# Label-free quantitative proteomics of Corynebacterium pseudotuberculosis isolates reveals differences between Biovars ovis and equi strains

Wanderson Silva, Edson L. Folador, Siomar C. Soares, Gustavo H. M. F.

Souza, Agenor V. Santos, Cassiana S. Sousa, Henrique Figueiredo, Anderson

Miyoshi, Yves Le Loir, Artur Silva, et al.

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## To cite this version:

Wanderson Silva, Edson L. Folador, Siomar C. Soares, Gustavo H. M. F. Souza, Agenor V. Santos, et al.. Label-free quantitative proteomics of Corynebacterium pseudotuberculosis isolates reveals differences between Biovars ovis and equi strains. BMC Genomics, 2017, 18 (1), open access. 10.1186/s12864-017-3835-y. http://hal-01593194

HAL Id: hal-01593194

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Submitted on 25 Sep 2017

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RESEARCH ARTICLE

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# Label-free quantitative proteomics of *Corynebacterium pseudotuberculosis* isolates reveals differences between *Biovars ovis* and *equi* strains

Wanderson M. Silva<fnref n="1" />,⁵,⁶, Edson L. Folador³, Siomar C. Soares<fnref n="1" />,⁸, Gustavo H. M. F. Souza⁴, Agenor V. Santos², Cassiana S. Sousa<fnref n="1" />, Henrique Figueiredo⁷, Anderson Miyoshi<fnref n="1" />, Yves Le Loir⁵,⁶, Artur Silva² and Vasco Azevedo<fnref n="1" />*

**Abstract**

**Background:** *Corynebacterium pseudotuberculosis* is a pathogen classified into two biovars: *C. pseudotuberculosis ovis*, the etiologic agent of caseous lymphadenitis and *C. pseudotuberculosis biovar equi*, which causes ulcerative lymphangitis. The available whole genome sequences of different *C. pseudotuberculosis* strains have enabled identify difference of genes related both virulence and physiology of each biovar. To evaluate be this difference could reflect at proteomic level and to better understand the shared factors and the exclusive ones of biovar *ovis* and biovar *equi* strains, we applied the label-free quantitative proteomic to characterize the proteome of the strains: 1002_ovis and 258_equi, isolated from goat (Brazil) and equine (Belgium), respectively.

**Results:** From this analysis, we characterized a total of 1230 proteins in 1002_ovis and 1220 in 258_equi with high confidence. Moreover, the core-proteome between 1002_ovis and 258_equi obtained here is composed of 1122 proteins involved in different cellular processes, which could be necessary for the free living of *C. pseudotuberculosis*. In addition, 120 proteins from this core-proteome presented change in abundant with statistically significant differences. Considering the exclusive proteome, we detected strain-specific proteins to each strain. When correlated, the exclusive proteome of each strain and proteome with change in abundant, the proteomic differences, between the 1002_ovis and 258_equi, this related to proteins involved in cellular metabolism, information storage and processing, cellular processes and signaling.

**Conclusions:** This study reports the first comparative proteomic study of the biovars *ovis* and *equi* of *C. pseudotuberculosis*. The results generated in this study provide information about factors which can contribute to understanding both the physiology and the virulence of this pathogen.

**Keywords:** *Corynebacterium pseudotuberculosis*, Caseous lymphadenitis, Ulcerative lymphangitis, Proteomic bacterial, Label-free proteomics, proteomic

<footnote>* Correspondence: vasco@icb.ufmg.br</footnote>

<footnote n="1">¹Departamento de Biologia Geral, Instituto de Ciências Biológicas,
Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brasil
Full list of author information is available at the end of the article</footnote>
## Background

*Corynebacterium pseudotuberculosis* is a Gram-positive facultative intracellular pathogen of the Corynebacterium, Mycobacterium, Nocardia, and Rhodococcus (CMNR) group. The CMNR group of pathogens has high G + C content in their genomes and shows a specific cell wall organization composed of peptidoglycan, arabinogalactan, and mycolic acids [1]. *C. pseudotuberculosis* is subdivided into two biovars: (i) *C. pseudotuberculosis* biovar *ovis* (nitrate negative) which is the etiologic agent of caseous lymphadenitis in small ruminants [2] and mastitis in dairy cattle [3] and (ii) *C. pseudotuberculosis* biovar *equi* (nitrate positive) that causes ulcerative lymphangitis and abscesses in internal organs of equines [4] and oedematous skin disease in buffalos [5]. *C. pseudotuberculosis* infection is reported worldwide and causes significant economic losses by affecting wool, meat, and milk production [6–9].

