First partial proteome of the poultry pathogen Mycoplasma synoviae

Veterinary Microbiology 145 (2010) 134–141

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First partial proteome of the poultry pathogen Mycoplasma synoviae Angela C.O. Menegatti a,b,1, Carolina P. Tavares a,b,1, Javier Vernal a,b, Catia S. Klein c, Luciano Huergo d, Herna´n Terenzi a,b,* a

Centro de Biologia Molecular Estrutural, Departamento de Bioquı´mica, CCB, Universidade Federal de Santa Catarina, 88040-900 Floriano´polis, SC, Brazil INBEB (Instituto Nacional de Cieˆncia e Tecnologia de Biologia Estrutural e Bioimagem), Brazil c Embrapa Suı´nos e Aves, Laborato´rio de Gene´tica e Sanidade Animal - Departamento de Bacteriologia de Suı´nos, Conco´rdia, SC, Brazil d Departamento de Bioquı´mica e Biologia Molecular, Universidade Federal do Parana´, CP 19046, 81531-990 Curitiba-PR, Brazil b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 16 December 2009 Received in revised form 3 March 2010 Accepted 8 March 2010

Mycoplasma synoviae is responsible for respiratory tract disease and synovitis in chickens and turkeys. In an attempt to identify the most prominent proteins expressed by this microorganism, a proteome map of M. synoviae was developed by using two-dimensional gel electrophoresis in combination with mass spectrometry. Based on the genome sequence of M. synoviae, a total of 30 different coding DNA sequences, including one hypothetical and one conserved-hypothetical protein, were experimentally verified with the identification of the corresponding protein products by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). The identified proteins were assigned according to the Clusters of Orthologous Groups of proteins functional classification. M. synoviae has 694 predicted CDSs. Overall, in this work 416 proteins spots were resolved in Coomassie Blue stained 2DE gels and were analyzed by mass spectrometry (MS). Altogether, we have achieved by MS the identification of 78 protein spots, corresponding to 30 different proteins. This is the first proteome map to be described in M. synoviae, and it is expected to be useful as a reference for comparative analysis. ß 2010 Elsevier B.V. All rights reserved.

Keywords: Mycoplasma synoviae Proteomics Mass spectrometry

1. Introduction The poultry industry has grown significantly reaching high productivity levels over the past decades and it is an important sector of the Brazilian agribusiness. Improvements in housing and equipment have led to important increases in animal production (Silva et al., 2008). However, incidence of many infectious diseases can affect poultry industry worldwide. One of these diseases is caused by Mycoplasma synoviae, responsible for respiratory tract disease and synovitis in chickens and turkeys.

* Corresponding author at: Centro de Biologia Molecular Estrutural, Departamento de Bioquı´mica, CCB, UFSC, 88040-900 Floriano´polis, SC, Brazil. Tel.: +55 48 3721 6426; fax: +55 48 3721 9672. E-mail address: [email protected] (H. Terenzi). 1 These authors contributed equally to this work. 0378-1135/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2010.03.006

Different strategies have been used to control this pathogen, for instance better management practices, improvement of housing conditions and application of antibiotics. However, the control by vaccination remains limited due to unavailability of an effective vaccine (Gerchman et al., 2008). M. synoviae is an important avian and extracellular pathogen, being responsible for significant economic impact in intensive production, due to decreased egg production, decreased growth and hatchability rates, and inefficient food conversion (Marois et al., 2002). The pathogen may cause chronic respiratory disease, infectious sinusitis, inflammatory synovitis and airsacculitis in chickens and turkeys. The clinical signs of infection are related to the great tropism of the agent for synovial membranes or the respiratory tract (May et al., 2007). Although the autoimmune mechanisms are not established, some of the systemic forms of infection resemble an

