497, Rv1436), respectively, all Table 2 Spot description of BCG Phipps vs. BCG Denmark comparison according to intensity changes between substrains. Pixel intensity*

Number of spotsy (n ¼ 544) BCG Phipps %x (n)

BCG Denmark %x (n)

Unique (>140.4-fold increase) Fold increase 1–1.5 1.51–2 2.1–5 5.1–10 10.1–140.3 Total No change

30.9 5.5 4 2.4 1.5 0.6 14 13.2

16.5 9.6 6.4 6.6 2.2 0.6 25.4 13.2

(168) (30) (22) (13) (13) (3) (81) (72)

(90) (52) (35) (36) (12) (3) (138) (72)

* Normalization of each spot according to the total density of the gel image to which it belongs. y 544 spots of the BCG Phipps vs. BCG Denmark comparison where included in the analysis. x Taking these 544 spots as 100%.

Table 3 Electrophoretic characteristics and fold increase of selected spots (marked in Figure 1). Spots with higher intensity in BCG Phipps Spot

H2 H4 H5 H9 H10 H6 H7 H21 H27 H28 H25 H24 H30

Experimentaly

Theoretical*

Fold increasex

MM (kDa)

pI

MM (kDa)

pI

21.56 34.9 32.4 42.98 55.87 32.7 56.72 17.25 25.4 32.2 28.2 24.4 36.0

4.3 4.9 4.64 4.73 4.74 4.7 4.56 4.1 4.4 4.4 5.7 5.6 5.1

21.7 33.1 36.1 51.2 61.3 44.1 68.1 16.89 25.4 32.2 28.3 24.4 36.1

4.5 4.34 4.74 4.72 4.9 4.18 4.0 4.34 4.7 4.6 5.6 5.5 5.2

1.3 1 >140.4yy 2.8 3.7 >140.4 2.8yy 5.1yy 1.5 1.8 1.7 2 1.3

Spots with higher intensity in BCG Denmark Spot

H8 H13 H29 H26

Experimentaly

Theoretical*

Fold increase**

MM (kDa)

pI

MM (kDa)

pI

56.72 56.1 37.1 29.3

4.56 4.73 4.9 5.8

62.4 31.6 37.5 29.2

4.55 4.77 5.1 5.7

4.2 1.2 1.1 1.8yy

* Theoretical Molecular Mass (MM) and isoelectric point (pI) were obtained from the BCGList World-Wide Web Server (http://genolist.pasteur.fr/BCGList/). y Experimental values of spot in gel according to PDQuest analysis. x Ratio of median value of pixel intensity in BCG Phipps substrain over the median value of pixel intensity in BCG Denmark. ** Ratio of the median value of pixel intensity in BCG Denmark substrain over the median value of pixel intensity in BCG Phipps. yy One-tailed p value <0.05, Mann–Whitney U-test.

belonging to FC 7. H27 was identified as transcription antitermination protein NusG (BCG0688, Rv0639). These last seven proteins showed increased intensity in BCG Phipps, while H29, a probable alcohol dehydrogenase NADP-dependent (BCG3069, Rv3045), was more intense in Denmark.

4. 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.13 Previous proteome analysis of different Mycobacterium spp. included cell-fraction, culture filtrate, and membrane proteins.4,6,7,21 All had well-established scenarios, and proteins identified integrate robust databases. However, spot-selection criteria from 2D-PAGE for protein identification are not always completely clear, and absence–presence condition is the most utilized. Statistical analysis represents an effective approach for proteome comparison. Thus, here we found that BCG Denmark possesses more spots with intensity changes, while Phipps has more unique spots (Table 2). This analysis could provide strong direction for which spots should be selected for protein identification. Nowadays the BCG Pasteur 1173P2 substrain could be considered the BCG-studies reference substrain, and its genome sequence

Table 4 Identification data of proteins with changes on intensity in BCG Phipps (A-1 and A-2) and BCG Denmark (B1 and B2). (A-1) Proteins with increased intensity in BCG Phipps, identified by MALDI-TOF–MS* Spot

Accession number

Gene namey

Protein Function (FCx)