Various studies at genome level have been carried out by our research group in order to explore the molecular basis of specific and shared factors among different strains of *C. pseudotuberculosis* that could contribute to such biovar specific pathogenicity. Our studies on whole-genome sequencing and analysis of several *C. pseudotuberculosis* strains belonging to biovar *ovis* and *equi*, isolated from different hosts showed an average genome size of approximately 2,3 Mb, a core-genome having approximately 1504 genes across several *C. pseudotuberculosis* species, and accessory genomes of biovar *equi* and *ovis* composed of 95 and 314 genes, respectively [10–12]. According with pan-genome analysis, *C. pseudotuberculosis* biovar *ovis* presented a more clonal-like behavior, than the *C. pseudotuberculosis* biovar *equi*. In addition, in this *in silico* study was observed a variability most interesting related to pilus genes, where biovar *ovis* strain presented high similarity, while, biovar *equi* strains have a great variability, suggesting that this variability could influence in the adhesion and invasion cellular of each biovar [10].

Apart from the structural genome informatics studies of *C. pseudotuberculosis*, some proteomic studies were conducted to explore the functional genome of this pathogen [13–19]. However, all these proteomic studies were performed using only strains belonging to biovar *ovis*. Until the present time, no proteomic studies were performed between biovar *equi* strains or between biovar *ovis* and biovar *equi* strains. Therefore, to provide insights on shared and exclusive proteins among biovar *ovis* and biovar *equi* strains and to complement the previous studies on functional and structural genomics of *C. pseudotuberculosis* biovars, using LC-MS<sup>E</sup> approach [13, 18] this study reports for the first time a comparative proteomic analysis of two *C. pseudotuberculosis* strains, 1002_ovis and 258_equi, isolated from caprine

(Brazil) and equine (Belgium), respectively. Our proteomic dataset promoted the validations of previous work *in silico* of *C. pseudotuberculosis*; in addition, the qualitative and quantitative differences in the proteins identified in this present work have potential to help understand the factors that might contribute for pathogenic process of biovar *ovis* and *equi* strains.

## Methods

### Bacterial strain and growth condition

*C. pseudotuberculosis* biovar *ovis* 1002, isolated from a goat in Brazil, and *C. pseudotuberculosis* biovar *equi* 258, isolated from a horse in Belgium, were maintained in brain-heart infusion broth or agar (1.5%) (BHI-HiMedia Laboratories Pvt. Ltd., India) at 37 °C. For proteomic analysis, overnight cultures (three biological replicate to each strain) in BHI were inoculated with a 1:100 dilution in fresh BHI at 37 °C and cells were harvested during the exponential growth at DO<sub>600</sub> = 0.8 (Additional file 1: Figure S1).

### Protein extraction and preparation of whole bacterial lysates for LC-MS/MS

After bacterial growth, the protein extraction was performed according to Silva et al. [18]. The cultures were centrifuged at 4000 x g at 4 °C for 20 min. The cell pellets were washed in phosphate buffered saline (PBS) and then resuspended in 1 mL of lysis buffer (7 M Urea, 2 M Thiourea, CHAPS 4% and 1 M DTT) and 10 µL of Protease Inhibitor Mix (GE Healthcare, Piscataway, NJ, USA) was added. The cells were broken by sonication at 5 × 1 min cycles on ice and the lysates were centrifuged at 14,000 x g for 30 min at 4 °C. Subsequently, samples were concentrated and lysis buffer was replaced by 50 mM ammonium bicarbonate at pH 8.0 using a 10 kDa ultra-filtration device (Millipore, Ireland). All centrifugation steps were performed at room temperature. Finally the protein concentration was determined by Bradford method [20]. A total of 50 µg proteins from each biological replicate of 1002_ovis and 258_equi were denatured by using RapiGEST SF [(0.1%) (Waters, Milford, CA, USA)] at 60 °C for 15 min, reduced with DTT [(10 mM) (GE Healthcare)], and alkylated with iodoacetamide [(10 mM) (GE Healthcare)]. For enzymatic digestion, trypsin [(0.5 µg/µL) (Promega, Sequencing Grade Modified Trypsin, Madison, WI, USA)] was added and placed in a thermomixer at 37 °C overnight. The digestion process was stopped by the addition of 10 µL of 5% TFA (Sigma-Aldrich, St. Louis, Missouri, USA) and glycogen phosphorilase (Sigma-Aldrich) was added to the digests to give 20 fmol.uL<sup>-1</sup> as an internal standard for scouting normalization prior to each replicate injection into label-free quantitation [21].
## LC-HDMS<sup>E</sup> analysis and data processing