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immune complex disease (Narat et al., 1998). Recently, the complete genome of the pathogenic M. synoviae strain 53 was obtained by The Brazilian National Genome Project Consortium. This genome consists of 779,476 DNA bp containing 694 predicted coding DNA sequences (CDSs) (Vasconcelos et al., 2005). In this work, we report the proteomic analysis of M. synoviae cells for the identification of the most abundantly expressed proteins. We applied a combination of twodimensional gel electrophoresis (2DE) with mass spectrometry. A preliminary 2DE map of M. synoviae is described and the relevant identified proteins are discussed. This is the first 2DE reference map of M. synoviae to be reported to date. 2. Materials and methods 2.1. Mycoplasma strains and growth conditions M. synoviae strain 53 (ATCC 25204) was grown in the Laborato´rio de Sanidade Animal EMBRAPA (Conco´rdia, Santa Catarina, Brasil). The pathogen was isolated from a broiler breeder. The cells were incubated in Frey broth (Frey et al., 1968) supplemented with: 12% swine serum, 0.1 g/l nicotinamide adenine dinucleotide (NAD), 0.1 g/l cysteine hydrochloride hydrate, 1,000,000 IU penicillin G and 0.25 g/l thalium acetate, to prevent opportunistic bacterial growth, at 37 8C/24 h and then an aliquot was grown in Frey broth and incubated at 37 8C/24 h until the culture reached mid-log phase as indicated by color change and turbidity. Mycoplasmas were pelleted by centrifugation and washed with ‘protein free’ Frey broth (glucose 3 g/ l, 0.1 g/l nicotinamide adenine dinucleotide (NAD), 0.1 g/l cysteine hydrochloride hydrate, 1,000,000 IU penicillin G and 0.25 g thalium acetate per liter) to reduce the level of contaminant proteins present in the growth medium. The cells were stored at 80 8C (Fiorentin et al., 2003). 2.2. Sample preparation Extracts of M. synoviae proteins were prepared as described (Pinto et al., 2007; Westermeier and Naven, 2002) with some modifications. M. synoviae cells from 2 l of culture were resuspended in 2 ml of 25 mM Tris–HCl, pH 7.2 in the presence of 40 mg/ml protease inhibitor (phenylmethylsulfonyl fluoride, Sigma Aldrich, St. Louis, MO). The cells were disrupted by sonication (25 Hz in a Fisher Scientific Model 100 Sonic Dismembrator) in an ice bath using six cycles of 25 s, with 1 min interval between pulses and centrifuged (1 h at 13,400  g, 4 8C). After centrifugation the supernatant and the pellet were separated. The supernatant was supplemented with 8 M urea, 4% (w/v) CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid), 2% IPG buffer pH 3–10 or pH 4–7, 40 mM dithiothreitol (DTT) and to the pellet, it was added 1 ml of 25 mM Tris–HCl, pH 7.2, 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 0.2% IPG buffer pH 3–10 or pH 4–7, 40 mM DTT by agitation for 1 h. The protein concentration was determined using the 2D Quant Kit (GE Healthcare, Uppsala, Sweden) and then precipitated by using 2D clean-up kit (GE Healthcare, Uppsala, Sweden). The samples containing the proteins were split into aliquots and frozen at 80 8C.

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2.3. Two-dimensional electrophoresis 2DE was performed using the method of Westermeier and Naven (2002) with minor modifications. Aliquots containing approximately 70 mg of protein were diluted in rehydration solution (7 M urea, 2 M thiourea, 4%, w/v; CHAPS, 18 mM DTT, 0.5% IPG buffer pH 3–10 or pH 4–7, bromophenol blue). Samples were applied to the IPG strips (7 cm, pH 3–10 or pH 4–7 linear; GE Healthcare, Uppsala, Sweden). IEF for the 2DE was carried out using the Ettan IPGphor 3 system (GE Healthcare, Uppsala, Sweden), at 20 8C in four steps with a total focusing of 5750 VH and a maximum current of 25 mA/strip. Focused proteins were reduced by DTT and alkylated by iodoacetamide prior to be submitted to the second dimension. The second dimension was run in homogeneous 12% acrylamide gels. The gels were fixed in 8% phosphoric acid and 50% ethanol and stained using Colloidal Coomassie Brillant Blue G-250 (BioRad, Hercules, USA). The stained gels were scanned with an ImageScanner III (GE Healthcare, Uppsala, Sweden). 2DGel image was analyzed using ImageMaster 2D Platinum 6.0 software (GE Healthcare, Uppsala, Sweden). The software automatically determined the spot molecular mass comparing the position of each spot with the standard protein marker used in the second dimension, and the isoelectric point was also automatically determined using pH gradient. Analysis of the gels was made in duplicate to confirm the reproducibility of results. 2.4. In-gel digestion, mass spectrometry and protein identification In-gel digestion of the protein spots was carried out as previously described (Shevchenko et al., 1996) with some modifications. Gel spots were destained by three washing steps with 300 ml, 25 mM ammonium bicarbonate in 50% acetonitrile and then dehydrated replacing the ammonium bicarbonate solution by 100 ml of 100% acetonitrile. The gel pieces were then dried under a vacuum system (Eppendorf, Hamburg, Germany). After drying, gel fragments were rehydrated on ice in 10 ml of sequencing grade modified trypsin (10 mg/ml in 25 mM ammonium bicarbonate) (Promega, Madison, WI) and incubated for 16 h at 37 8C. Following incubation, the tryptic peptides were extracted with three washing steps containing 30 ml of 50% acetonitrile in 5% trifluoroacetic acid (TFA) for 30 min. Finally, the concentrated peptide extracts were dried under a vacuum system and stored at 20 8C. MS analysis was performed on a MALDI-TOF Autoflex spectrometer (Bruker Daltonics, Bremen, Germany). Extracted peptides were resuspended in 10 ml 0.1% TFA and a sample of 1 ml of concentrated digest was mixed with 1 ml of a saturated matrix solution of a-cyano 4-hydroxycinnamic acid (10 mg/ml in 0.1% TFA in 1:1 acetonitrile/methanol). Half of this mixture was spotted on the MALDI target plate (Bruker Daltonics, Bremen, Germany) and allowed to crystallize at room temperature. The spectra were acquired in positive ion mode, using an accelerating voltage of 20 kV and laser frequency of 50 Hz. External calibration was performed using a peptide mix with 200 shots for each spectrum (4 steps of 50 shots, in different places in the