BCG

MTb H37Rv orthologue

1826c

1794c

0479

0440

3487c

3417c

1453

1392

3033c

3011c

H5

NP_216310.1

Conserved hypothetical protein

Unknown (10)

H7

NP_214954.1

GroEL2 [Cpn62-2] (65 kDa antigen)

Prevents misfolding and promotes the refolding and proper assembly of unfolded polypeptides generated under stress conditions (0)

H10

NP_217934.1

GroEL1 [Cpn60-1] (60 kDa chaperonin 1)

Prevents misfolding and promotes the refolding and proper assembly of unfolded polypeptides generated under stress conditions (0)

H9 (1)yy

NP_335888.1

MetK (probable S-adenosylmethionine synthase)

Catalyses the formation of S-adenosylmethionine from methionine and ATP (7)

H9 (2)yy

NP_217527.1

GatA (probable glutamyltRNA(Gln) amidotransferase [sub-unit A])

Component of the translational apparatus (2)

Matched peptides position start–end/ sequence

Global scorexx

Sequence coverage (%)

58–74/EQGIVVNDAVNEQVAAR 94–109/LLYGVIDDENQPPGSR 110–117/DIPDNEFR 123–132/RGQHWVSAVR 124–132/GQHWVSAVR 185–200/SWQESGFNVSGGDLR 252–259/IALYQQAR 284–295/TVLDTLPYGEWK

129

29

3–11/TIAYDEEAR 3–12/TIAYDEEARR 17–26/GLNALADAVK 42–56/WGAPTITNDGVS 57–66 EIELEDYEK 78–99/KTDDVAGDGTTTA 79–99/TDDVAGDGTTTA 104–115/NVAAGANPLGLK 195–207/GYISGYFVTDPE 208–223/QEAVLEDPYILL 229–236/DLLPLLEK 325–342/DETTIVEGAGDT 449–469/QIAFNSGLEPGV

168

27

4–12 (LIEYDETAR) 4–13 (LIEYDETARR) 42–57 (AFGGPTVTNDGV) 141–152 (TGIAQVATVSSR) 276–282 (GPYFGDR) 309–319 (EVGLEVLGSAR) 326–343 (DDTVIVDGGGTA) 349–360 (AEIDKSDSDWDR) 403–422/AAVEEGIVPGGG 502–512/SAVLNASSVAR 528–539/AEDHDHHHDHAH

163

23

7–20/LFTSESVTEGHPDK 65–75/EAFADITNTVR 65–77/EAFADITNTVRAR 214–220/TLDPDIR 251–265/FVLGGPMGDAGLTGR 266–277/KIIVDTYGGWAR 267–277/IIVDTYGGWAR 305–314/NVVAAGLAER 326–345/AAPVGLFVETFGTETEDPVK 384–395/TDVELPWEQLDK

101

25

52

35

103–112/SPYDATLTAR 145–151/NPWNLDR 239–255/DSTSVDAEVPDVVGAAR 257–365/AAGFGPEVK 397–403/DLDAAYRS 404–419/VDVLVSPTTPTTAFR 479–487/VGAAYEAAR

129

(continued on next page)

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Name

130

Table 4 (continued) (A-2) Proteins with increased intensity in BCG Phipps, identified by LC–/ESI-MS/MS** Spot

Accession number

Protein Name

Function (FCx)

Gene namey

Matched peptides

BCG

Position/sequence

MTb H37Rv orthologue

Score

Global scorexxx

Sequence coverage (%)

NP_216944.1

AhpC (alkylhydroperoxidereductase C)

Involved in oxidative stress response (0)

2447

2428

30–46/QPGDYFTTITSDEHPGK 49–55/VVFFWPK 49–55/VVFFWPK 56–70/DFTFVCPTEIAAFSK 71–78/LNDEFEDR 105–115/TLPFPMLSDIK 117–133/ELSQAAGVLNADGVADR 117–133/ELSQAAGVLNADGVADR 159–163/DEVLR 164–179/VLDALQSDELCACNWR 164–179/VLDALQSDELCACNWR 180–192/KGDPTLDAGELLK

52 29 40 61 50 55 97 87 26 68 62 58

442

55

H6

YP_177849.1

Antigen Mpt32 (45-kDa glycoprotein)

Unknown (3)