Qualitative and quantitative analysis were performed using 2D RPxRP (two-dimensional reversed phase) nanoUPLC-MS (Nano Ultra Performance Liquid Chromatography Mass Spectrometry) approach with multiplexed Nano Electrospray High Definition Mass Spectrometry (nanoESI-HDMS<sup>E</sup>). To ensure that all samples were injected with the same amount into the columns and to ensure standardized molar values across all conditions, stoichiometric measurements based on scouting runs of the integrated total ion account (TIC) were performed prior to analysis. The experiments were conducted using both a 1 h reversed phase gradient from 7% to 40% (v/v) acetonitrile (0.1% v/v formic acid) and a 500 nL.min<sup>-1</sup> on a 2D nanoACQUITY UPLC technology system [22]. A nanoACQUITY UPLC HSS (High Strength Silica) T3 1.8 µm, 75 µm × 15 cm column (pH 3) was used in conjunction with a reverse phase (RP) XBridge BEH130 C18 5 µm 300 µm × 50 mm nanoflow column (pH 10). Typical on-column sample loads were 250 ng of total protein digests for each 5 fractions (250 ng/fraction/load). For all measurements, the mass spectrometer was operated in the resolution mode with a typical m/z resolving power of at least 35,000 FMHW and an ion mobility cell filled with nitrogen gas and a cross-section resolving power at least 40 Ω/ΔΩ. All analyses were performed using nano-electrospray ionization in the positive ion mode nanoESI (+) and a NanoLockSpray (Waters, Manchester, UK) ionization source.

The lock mass channel was sampled every 30 s. The mass spectrometer was calibrated with a MS/MS spectrum of [Glu1]-Fibrinopeptide B human (Glu-Fib) solution (100 fmol.uL<sup>-1</sup>) delivered through the reference sprayer of the NanoLockSpray source. The doubly-charged ion ([M + 2H]<sup>2+</sup> = 785.8426) was used for initial single-point calibration and MS/MS fragment ions of Glu-Fib were used to obtain the final instrument calibration. Multiplexed data-independent (DIA) scanning with added specificity and selectivity of a non-linear 'T-wave' ion mobility (HDMS<sup>E</sup>) experiments were performed with a Synapt G2-S HDMS mass spectrometer (Waters), which was automatically planned to switch between standard MS (3 eV) and elevated collision energies HDMS<sup>E</sup> (19–45 eV) applied to the transfer 'T-wave' CID (collision-induced dissociation) cell with argon gas. The trap collision cell was adjusted for 1 eV, using a milliseconds scan time previously adjusted based on the linear velocity of the chromatography peak delivered through nanoACQUITY UPLC to get a minimum of 20 scan points for each single peak, both in low energy and at high-energy transmission at an orthogonal acceleration time-of-flight (oa-TOF) from m/z 50 to 2000. The RF offset (MS profile) was adjusted to such a way that

the nanoUPLC-HDMS<sup>E</sup> data are effectively acquired from m/z 400 to 2000, which ensured that any masses observed in the high energy spectra with less than m/z 400 arise from dissociations in the collision cell.

## Database searching and quantification

Following the identification of proteins, the quantitative data were packaged using dedicated algorithms [23, 24] and searching against a database with default parameters to account for ions [25]. The databases used were reversed "on-the-fly" during the database queries and appended to the original database to assess the false positive rate (FDR) during identification. For proper spectra processing and database searching conditions, the Protein Lynx Global Server v.2.5.2 (PLGS) with Identity<sup>E</sup> and Expression<sup>E</sup> informatics v.2.5.2 (Waters) were used. UniProtKB (release 2013_01) with manually reviewed annotations was used, and the search conditions were based on taxonomy (Corynebacterium pseudotuberculosis). We have utilized a database from genome annotation of 1002_ovis CP001809.2 version and 258_equi CP003540.2 version. These databases were randomized within PLGS v.2.5.2 for generate a concatenated database from both genomes. Thus, the measured MS/MS spectra from proteomic datasets of 1002_ovis and 258_equi were searched against this concatenated database. The maximum allowed missed cleavages by trypsin were up to one, and variable modifications by carbamidomethyl (C), acetyl N-terminal, phosphoryl (STY) and oxidation (M) were allowed and peptide mass tolerance value of 10 ppm was used [26]. Peptides as source fragments, peptides with a charge state of at least [M + 2H]<sup>2+</sup> and the absence of decoys were the factors we considered to increase the data quality. The collected proteins were organized by the PLGS Expression<sup>E</sup> tool algorithm into a statistically significant list that corresponded to higher or lower regulation ratios among the different groups. For protein quantitation, the PLGS v2.5.2 software was used with the Identity<sup>E</sup> algorithm using the Hi3 methodology. The search threshold to accept each spectrum was the default value in the program with a false discovery rate value of 4%. The quantitative values were averaged over all samples, and the standard deviations at *p* < 0.05 were determined using the Expression software. Only proteins with a differential expression log2 ratio between the two conditions greater than or equal to 1.2 were considered [26].

## Bioinformatics analysis

The identified proteins in 1002_ovis and 258_equi were subjected to the bioinformatics analysis using the various prediction tools. SurfG+ v1.0 [27] was used to predict sub-cellular localization, SignalP 4.1.0 server [28] to
predict the presence of N-terminal signal peptides for secretory proteins, SecretomeP 2.0 server [29] to identify exported proteins from non-classical systems (positive prediction score greater than to 0.5), LipoP server [30] to determine lipoproteins, Blast2GO [31] and COG database [32] were used for functional annotations. The protein-protein interaction network was generated using Cytoscape version 2.8.3 [33] with a spring-embedded layout.