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Fig. 1. Representative 2D electrophoresis map of soluble fraction from M. synoviae. (A) Total protein extract (70 mg) was separated on a 7 IPG strip (3–10) followed by SDS-PAGE on 12% gels and stained with Colloidal Coomassie Blue G250. (B) Total protein extract (70 mg) was separated on a 7 IPG strip (4–7) followed by SDS-PAGE on 12% gels and stained with Colloidal Coomassie Blue G250. The pI gradient is indicated at the top of the gel and the estimated molecular weights are shown on the left of the gel. The spot numbers correspond to identified proteins (see Tables 1 and 3 for details).

internal calibration sample) whenever possible. The samples spectra were obtained by accumulation data from 200 shots. The spectra generated were analyzed by the FlexAnalysis 3.0 software (Bruker Daltonics, Bremen, Germany). The proteins were identified using peptide mass fingerprinting and the mass list derived from the tryptic peptides were searched against the NCBInr using the MASCOT search program (http://www.matrixscience.com/ cgi/protein-view). The search parameters were as follow: mycoplasma category, one missed cleavage, 100 ppm measurement tolerance, cysteines were assumed to be carbamidomethylated and methionines oxidated. The probability score for the match, molecular weight (Mw), isoelectric point (pI), number of peptide matches and the percentage of the total translated ORF sequence covered by

the peptides were all used as confidence factors in protein identification. 3. Results and discussion 3.1. Two-dimensional gel electrophoresis M. synoviae, the causative agent of avian synovitis, is an economically important pathogenic agent. However, no proteomic studies have been performed to date in this organism. The main objective of this work was to catalog the most prominent expressed proteins of M. synoviae. M. synoviae has 694 predicted coding DNA sequences (CDSs), 464 of them corresponding to known proteins, 167 to conserved hypothetical proteins and 63 to hypothetical

Fig. 2. Representative 2D electrophoresis map of CHAPS-soluble fraction from M. synoviae. (A, B) Total CHAPS-soluble protein extract (70 ml) was separated on a 7 IPG strip (3–10) followed by SDS-PAGE on 12% gels and stained with Colloidal Coomassie Blue G250. The pI gradient is indicated at the top of the gel and the estimated molecular weights are shown on the left of the gel. The spot numbers correspond to identified proteins (see Table 2 for details).