1896

1860

146–164/TTGDPPFPGUPPPVANDTR 229–268/FSDPSKPNGQIWTGVIGSPAANAPDAGPPQR

39 13

52

17

H4

NP_214639.1

Pbe serine proteinase A (PepA, Mtb32A)

Unknown (7)

0159

0125

124–134/TQDVAVLQLR 124–134/TQDVAVLQLR 256–284/SGGGSPTVHIGPTAFLGLGVVDNNGNGAR

44 44 31

75

10

H21

NP_216343.1

Hypothetical CFP17

Unknown (10)

1862

1827

67–78/FLLDQAITSAGR 79–93/HPDSDIFLDDVTVSR 141–148/LVFLTGPK

84 69 46

198

22

H24

NP_215146.1

Enoyl-CoA-hydratase

Could possibly oxidizes fatty acids using specific components (1)

0679c

0632c

57–65/VFSGGFDLK 66–80/ILTSGEDQPAIDMLR 81–88/GGFELAYR 81–88/GGFELAYR 89–94/LLSYPK 89–94/LLSYPK 152–162/SAYQQATGLAK 220–231/AGIDGIAAEFGL

62 93 37 68 28 38 83 29

242

26

H25

NP_217917.1

Probable hydrolase

Unknown (7)

3470

3400

64–76/FVPFDPAADYHTY 64–80/FVPFDPAADYHTYVDGK 162–171/DVLATTGLDR 191–198/PAPDSFLR 225–236/AGNFAVVVGINR 249–262/HGADVVVTDLAELL 249–262/HGADVVVTDLAELL

65 17 42 54 79 106 3

245

23

H27

NP_215153.1

Transcription antitermination protein NusG

Influences transcription termination and antitermination (2)

0688

0639

104–112/VLPGYILVR 104–112/VLPGYILVR 125–132/TPGVTGF 125–138/NTPGVTGFVGATSR 139–149/PSALALDDVVK 139–149/PSALALDDVVK 150–154 (FLLPR 215–237/LKVLVSIFGREPPVELTFGQVSK 217–222/VLVSIF 217–224/VLVSIFGR 217–224/VLVSIFGR 219–224/VSIFGR 225–237/ETPVELTFGQVSK 225–237/ETPVELTFGQVSK

6 67 42 65 67 89 27 55 32 67 60 45 42 66

227

26

M. Rodrı´guez-Alvarez et al. / Tuberculosis 89 (2009) 126–135

H2

NP_215840.1

Possible thioredoxin

Participates in redox reactions through its reversible oxidation (7)

1386

1324

2–22/TRPRPPLGPAMAGAVDLSGIK 61–70/SDEVPVVVLL 96–108/WSLASVNVDVAPR 145–156/WVDSLLSATAGK 191–206/SYQAILDANPGSVEAK 211–217/QIEFLIR 211–217/QIEFLIR

28 64 109 82 85 38 42

318

25

H30

NP_215952.1

Glyceralde-hyde-3phosphate deshydrogenase

Involved in second phase of glycolysis (7)

1497

1436

5–12/VGINGFGR 52–58/FDSILGR 59–75/LPCDVGLEGDDTIVVGR 169–178/VLDDEFGIVK 206–220/AAALNIVPTSTGAAK 221–230/AIGLVMPQLK 240–257/VPIPTGSVTDLTVDLSTR 258–268/ASVDEINAAFK 328–337/LVDLVTLVGK

69 46 87 71 73 49 95 106 62

575

31

(B-1) Proteins with increased intensity in BCG Denmark, identified by MALDI-TOF–MS* Spot

Accession number

Gene namey

Protein Name

Function (FCx)

BCG

MTb H37Rv orthologue

Matched peptides position start–end/sequence

Global scorexx

Sequence coverage (%)

H8

NP_214954.1

GroEL2 [Cpn62-2] (65 kDa antigen)

Prevents misfolding and promotes the refolding and proper assembly of unfolded polypeptides generated under stress conditions (0)

0479

0440

57–66/EIELEDPYEK 104–115/NVAAGANPLGLK 195–207/GYISGYFVTDPER 325–342/DETTIVEGAGDTDAIAGR 449–464/QIAFNSGLEPGVVAEK

77

12

H13

NP_214534.1

Hypothetical TB39.8

Unknown (10)