## Results and discussion

### Characterization of the proteome of *C. pseudotuberculosis* biovar *ovis* and *equi*

In this study, we applied the 2D nanoUPLC-HDMS<sup>E</sup> approach to characterize the proteome of the strains 1002_ovis and 258_equi. Both strains were grown in BHI media, subsequently proteins were extracted and digested in solution, and then the peptides were analyzed by LC/MS<sup>E</sup>. Our proteomic analysis identified a total of 1227 non-redundant proteins in 1002_ovis (Additional file 2: Table S1 and Additional file 3: Table S2) and 1218 in 258_equi (Additional file 2: Table S1 and Additional file 4: Table S3) (Fig. 1a). The information about sequence coverage and a number of identified peptides for each protein sequence identified, as well as

the information about the native peptide are available at Additional file 5: Table S4 and Additional file 6: Table S5. Altogether from the proteome of these two biovars, we identified a total of 1323 different proteins of *C. pseudotuberculosis* with high confidence (Fig. 1a) and characterized approximately 58% of the predicted proteome of 1002_ovis [11] (Fig. 1b). In the case of 258_equi, we characterized approximately 57% of the predicted proteome [12] (Fig. 1b). The proteins identified in both proteomes were analyzed by SurfG+ tool [27] to predict the subcellular localization into four categories: cytoplasmic (CYT), membrane (MEM), potentially surface-exposed (PSE) and secreted (SEC) (Fig. 1c). Further, we identified 83% (43 proteins) of the lipoproteins predicted in 1002_ovis and 79% (41 proteins) in 258_equi. Considering proteins with LPxTG motif which are involved in covalent linkage with peptidoglycan, we identified 6 proteins in 1002_ovis and 4 proteins in 258_equi that correspond to approximately 38% and 34% of the LPxTG proteins predicted in each strain, respectively.

### The biovar *equi* and biovar *ovis* core proteome

The core-proteome, between 258_equi and 1002_ovis is composed of 1122 proteins (Fig. 1) (Additional file 2: Table S1). Interestingly, when correlated these 1122

<figure><img src="image_4.png" /><figcaption>**Fig. 1** Characterization of the proteome of *C. pseudotuberculosis* and correlation with *in silico* data. **a** Distribution of the proteins identified in the proteome of 1002_ovis and 258_equi, represented by Venn diagram. **b** Correlation of the proteomic results with *in silico* data of the genomes of 1002_ovis and 258_equi. **c** Subcellular localization of the identified proteins and correlation with the *in silico* predicted proteome. CYT, cytoplasmic; MEM, membrane; PSE, potentially surface-exposed and SEC, secreted</figcaption></figure>
proteins with in silico data of the *C. pseudotuberculosis* core-genome [10], we observed that 86% (960 proteins) of the *Open Reading Frame* (ORF) that encodes these proteins are part of the core-genome (Additional file 2: Table S1), what represents approximately 64% of the predicted core-genome of this pathogen. In addition, these data show a set of proteins involved in different cellular processes which could be necessary for the free living of *C. pseudotuberculosis*. The other 14% (262 proteins) of the proteins that constitute the core-proteome are shared by at least one of the 15 strains used in the core-genome study. According to Gene Ontology analysis [31, 32], the 1122 proteins were classified into four important functional groups: (i) metabolism, (ii) information storage and processing, (iii) cellular processes and signaling, and (iv) poorly characterized (Fig. 2a). As observed in the study of *C. pseudotuberculosis* [10] core genome in the categories “metabolism” and “information storage and processing” were detected a large number of proteins.

The label-free quantification was applied to evaluate the relative abundance of the core-proteome of 258_equi and 1002_ovis. The ProteinLynx Global Server (PLGS) v2.5.2 software with Expression<sup>E</sup> algorithm tool was used to identify proteins with *p* ≤ 0.05 (Additional file 2: Table S1). Among these proteins, 120 proteins between 258_equi and 1002_ovis showed difference in level of abundance (log<sub>2</sub> ratios equal or greater than a factor of 1.2) [26] (Table 1). In this group of proteins that have presented different abundance level (258_equi:1002_ovis), 49 proteins were more abundant and 71 less abundant (Table 1). To visualize this differential distribution of the core-proteome a volcano plot of the log<sub>2</sub> ratio of 258_equi/1002_ovis versus Log (e) Variance was generated (Fig. 2b). Interestingly, the Phospholipase D (Pld), the major virulence factor of *C. pseudotuberculosis*, was more abundant in 258_equi, than in 1002_ovis (Table 1). The Pld have an important play role in the pathogenic process of *C. pseudotuberculosis*, due to the sphingomyelinase activity of the Pld, this exotoxin increases