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proteins (Vasconcelos et al., 2005). Overall, in this work 416 different protein spots were resolved in Coomassie Blue stained 2DE gels with molecular weights ranging from 6 to 90 kDa. Protein spots were analyzed from different gels (including redundant spots), and were resolved in the pH range 3–10 and in the pH range 4–7 (Figs. 1 and 2, Additional file Fig. 1). 3.2. Mass spectrometry and data analysis Altogether, we have achieved by MS the identification of 78 protein spots. Fifty-two protein spots were resolved with pH 3–10 IPG (IPG 3–10) strips and 26 protein spots were resolved with pH 4–7 IPG (IPG 4–7) strips. The 78 protein spots identified corresponded to 30 different proteins and several spots resulted in the same protein identification, suggesting the presence of numerous isoforms. The molecular weight (Mw) and the isoelectric point (pI) of each protein spot were experimentally determined and compared with the gene deduced Mw/ pI coordinates obtained from MASCOT database. We observed that 53 out of 78 gel-estimated Mw/pI corresponded very well to the theoretical values. For the other 25 spots (indicated in the Tables 1–3), experimentally determined Mw and/or pI values were significantly distinct from the predicted ones, suggesting the occurrence of posttranslational modifications. A careful examination of elongation factor Tu, thiol peroxidase, pyruvate dehydrogenase E1 component, molecular chaperone DnaK, phosphoglycerate kinase and a conserved hypothetical protein MS53_0334 and an hypothetical protein MS53_0538 indicated pI and/or molecular weight modifications in comparison to theoretical values. In most cases, two or three spots were identified for a given protein, but in the cases of elongation factor Tu, molecular chaperone DnaK and thiol peroxidase, a larger number of corresponding spots were found. The high abundance of

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these proteins suggests a constitutive expression and this observation was similar to other mycoplasmas proteomes analyzed (Demina et al., 2009; Ferrer-Navarro et al., 2006; Li et al., 2009; Pinto et al., 2007). The 30 proteins identified were classified according to the Clusters of Orthologous Groups of proteins (COGs) functional classification. The identified proteins belong to the following categories: (C) energy production and conversion (7); (D) cell cycle control, cell division, chromosome partitioning (1); (E) amino acid transport and metabolism (1); (F) nucleotide transport and metabolism (1); (G) carbohydrate transport and metabolism (5); (H) coenzyme transport and metabolism (1); (I) lipid transport and metabolism (1); (J) translation, ribosomal structure and biogenesis (4); (L) replication, recombination and repair (2); (O) posttranslational modification, protein turnover, chaperones (4); (V) defense mechanisms (1); (R) general function prediction only (2). Most of the identified proteins belong to categories (C), (G), (J) and (O). Among the identified protein products, one was an hypothetical protein and another was a conserved hypothetical protein. Our results are in agreement with the proteomic study of other mycoplasmas. In Mycoplasma penetrans most of the proteins identified were classified belonging to categories (O) and (J) (Ferrer-Navarro et al., 2006). The Mycoplasma genitalium proteome showed that the majority of the proteins identified belong to class (J) (Wasinger et al., 2000). In contrast, the Mycoplasma hyopneumoniae proteome clearly depicted that the greater part of the identified proteins could not be assigned to any functional category (Pinto et al., 2007). We identified 7 proteins assigned in category (C): the pyruvate dehydrogenase E1 component alpha and beta subunits, the F0F1-ATP synthase subunits alpha and beta (F0F1-ATPases) (2 CDSs were detected for this protein), phosphotransacetylase, an acetate kinase and dihydrolipoamide dehydrogenase. As M. synoviae has a limited

Table 1 Identified proteins from soluble fraction from M. synoviae (pI 3–10). Spot

Protein namea

Gene locus tag

pI/Mr theoretical

pI/Mr observed

MASCOT score

Sequence coverage %

Match peptides

COGb

A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13 A14 A15 A16 A17

Dihydrolipoamide dehydrogenase F0F1 ATP synthase subunit beta Cell division protein Fructose-bisphosphate aldolase Fructose-bisphosphate aldolase Elongation factor EF-Ts Elongation factor Tu Elongation factor Tu Elongation factor Tu Elongation factor Tu Single stranded binding protein Molecular chaperone DnaK Thiol peroxidase Thiol peroxidase Thiol peroxidase Thiol peroxidase Thioredoxin reductase

pdhD MS53_0275 AtpD MS53_0405 FtsZ MS53_0340 Fba MS53_0354 Fba MS53_0354 Tsf MS53_0414 tufA MS53_0667 tufA MS53_0667 tufA MS53_0667 tufA MS53_0667 Ssb MS53_0533 dnaK MS53_0351 Tpx MS53_0368 Tpx MS53_0368 Tpx MS53_0368 Tpx MS53_0368 trxB MS53_0596