0050c

0020c

164–178/GGQGQGRPDEYYDDR 179–187/YARPQEDPR 188–199/GGPDPQGGSDPR 362–372/QDYGGGADYTR 511–519/LGHSEIIVR

53

10

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H28

(continued on next page)

131

132

Table 4 (continued) (B-2) Proteins with increased intensity in BCG Denmark, identified by LC–/ESI-MS/MS** Spot

Gene namey

Protein Name

Function (FC )

H29

NP_217561.1

Probable alcohol dehydrogenase NADP-dependent

Generates aldehyde or ketone from alcohol (7)

H26

NP_337321.1

Conserved 35 kDa alanine-rich protein

Unknown (10)

x

BCG

Matched peptides MTb H37Rv orthologue

3069

3045

2760c

2744c

Position/sequence 24–36/RDPGPHDVAIDIK 24–36/RDPGPHDVAIDIK 24–36/RDPGPHDVAIDIK 25–36/DPGPHDVAIDIK 25–36/DPGPHDVAIDIK 26–36/PGPHDVAIDIK 26–36/PGPHDVAIDIK 37–49/FAGICHSDIHTVK 37–49/FAGICHSDIHTVK 85–95/VGVGCFVDSCR 85–95/VGVGCFVDSCR 210–220/KMEDGLRLGAK 221–232/SYYATADPDTFR 224–232/ATADPDTFR 330–336/VLASDVR 330–336/VLASDVR 20–27/IDEHADPK 65–71/QLAVIEK 72–94/LQVNVRQALTLADQATAAGDAAK 78–94/QALTLADQATAAGDAAK 154–162/LLSQLEQAK 197–214/YANAIGSAELAESSVQGR

Global scorexxx

Sequence coverage (%)

55 73 47 75 54 63 63 43 68 49 43 37 57 57 51 39

358

19

1 32 82 95 48 112

161

24

Score

* For protein identification by MALDI-TOF–MS, we also considered 5 matching peptides, and 10% sequence coverage. y Annotation from BCGList World-Wide Web Server (http://genolist.pasteur.fr/BCGList/). x FC ¼ functional category. 0: virulence, detoxification, adaptation; 1: lipid metabolism; 2: information pathways; 3: cell wall and cell processes; 4: stable RNAs; 5: insertion sequences and phages; 6: Pe/PPE; 7: intermediary metabolism and respiration; 8: unknown; 9: regulatory protein; 10: conserved hypothetical (from BCGList World-Wide Web Server http://genolist.pasteur.fr/BCGList/). ** For LC/NSI-MS/MS we considered 2 matching peptides, a minimum individual peptide score of 25, 10% sequence coverage, and 3 consecutive matches in the y and b ion series. yy Spot H9 was found to be a mixture of two proteins, each described in a row of the table. xx According to Mascot Search Results, protein scores >22 indicate identity or extensive homology (p < 0.05). In addition to Mascot score. xxx According to Mascot Search Results, protein scores >25 are significant (p < 0.05).

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Figure 1. Representative 2D-PAGE of 80 mg of cell proteins of BCG Denmark (A) and Phipps (B) substrains at mid-log phase culture in the pH range 4–7. Each sample was isoelectrofocused in IPG-strips and run on sodium dodecyl sulfate (SDS) 12.5% polyacrylamide gel. Gels were silver-stained and analyzed with PDQuest 2D Analysis V8.0 (Bio-Rad). Spots in circles showed intensity changes over the three experiments in one BCG substrain and were selected for identification by MS-based techniques. Spots in squares were taken as methodological control in both BCG Denmark and Phipps.