<figure><img src="image_5.png" /><figcaption>**Fig. 2** Representative results of the core-proteome 1002_ovis and 258_equi. a Functional distribution of the proteins identified in the core-proteome. b Volcano plot generated by differentially expressed proteins, log2 ratio of 258_equi/1002_ovis. c Biological processes differential between 258_equi and 1002_ovis</figcaption></figure>
<table><caption>**Table 1** Differentially regulated proteins between 258\_equi and 1002\_ovis</caption><thead><tr><th>Accession</th><th>Description</th><th>Score</th><th>258:1002 Log(2)Ratio<sup>(a)</sup></th><th>p_value<sup>(a)</sup></th></tr></thead><tbody><tr><td>258 equi</td><td>1002 ovis</td><td></td><td></td><td></td></tr><tr><td colspan="5">Cellular processes and signaling</td></tr><tr><td colspan="5">Adhesion and motility cell</td></tr><tr><td>I3QUW8_CORPS</td><td>D9Q5S4_CORP1 Periplasmic zinc binding protein troA</td><td>4245,52</td><td>-1,32</td><td>0</td></tr><tr><td colspan="5">Cell wall/Membrane and Envelope biogenesis</td></tr><tr><td>I3QYI6_CORPS</td><td>D9Q3G9_CORP1 Phospho N acetylmuramoyl pentapeptide</td><td>166,05</td><td>1,22</td><td>1</td></tr><tr><td>I3QZH4_CORPS</td><td>D9Q4F8_CORP1 Corynomycolyl transferase</td><td>3886,67</td><td>-1,45</td><td>0</td></tr><tr><td>I3R044_CORPS</td><td>D9Q526_CORP1 Peptidoglycan recognition proteino</td><td>5283,55</td><td>-2,06</td><td>0</td></tr><tr><td>D9Q4M0_CORP1</td><td>Cell wall channel</td><td>2220,85</td><td>-2,14</td><td>0</td></tr><tr><td>I3QYK8_CORPS</td><td>D9Q3J1_CORP1 Cell wall peptidase NlpC P60 protein</td><td>1207,7</td><td>-2,78</td><td>0</td></tr><tr><td colspan="5">Defense mechanism</td></tr><tr><td>I3QW82_CORPS</td><td>D9Q743_CORP1 Cold shock protein</td><td>6171,9</td><td>1,37</td><td>1</td></tr><tr><td>I3R0B7_CORPS</td><td>D9Q597_CORP1 DNA protection during starvation protein</td><td>70,504,73</td><td>-1,48</td><td>0</td></tr><tr><td>I3QV66_CORPS</td><td>D9Q632_CORP1 Cold shock protein</td><td>31,703,46</td><td>-2458</td><td>0</td></tr><tr><td>I3QZX1_CORPS</td><td>D9Q4V4_CORP1 Protein GrpE</td><td>57,665,59</td><td>-2,64</td><td>0</td></tr><tr><td>I3QZW9_CORPS</td><td>D9Q4V2_CORP1 Heat shock protein HspR</td><td>929,96</td><td>-3,43</td><td>0</td></tr><tr><td colspan="5">Intracellular trafficking secretion and vesicular transport</td></tr><tr><td>I3QVC7_CORPS</td><td>D9Q697_CORP1 ABC type transporter</td><td>376,36</td><td>2,91</td><td>1</td></tr><tr><td>I3QZ34_CORPS</td><td>D9Q431_CORP1 ABC transporter ATP binding protein</td><td>6339,11</td><td>1,54</td><td>1</td></tr><tr><td>I3QZQ5_CORPS</td><td>D9Q4N9_CORP1 ABC superfamily ATP binding cassette</td><td>25,578,26</td><td>-1,38</td><td>0</td></tr><tr><td>I3R0D8_CORPS</td><td>D9Q5B9_CORP1 Oligopeptide transport system permease</td><td>705,01</td><td>-1,45</td><td>0,01</td></tr><tr><td colspan="5">Post-translational modification, protein turnover, chaperones</td></tr><tr><td>I3QWY7_CORPS</td><td>D9Q7U6_CORP1 Thioredoxin TrxA</td><td>1832,12</td><td>3,15</td><td>1</td></tr><tr><td>I3QVC2_CORPS</td><td>D9Q692_CORP1 Thiol disulfide isomerase thioredoxin</td><td>157,88</td><td>1,80</td><td>1</td></tr><tr><td>I3QXH3_CORPS</td><td>D9Q8C5_CORP1 Proteasome accessory factor PafA2</td><td>305,06</td><td>-1,32</td><td>0</td></tr><tr><td>I3QZA4_CORPS</td><td>D9Q493_CORP1 Glutaredoxin like protein nrdH</td><td>3140,61</td><td>-1,34</td><td>0</td></tr><tr><td>I3QUL7_CORPS</td><td>D9Q5I3_CORP1 