5.87/66333 5.76/50831 4.38/59956 6.27/31276 6.27/31276 5.72/31919 5.61/43317 5.61/43203 5.61/43319 5.61/43319 4.50/21284 5.18/65056 5.93/18310 5.93/18309 5.93/18310 5.93/18310 5.37/27538

6.6/33463 6.23/48444 3.38/72924 7.17/23255 6.9/23351 5.00/61621 5.95/42366 6.10/42336 5.7/40150 5.82/42658 4.30/17218 5.33/61199 6.10/37817 4.7/17218 6.06/17218 5.88/20959 6.50/27760

70 111 215 88 109 73 94 216 92 123 65 137 81 159 117 141 79

28 32 33 34 25 33 26 53 23 39 39 25 34 56 38 45 23

10 10 14 7 8 6 8 17 8 11 5 14 8 13 9 12 8

C C D G G J J J J J L O O O O O O

Proteins with experimentally determined Mw and/or pI values significantly distinct from the predicted ones are indicated in bold. a Protein identification according to MASCOT database. b COG database functional classes: (C) energy production and conversion; (D) cell cycle control, cell division, chromosome partitioning; (G) carbohydrate transport and metabolism; (J) translation, ribosomal structure and biogenesis; (L) replication, recombination and repair; (O) posttranslational modification, protein turnover, chaperones.

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Table 2 Identified proteins from CHAPS-soluble fraction from M. synoviae (pI 3–10). Spot

Protein namea

Gene locus tag

pI/Mr theoretical

pI/Mr observed

MASCOT score

Sequence coverage %

Match peptides

COGb

B1 B2 B3 B4

Acetate kinase F0F1 ATP synthase subunit beta F0F1 ATP synthase subunit beta Pyruvate dehydrogenase E1 component, alpha subunit Pyruvate dehydrogenase E1 component, alpha subunit Pyruvate dehydrogenase E1 component, beta subunit Pyruvate dehydrogenase E1 component, beta subunit Cell division protein Putative lipoprotein Ribonucleotide diphosphate reductase subunit beta Fructose-bisphosphate aldolase N-acetylmannosamine kinase Phosphoglycerate kinase Phosphoglycerate kinase Phosphopyruvate hydratase Acyl carrier protein phosphodiesterase Acyl carrier protein phosphodiesterase 30S ribossomal protein S9 50S ribosomal protein L3 Elongation factor Tu Elongation factor Tu Elongation factor Tu Elongation factor Tu Elongation factor Tu Elongation factor Tu DNA polymerase III beta subunit Molecular chaperone DnaK Molecular chaperone DnaK Molecular chaperone DnaK Putative trigger factor Thiol peroxidase Thiol peroxidase Hypothetical protein MS53_0538 Hypothetical protein MS53_0538 NADH oxidase

ackA MS53_0652 atpD-2 MS53_0160 atpD-2 MS53_0160 pdhA MS53_0272

7.10/44601 5.75/52574 5.76/50831 6.20/42105

7.57/38926 6.02/52668 6.11/52123 6.75/43671

62 138 119 142

19 32 34 41

6 11 14 14

C C C C

pdhA MS53_0272

6.20/42219

4.44/21999

61

17

6

C

pdhB MS53_0273

6.73/35831

7.30/31723

97

40

11

C

pdhB MS53_0273

6.73/35831

7.25/36525

122

38

12

C

ftsZ MS53_0340 MS53_0190 nrdF MS53_0399

4.38/59953 5.13/10067 5.09/39273

4.44/60081 5.10/68776 4.84/70221

71 60 80

12 14 14

6 10 5

D E F

fba MS53_0354 nagC MS53_0195 pgk MS53_0114 pgk MS53_0114 eno MS53_0009 acpD MS53_0088 acpD MS53_0088 rpsI MS53_0077 rplC MS53_0643 tufA MS53_0667 tufA MS53_0667 tufA MS53_0667 tufA MS53_0667 tufA MS53_0667 tufA MS53_0667 dnaN MS53_0002 dnaK MS53_0351 dnaK MS53_0351 dnaK MS53_0351 MS53_0603 tpx MS53_0368 tpx MS53_0368 MS53_0538 MS53_0538 nox MS53_0522