has been recently published.10 BCG Phipps apparently derived from BCG Pasteur in 1938 and was employed in two U.S. clinical trials, showing protection levels between 40 and 83%.2 Since then, BCG Phipps has only been preserved in culture collections for basic research.5 Regarding genetic vs. protein differences, data from the 1990s (mainly microarray technology) deciphered genetic differences among BCG substrains (i.e., deletions, insertions, and duplications). Recently, specific duplicated units (DU) and regions deleted (RD) among BCG substrains have been elucidated.10 BCG Denmark and Phipps share three major changes in their genomes: RD1; RD2, and the loss of a insertion sequence (IS) 6110 upstream to phoP, and two mutations in sigK and crp genes10; altogether, these genetic changes affected 22 (open reading frames) ORFs. The two best-known genetic differences between these substrains are nRD18 and RD-Glaxo-Denmark (RDgd) (involving 35 ORFs). Nonetheless, these minimal differences do not explain dissimilarities in protective efficacy among BCG substrains in clinical trials and in murine model.13 None of the proteins identified in the present study is encoded by genes located in nRD18 or RDgd. Four proteins identified herein participate in virulence, detoxification, or adaptation (FC 0).19 Three (H10, H7, and H2: GroEL1; GroEL2, and AhpC, respectively) are highly intense in BCG Phipps and one (H8: GroEL2) in Denmark. GroEL1 (BCG3487c, Rv3417c) is a chaperone involved in protein-folding and heat-shock response, and induces several cytokines, including interleukins (IL) IL-1b, IL6, IL-8, and IL-12, as well as tumor necrosis factor-a (TNF-a) and granulocyte–macrophage colony-stimulating factor (GM-CSF) in peripheral blood mononuclear cells (PBMC) of healthy donors.22 GroEL2 (BCG0479, Rv0440), also denominated 65 kDa antigen, is also a heat-shock protein that participates in folding of newly synthesized, imported, unfolded, or poorly folded proteins; it represents a key mycobacterial-survival element inside macrophages and is well recognized by the immune system, in vitro and in vivo.23 There is only one copy of BCG0479 gene in BCG genome; thus, differences in electrophoretic mobility of this protein in our 2D-PAGE (H7 and H8) could be attributable to post-translational modification or association with other proteic elements, and not to protein isoforms.6 Stimulation of IgG2a and gamma-interferon (IFN-g) production was found in BALB/c mice vaccinated with DNA encoding GroEL2 and challenged with M. tuberculosis H37Rv; in addition, specific immune response to recombinant GroEL of M. tuberculosis H37Rv induced TNF-a and IL-10 production in PBMC of BCG-vaccinated and purified protein derivative (PPD)-negative healthy subjects.

AhpC was widely described as involved in oxidative stress response, mainly against RNI and isoniazid resistance. AhpC gene is present in several mycobacteria, including M. tuberculosis H37Rv, M. bovis, and M. bovis BCG (reviewed in Ref. 24). AhpC was overexpressed in BCG Chicago compared with M. tuberculosis H37Rv under standard culture conditions6; when two virulent M. tuberculosis strains (H37Rv and CDC1551) proteomes were compared, this protein was downregulated in CDC1551 at 5, 8, and 12 days of culture.25 AhpC induced strong IFN-g expression in Mycobacterium avium sub specie Pseudotuberculosis-infected goats and has been described as a M. bovis virulence factor in guinea pig.24 We reported that BCG Phipps conferred best protection level in pulmonary-tuberculosis mouse model.13 A rise of AhpC and GroELs in this substrain (Phipps) could be a survival mechanism for this mycobacteria, prolonging its intracellular stay in macrophages and allowing these to perform more efficacious antigen processing and presentation, concomitantly with increased protection as result of prolonged immune-system activation. Four proteins herein were identified as conserved hypotheticals; their functions have not been completely elucidated. Nevertheless, previous reports described their participation in cellular processes. For instance, H13, conserved hypothetical TB39.9 (BCG0050c, Rv0020c) protein increased in BCG Denmark in this study; in M. tuberculosis H37Rv, it is encoded by Rv0020c, within pknA/pknB/ ppp gene cluster, which includes the essential protein PknB.26 Although reported as present in BCG-substrain culture supernatant,4 here we found this protein in the cell-fraction. Also highly intense in BCG Denmark, H26 was identified as conserved 35 kDa alanine-rich protein (BCG2760c, Rv2744c); in M. tuberculosis H37Rv it is encoded by Rv2744c, a gene expressed as an operon together with Rv2745c, a possible transcriptional regulator. This is the first report regarding this protein in BCG. H5, conserved hypothetical protein BCG1826 (Rv1794), was unique for BCG Phipps. It has been described in the cell-fraction of both virulent M. tuberculosis strains H37Rv and Erdman, as well as in two BCG substrains, Chicago and Copenhagen (at http://web. mpiib-berlin.mpg.de/cgi-bin/pdbs/2d-page/extern/menu_frame.cgi); however, no differences at protein level have been reported to date. Rv1794 is up-regulated under natural infection in animal model, and transcripts of BCG1826 in BCG Denmark were equivalent to that in two virulent strains of M. bovis, and twice as that in BCG Japan, one of the earliest substrains.10 H21, more intense in BCG Phipps, was identified as hypothetical CFP17 (BCG1862, Rv1827). This protein possesses FHA domains that bind phosphothreonine, participates in glycogen regulation and