Peptidyl prolyl cis trans isomerase</td><td>49,161,11</td><td>-1,44</td><td>0</td></tr><tr><td>I3QWQ3_CORPS</td><td>D9Q7L6_CORP1 Ferredoxin</td><td>54,332,67</td><td>-1,48</td><td>0</td></tr><tr><td>I3QW91_CORPS</td><td>D9Q753_CORP1 Peptidyl prolyl cis trans isomerase</td><td>19,736,36</td><td>-1,63</td><td>0</td></tr><tr><td>I3QV23_CORPS</td><td>D9Q5Y2_CORP1 Catalase</td><td>52,016,22</td><td>-1,70</td><td>0</td></tr><tr><td>I3QUX7_CORPS</td><td>D9Q5T5_CORP1 Glyoxalase Bleomycin resistance protein</td><td>18,489,51</td><td>-1,99</td><td>0</td></tr><tr><td>I3QVR1_CORPS</td><td>D9Q6M3_CORP1 10 kDa chaperonin</td><td>90,387,73</td><td>-2,78</td><td>0</td></tr><tr><td colspan="5">Signal transduction mechanisms</td></tr><tr><td>I3QY22_CORPS</td><td>D9Q8W9_CORP1 Phosphocarrier protein HPr</td><td>38,569,08</td><td>-2,92</td><td>0</td></tr><tr><td colspan="5">Information storage and processing</td></tr><tr><td colspan="5">DNA Metabolism: replication, recombination and repair</td></tr><tr><td>I3QV41_CORPS</td><td>D9Q606_CORP1 Metallophosphoesterase</td><td>529,63</td><td>1,88</td><td>1</td></tr><tr><td>I3QWM2_CORPS</td><td>D9Q606_CORP1 Exodeoxyribonuclease 7 small subunit</td><td>10,725,45</td><td>-2,00</td><td>0</td></tr><tr><td colspan="5">Transcription: regulation, degradation and RNA processing</td></tr><tr><td>I3QX71_CORPS</td><td>D9Q817_CORP1 SAM dependent methyltransferase y</td><td>397,16</td><td>1,25</td><td>0,96</td></tr><tr><td>I3QWD6_CORPS</td><td>D9Q7A0_CORP1 TetR family regulatory protein</td><td>5685,08</td><td>-1,38</td><td>0</td></tr><tr><td>I3QXS5_CORPS</td><td>D9Q8M5_CORP1 N utilization substance protein B homol</td><td>16,977,04</td><td>-1,42</td><td>0</td></tr><tr><td>I3QWH0_CORPS</td><td>D9Q7D4_CORP1 Transcriptional regulatory protein PvdS</td><td>7456,32</td><td>-1,48</td><td>0</td></tr><tr><td>I3QYU0_CORPS</td><td>D9Q3T4_CORP1 Ferric uptake regulatory protein</td><td>7805,46</td><td>-1,76</td><td>0</td></tr></tbody></table>
<table><caption>**Table 1** Differentially regulated proteins between 258\_equi and 1002\_ovis (Continued)</caption><tbody><tr><td>I3QWK3_CORPS</td><td>D9Q7G7_CORP1</td><td>Transcription elongation factor GreA</td><td>77,246,3</td><td>-1,87</td><td>0</td></tr><tr><td>I3QZJ2_CORPS</td><td>D9Q4H4_CORP1</td><td>Transcriptional regulator</td><td>10,476,01</td><td>-1,93</td><td>0</td></tr><tr><td>I3QUZ7_CORPS</td><td>D9Q5V6_CORP1</td><td>Nucleoid associated protein ybaB</td><td>81,447,09</td><td>-3,04</td><td>0</td></tr><tr><td>I3QUU4_CORPS</td><td>D9Q5Q1_CORP1</td><td>YaaA protein</td><td>25,362,05</td><td>-3,14</td><td>0</td></tr><tr><td colspan="6">Translation, ribosomal structure and biogenesis</td></tr><tr><td>I3R0I2_CORPS</td><td>D9Q5F9_CORP1</td><td>Ribosomal RNA small subunit methyltrans</td><td>395,99</td><td>1,29</td><td>1</td></tr><tr><td>I3QWD9_CORPS</td><td>D9Q7A3_CORP1</td><td>30S ribosomal protein S14</td><td>4756,75</td><td>-1,47</td><td>0</td></tr><tr><td colspan="6">Metabolism</td></tr><tr><td colspan="6">Amino acid transport and metabolism</td></tr><tr><td>I3QX11_CORPS</td><td>D9Q3B4_CORP1</td><td>Glutamate dehydrogenase</td><td>1534,86</td><td>3,56</td><td>1</td></tr><tr><td>I3QX11_CORPS</td><td>D9Q8D2_CORP1</td><td>Aspartate ammonia lyase</td><td>2326,21</td><td>1,60</td><td>1</td></tr><tr><td>I3QWF9_CORPS</td><td>D9Q7C4_CORP1</td><td>Glycine