6.27/31218 9.34/32163 6.13/43491 6.13/43491 6.59/49358 7.74/22732 7.74/22731 11.1/14790 9.68/28810 5.61/43203 5.61/43317 5.61/43317 5.61/43317 5.61/43317 5.61/43317 7.63/43489 5.18/65056 5.18/65056 5.18/65056 5.37/54529 5.93/18310 5.93/18310 6.30/20617 6.30/20619 6.44/50670

7.0/31376 5.93/42055 7.16/60495 7.38/63507 7.17/56185 8.51/20000 8.28/20041 5.95/32925 5.78/20630 5.72/41792 5.70/40205 5.97/47205 5.53/36063 5.78/34135 5.94/47365 5.15/21909 5.23/60709 5.16/61343 5.41/56058 5.40/57834 6.19/20418 6.39/18564 5.18/66664 6.95/20173 6.96/50174

82 46 52 51 174 66 68 46 51 164 106 73 124 85 173 69 98 122 90 93 85 60 46 110 80

35 18 20 18 35 37 29 37 17 37 24 25 35 19 42 19 22 20 21 20 34 46 27 41 25

9 5 4 5 13 8 11 4 4 17 7 9 22 12 23 6 12 12 10 9 7 7 3 7 9

G G G G G I I J J J J J J J J L O O O O O O R R R

B5 B6 B7 B8 B9 B10 B11 B12 B13 B14 B15 B16 B17 B18 B19 B20 B21 B22 B23 B24 B25 B26 B27 B28 B29 B30 B31 B32 B33 B34 B35

Proteins with experimentally determined Mw and/or pI values significantly distinct from the predicted ones are indicated in bold. a Protein identification according to MASCOT database. b COG database functional classes: (C) energy production and conversion; (D) cell cycle control, cell division, chromosome partitioning; (E) amino acid transport and metabolism; (F) nucleotide transport and metabolism; (G) carbohydrate transport and metabolism; (I) lipid transport and metabolism; (J) translation, ribosomal structure and biogenesis; (L) replication, recombination and repair; (O) posttranslational modification, protein turnover, chaperones; (R) general function prediction only.

metabolic capacity, this pathogen lacks a functional citric acid cycle (Manolukas et al., 1988; Razin et al., 1998) and scavenges nutrients from host cells to survive (Dybvig and Voelker, 1996). Thus, mycoplasmas have evolved adenosine triphosphate (ATP) conservation mechanisms to counteract the lack of a citric acid cycle, including a pathway known as the ‘‘pyruvate roundhouse’’ (Matic et al., 2003). Contrary to what happens in other organisms, M. synoviae conversion of pyruvate directly to oxaloacetate using pyruvate carboxylase requires the utilization of ATP. Hence the alternative pathway facilitates oxaloacetate formation without ATP consumption. The pyruvate dehydrogenase (PDH) complex catalyses the first step of the roundhouse, the conversion of pyruvate to acetyl-CoA. This finding justifies a large number of the identified proteins involved in energy metabolism. To complete energy production, the F0F1-ATPase enzyme complex catalyzes

the terminal step in oxidative phosphorylation and is located in the bacterial membranes (Weber and Senior, 2000). Mycoplasmas differ from the majority of the bacteria by the deficiency of a cytochrome-containing electron transport chain; therefore their F0F1-ATPase function seems restricted to maintain a proton gradient (Pyrowolakis et al., 1998). Included in category (E) we identified a putative lipoprotein MS53_0190. Lipoproteins represent an abundant group of proteins in mycoplasma membranes and some exert important role in adhesion and transport (Calcutt et al., 1999). Mycoplasmas tolerate an extracellular existence exposed to the host defenses. For that reason, critical systems for transport and other metabolic pathways related with periplasmic roles are found on the membrane surface of these organisms, such as in other eubacteria.