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storage, and is the physiological substrate of PknB.26 It is present in the culture filtrate, cell-fraction, and cell wall of M. tuberculosis H37Rv21; together with CFP21, is recognized as a highly IFN-g inducer in C57BL/10 mice, and recombinant CFP17 induces high IFN-g level in mice and human TB patients.27 Due to its unclear function and that herein we report differences of these at proteome level, further studies must be conducted in hypothetical proteins to understand their role in mycobacterial biology and pathogenesis. Theoretically, 742 BCG proteins belong to mycobacterial FC 3 (cell wall or cell processes) (w19% of the genome) (at http:// genolist.pasteur.fr/BCGList). Here, one of these proteins was identified: H6, unique in BCG Phipps, Antigen Mpt32 (glycoprotein 45 kDa) (BCG1896, Rv1860). Although its function remains unclear, it is probably implicated on mycobacterial persistence and antigenicity; in guinea pig, Mpt32 was recognized by antibodies and interacted with T-lymphocyte receptors; as a recombinant vaccine in animal model, it demonstrated IFN-g- and IL-2-mediated protection.28 Nine hundred (w23% of the genome) proteins are involved in intermediary metabolism and respiration (FC 7). We described six of these with different intensities between BCG substrains: five from Phipps (H4, H9, H25, H30, and H28), and one from Denmark (H29). H9, a probable S-adenosylmethionine synthase (MetK, BCG1453, Rv1392), participates in amino acid synthesis. H25 is a probable hydrolase (BCG3470, Rv3400); its function remains unknown, and it is probably involved in cellular metabolism. H30, glyceraldehyde-3-phosphate dehydrogenase (BCG1497, Rv1436), participates in glycolysis; BCG Pasteur proteome comparison from standing vs. shaking culture condition showed presence of this protein only in standing culture condition, suggesting that its upregulation during low oxygen tension is required for survival.29 H28, a possible thioredoxin (BCG1386, Rv1324), participates in several redox reactions; this protein was already described in M. tuberculosis H37Rv,21 and transcript analysis of its mRNA found it downregulated at 4, 24 and 96 h after starvation.30 The probable alcohol dehydrogenase NADP-dependent (BCG3069, Rv3045) (H29 in this study) was described as present only understanding culture in BCG proteomes.29 Even when grown under enriched standard culture conditions in our experiments, BCG Phipps has more expressed several proteins related to survival under oxidative stress, as well as some relevant metabolic proteins, in comparison with BCG Denmark. This could represent a Phipps-substrain advantage for adaptation to adverse milieu and metabolic substrate changes, nevertheless, further studies most be done in order to elucidate this. Data presented do not support the hypothesis that BCG substrains with similar genomic content will encode similar protein numbers. The important contribution was a difference in the number of unique proteins along with changes of intensity of proteins for BCG Phipps or Denmark. In fact the immune response could not only be directly related to higher or lower genomic content rather but also it is related to proteins synthesized. Therefore, we stress that it is not appropriate to assume that BCG with similar genomic content will regulate the same level of protein production.31 The data presented herein related with phenotypical differences in hypothetical, cell wall, metabolic, and virulence proteins between Phipps and Denmark BCG substrains, together with our previous findings on BCG protective efficacy, provide new elements for understanding BCG-substrain differences. In vivo proteomic assays are ongoing at our laboratory for comparison analysis. The biological relevance of protein expression among distinct BCG substrains renders them definitive molecules, particularly in pathogenesis and immune-response scenarios.

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