betaine transporter</td><td>136,23</td><td>1,21</td><td>1</td></tr><tr><td>I3QV21_CORPS</td><td>D9Q5Y0_CORP1</td><td>Aspartate semialdehyde dehydrogenase</td><td>8778,47</td><td>-1,28</td><td>0</td></tr><tr><td>I3QWZ5_CORPS</td><td>D9Q7V4_CORP1</td><td>Cysteine desulfurase</td><td>1813,31</td><td>-1,31</td><td>0</td></tr><tr><td>I3QXT1_CORPS</td><td>D9Q8N1_CORP1</td><td>Chorismate synthase</td><td>5341,49</td><td>-1,41</td><td>0</td></tr><tr><td>I3QXI5_CORPS</td><td>D9Q8D5_CORP1</td><td>Phosphoribosyl ATP pyrophosphatase</td><td>25,184,13</td><td>-1,90</td><td>0</td></tr><tr><td>I3QXL8_CORPS</td><td>D9Q8G8_CORP1</td><td>UPF0237 protein Cp258 1096</td><td>16,011,21</td><td>-1,96</td><td>0</td></tr><tr><td>I3QZZ5_CORPS</td><td>D9Q4X5_CORP1</td><td>Urease subunit beta</td><td>4349,97</td><td>-2,12</td><td>0</td></tr><tr><td colspan="6">Carbohydrate transport and metabolism</td></tr><tr><td>I3R0E6_CORPS</td><td>D9Q5C6_CORP1</td><td>Aldose 1 epimerase</td><td>221,55</td><td>2,78</td><td>1</td></tr><tr><td>I3QV93_CORPS</td><td>D9Q660_CORP1</td><td>Formate acetyltransferase 1</td><td>9381,31</td><td>2,00</td><td>1</td></tr><tr><td>I3QZB7_CORPS</td><td>D9Q4A5_CORP1</td><td>Phosphoglucomutase</td><td>359,43</td><td>1,77</td><td>1</td></tr><tr><td>I3QWW1_CORPS</td><td>D9Q7S1_CORP1</td><td>L lactate permease</td><td>103,53</td><td>1,38</td><td>0,99</td></tr><tr><td>D9Q8W7_CORP1</td><td></td><td>PTS system fructose specific EIABC</td><td>191,71</td><td>1,35</td><td>1</td></tr><tr><td>I3QY20_CORPS</td><td>D9Q8W6_CORP1</td><td>1 phosphofructokinase</td><td>2879,52</td><td>1,32</td><td>1</td></tr><tr><td>I3R064_CORPS</td><td>D9Q545_CORP1</td><td>L lactate dehydrogenase</td><td>5695,04</td><td>1,32</td><td>1</td></tr><tr><td>I3R051_CORPS</td><td>D9Q691_CORP1</td><td>Probable phosphoglycerate mutase</td><td>2014,4</td><td>-1,24</td><td>0</td></tr><tr><td>D9Q396_CORP1</td><td></td><td>PTS system fructose specific IIABC</td><td>507,53</td><td>-1,25</td><td>0</td></tr><tr><td>I3QWR8_CORPS</td><td>D9Q7N1_CORP1</td><td>Sucrose 6 phosphate hydrolase</td><td>6075,08</td><td>-1,34</td><td>0</td></tr><tr><td>I3QYN0_CORPS</td><td>D9Q3L2_CORP1</td><td>Glycine cleavage system H proteino</td><td>91,529,52</td><td>-1,38</td><td>0</td></tr><tr><td>I3QWH7_CORPS</td><td>D9Q7D8_CORP1</td><td>Glyceraldehyde 3 phosphate dehydrogenase</td><td>9529,4</td><td>-2,68</td><td>0</td></tr><tr><td colspan="6">Coenzyme metabolism</td></tr><tr><td>I3QUY1_CORPS</td><td>D9Q862_CORP1</td><td>ATP dependent dethiobiotin synthetase B</td><td>104,92</td><td>-1,24</td><td>1</td></tr><tr><td>I3QXF8_CORPS</td><td>D9Q5T9_CORP1</td><td>Pyridoxal biosynthesis lyase PdxSo</td><td>1029,15</td><td>-1,42</td><td>0</td></tr><tr><td>I3QXE1_CORPS</td><td>D9Q8B1_CORP1</td><td>Precorrin 8X methyl mutase</td><td>34,981,98</td><td>-1,77</td><td>0</td></tr><tr><td></td><td>D9Q893_CORP1</td><td>Hemolysin related protein</td><td>3609,76</td><td>1,35</td><td>0</td></tr><tr><td colspan="6">Energy metabolism</td></tr><tr><td>I3QUS5_CORPS</td><td>D9Q5N2_CORP1</td><td>NADH dehydrogenase</td><td>3257,66</td><td>3,14</td><td>1</td></tr><tr><td>I3QZ12_CORPS</td><td>D9Q411_CORP1</td><td>Malate dehydrogenase</td><td>11,220,59</td><td>1,22</td><td>1</td></tr><tr><td>I3QXN4_CORPS</td><td>D9Q8I5_CORP1</td><td>Cytochrome oxidase assembly protein</td><td>297,19</td><td>-1,86</td><td>0,09</td></tr><tr><td>I3QYB6_CORPS</td><td>D9Q3A4_CORP1</td><td>Nitrogen