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Table 3 Identified proteins from soluble fraction from M. synoviae (pH 4–7). Spot

Protein namea

Gene locus tag

pI/Mr theoretical

pI/Mr observed

MASCOT score

Sequence coverage %

Match peptides

COGb

C1c C2c C3c C4

F0F1 ATP synthase subunit alpha F0F1 ATP synthase subunit beta Phosphotransacetylase Pyruvate dehydrogenase E1 component, alpha subunit Pyruvate dehydrogenase E1 component, beta subunit Phosphoglyceromutase Phosphopyruvate hydratase Conserved hypothetical protein Conserved hypothetical protein Elongation factor EF-Ts Elongation factor Tu Elongation factor Tu Elongation factor Tu Elongation factor Tu Molecular chaperone DnaK Molecular chaperone DnaK Molecular chaperone DnaK Molecular chaperone DnaK Molecular chaperone DnaK Molecular chaperone DnaK Putative trigger factor Thiol peroxidase Thiol peroxidase Thiol peroxidase Thioredoxin reductase Putative ABC transporter, ATP-binding protein

atpD-2 MS53_0159 atpD MS53_0405 eutD MS53_0653 pdhA MS53_0272

7.66/57947 5.76/50831 6.06/34978 6.20/42221

5.98/50000 6.06/49101 6.36/31744 6.54/39784

142 133 192 73

29 34 52 23

10 14 14 10

C C C C

pdhB MS53_0273

6.73/35831

6.98/24854

157

56

13

C

Pgm MS53_0656 Eno MS53_0009 MS53_0334 MS53_0334 Tsf MS53_0414 tufA MS53_0667 tufA MS53_0667 tufA MS53_0667 tufA MS53_0667 dnaK MS53_0351 dnaK MS53_0351 dnaK MS53_0351 dnaK MS53_0351 dnaK MS53_0351 dnaK MS53_0351 MS53_0603 Tpx MS53_0368 Tpx MS53_0368 Tpx MS53_0368 trxB MS53_0596 MS53_0130

5.91/56365 6.59/49358 8.72/83696 8.72/83696 5.72/31919 5.61/43319 5.61/43319 5.61/43319 5.61/43319 5.18/65056 5.18/65056 5.18/65056 5.18/65056 5.18/65056 5.18/65056 5.37/54529 5.93/ 18310 5.93/18310 5.93/18309 5.92/33513 9.21/63413

6.26/53247 6.93/39069 6.92/64308 6.88/73443 4.95/80593 5.66/38928 5.97/43561 5.84/42934 6.10/42623 5.42/65671 5.25/66596 5.20/63060 5.18/69936 5.14/71919 5.35/69449 5.39/56310 6.16/22502 5.99/24683 6.25/20747 6.27/28619 6.63/38290

62 135 67 140 94 77 151 112 128 94 70 121 80 76 91 95 109 90 71 69 49

14 34 15 27 28 26 42 30 37 15 19 29 17 11 13 19 57 34 31 27 18

6 12 8 17 7 9 16 10 14 6 8 17 7 5 6 8 12 9 8 7 4

G G H H J J J J J O O O O O O O O O O O V

C5c C6c C7c C8c C9c C10c C11c C12c C13c C14 C15 C16c C17c C18c C19c C20c C21c C22 C23 C24 C25c C26c

Proteins with experimentally determined Mw and/or pI values significantly distinct from the predicted ones are indicated in bold. a Protein identification according to MASCOT database. b COG database functional classes: (C) energy production and conversion; (G) carbohydrate transport and metabolism; (H) coenzyme transport and metabolism; (J) translation, ribosomal structure and biogenesis; (O) posttranslational modification, protein turnover, chaperones; (V) defense mechanisms. c See Additional file Fig. 1.

We identified a class II ribonucleotide diphosphate reductase subunit beta (RNR) classified as belonging to (F) category. This enzyme performs an essential role in the cycling of nucleotides in the cell and during replication of the chromosome in all organisms (Dawes et al., 2003). The fact that this protein is expressed by M. synoviae is a very interesting finding due to the low number of genes and enzymes responsible for purine and pyrimidine synthesis in the mycoplasmas (Razin et al., 1998). The enzyme ribonucleotide reductase was already used as a recombinant product with b-galactosidase as an experimental vaccine for enzootic pneumonia caused by M. hyopneumoniae. It was reported that gross lung pathology of vaccinated animals, irrespective of adjuvant treatment, was significantly reduced compared with that of unvaccinated control (Fagan et al., 1996). Two protein spots identified corresponded to a phosphopyruvate hydratase, a enolase that occurs in the cytosol of prokaryotic and eukaryotic cells and it is reported that this protein is multifunctional. Besides the catalytic functions in various types of cells, enolases exposed on the surface of cells may be receptors for certain ligands in pathological mechanisms (Yavlovich et al., 2007). One of the most abundant protein spots identified in our analysis was the elongation factor Tu (14 spots). This factor participates in the formation of a ternary complex

with tRNA, which is necessary to deliver the aminoacyltRNAs, the primary substrate of the ribosome (Nilsson and Nissen, 2005). It was described by Bercic et al. (2008) that elongation factor Tu, an abundant cytoplasmic 43 kDa protein, which might cross-react with antibodies against the P1 adhesin of M. pneumoniae, does not seem to be immunogenic for chicken infected with M. synoviae. Both elongation factors (EF-Tu and EF-Ts) participate in the elongation steps of protein synthesis and they are present in most prokaryotes like E. coli, Pseudoalteromonas haloplanktis and mycoplasmas (Raimo et al., 2004). Further the primary function in protein biosynthesis the elongation factor Tu can be involved in the mediation of pathogen binding to fibronectin, as demonstrated in M. pneumoniae Dallo et al. (2002). This fact could explain the abundance of this protein in our analysis. We identified 4 proteins belonging to (O) category: the molecular chaperone dnaK, a putative trigger factor, a thiol peroxidase, a thioredoxin reductase. In the M. synoviae proteome was observed a large number of protein spots corresponding to DnaK. We identified a putative ABC transporter/ATP-binding protein belonging to category V (defense mechanismsMS53_0130). In the M. synoviae genome was observed that 11.2% of the CDSs identified encode membrane transport proteins and from these, 85% are ABC system proteins like the putative ABC transporter/ATP-binding

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protein identified in this work. The ABC transporter can be involved in pathogenesis through the efflux transport of proteins such as hemolysin, heme-binding protein and alkaline protease (Nicola´s et al., 2007; Zhao et al., 2004). 4. Conclusions The results presented in this work constitute an analysis that focused primarily on the most abundant proteins in the 2DE map of M. synoviae. We described a preliminary proteome map of M. synoviae, whose entire genome sequence has been reported (Vasconcelos et al., 2005). According to the results obtained by Bercic et al. (2008), the major proteins from the M. synovaie proteome are involved in metabolism and protein processing, such phosphopyruvate hydratase, chaperones (DnaK and trigger factor), elongation factors (EF-Tu/EF-G) as well as a lipoprotein. We identified some of these proteins, like elongation factor Tu, phosphopyruvate hydratase, chaperone DnaK, NADH oxidase and trigger factor. It seems that infection of chickens with M. synoviae induces the production of antibodies against M. synoviae enolase (phosphopyruvate hydratase) (Bercic et al., 2008). These antibodies against bacterial enolase are involved in a variety of autoimmune diseases, because they can crossreact with the host enolase, suggesting that M. synoviae enolase may be involved in the appearance of autoimmune processes (Wegner et al., 2009). As shown in Tables 1–3, we found multiple isoforms for elongation factor Tu, molecular chaperone DnaK and thiol peroxidase. Several Mw/pI values observed showed variations when compared with the theoretical values. These variations and isoforms found may suggest a relatively high level of posttranslational modifications. In some bacteria, including mycoplasmas was observed that the phosphorylation of heat shock proteins, EF-Tu and pyruvate dehydrogenase are regulated by environmental stress (Su and Hutchison, 2007). We report here the first proteome analysis of M. synoviae, which is expected to provide the basis for more extensive proteome studies addressing mycoplasmal biology and will be a useful reference for future comparative studies. The identification of novel antigenic proteins will further increase the repertoire of antigens available for the formulation of immunodiagnostic reagents and vaccines to this pathology. Future studies will focus on the identification of low abundance proteins by using narrower pH ranges, more sensitive techniques to improve solubilization of proteins and protein spots detection. Authors’ contributions ACOM, CPT, JV and HT participated in the design of the study. ACOM and CPT carried out the experiments, data analysis and drafted the manuscript and contributed equally to this work. CSK performed the M. synoviae cultivation. LH helped with the MS analysis. JV revised and improved the manuscript. HT coordinated the study and

revised the manuscript. All the authors read and approved the final manuscript. Acknowledgements This work was supported by grants from CNPq, CAPES, Ministe´rio de Cieˆncia e Tecnologia, FAPESC, FINEP.

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