regulatory protein P II</td><td>16,165,93</td><td>-2,29</td><td>0</td></tr><tr><td colspan="6">Inorganic Ion Transport and Metabolism</td></tr><tr><td>I3R097_CORPS</td><td>D9Q575_CORP1</td><td>Cation transport protein</td><td>882,31</td><td>-4,64</td><td>1</td></tr><tr><td>I3QZG5_CORPS</td><td>D9Q4F0_CORP1</td><td>Trk system potassium uptake protein trk</td><td>2973,22</td><td>1,25</td><td>1</td></tr><tr><td>I3QVU4_CORPS</td><td>D9Q6Q3_CORP1</td><td>Hemin binding periplasmic protein hmuT</td><td>607,64</td><td>1,71</td><td>1</td></tr></tbody></table>
<table><caption>**Table 1** Differentially regulated proteins between 258\_equi and 1002\_ovis (Continued)</caption><thead><tr><th></th><th></th><th></th><th></th><th></th><th></th></tr></thead><tbody><tr><td>I3QVT3_CORPS</td><td>D9Q6P2_CORP1</td><td>Manganese ABC transporter substrate binding</td><td>3917,5</td><td>1,51</td><td>0</td></tr><tr><td colspan="6">Lipid transport and metabolism</td></tr><tr><td>I3QUM7_CORPS</td><td>D9Q5J0_CORP1</td><td>Phospholipase D</td><td>25,847,67</td><td>3,27</td><td>1</td></tr><tr><td>I3QZM9_CORPS</td><td>D9Q4L5_CORP1</td><td>Secretory lipase</td><td>1254</td><td>3,08</td><td>1</td></tr><tr><td colspan="6">Nucleotide metabolism</td></tr><tr><td>I3QZR5_CORPS</td><td>D9Q4P8_CORP1</td><td>Purine phosphoribosyltransferase</td><td>227,51</td><td>4,45</td><td>1</td></tr><tr><td>I3QZF7_CORPS</td><td>D9Q4E2_CORP1</td><td>Phosphoribosylformylglycinamidine synth</td><td>978,57</td><td>1,45</td><td>1</td></tr><tr><td>I3QXV6_CORPS</td><td>D9Q8Q6_CORP1</td><td>Adenine phosphoribosyltransferase</td><td>907,16</td><td>1,44</td><td>1</td></tr><tr><td>I3QZ07_CORPS</td><td>D9Q406_CORP1</td><td>Nucleoside diphosphate kinase</td><td>14,996,33</td><td>1,35</td><td>1</td></tr><tr><td>I3QZG8_CORPS</td><td>D9Q4F3_CORP1</td><td>HIT family protein</td><td>4039,81</td><td>1,21</td><td>1</td></tr><tr><td colspan="6">Secondary metabolites biosynthesis, transport and catabolism</td></tr><tr><td>I3QWA4_CORPS</td><td>D9Q766_CORP1</td><td>Multidrug resistance protein norMo</td><td>208,76</td><td>1,35</td><td>0,98</td></tr><tr><td colspan="6">Poorly characterized</td></tr><tr><td colspan="6">General function prediction only</td></tr><tr><td>I3QXE6_CORPS</td><td>D9Q898_CORP1</td><td>Methyltransferase type 11</td><td>102,29</td><td>2,84</td><td>0,99</td></tr><tr><td>I3QWH3_CORPS</td><td>D9Q7D6_CORP1</td><td>Enoyl CoA hydratase</td><td>225,9</td><td>2,14</td><td>1</td></tr><tr><td></td><td>D9Q5M7_CORP1</td><td>Unknown function</td><td>2201,88</td><td>1,93</td><td>1</td></tr><tr><td>I3QZJ7_CORPS</td><td>D9Q4I2_CORP1</td><td>Carbonic anhydrase</td><td>550,46</td><td>1,81</td><td>1</td></tr><tr><td>I3QWR6_CORPS</td><td>D9Q7M9_CORP1</td><td>Unknown function</td><td>246,05</td><td>1,24</td><td>0,98</td></tr><tr><td>I3QX38_CORPS</td><td></td><td>Cutinase</td><td>11,245,11</td><td>-1,48</td><td>0</td></tr><tr><td>I3QXY3_CORPS</td><td>D9Q8T2_CORP1</td><td>Chlorite dismutase</td><td>32,986,51</td><td>-1,51</td><td>0</td></tr><tr><td>I3QW56_CORPS</td><td>D9Q720_CORP1</td><td>Methylmalonyl CoA carboxyltransferase 1</td><td>4283,03</td><td>-1,52</td><td>0</td></tr><tr><td>I3QWE6_CORPS</td><td>D9Q7B0_CORP1</td><td>Serine protease</td><td>5615,41</td><td>-1,54</td><td>0</td></tr><tr><td>I3QX14_CORPS</td><td>D9Q7X2_CORP1</td><td>Aldo keto reductase</td><td>6121,73</td><td>-1,63</td><td>0</td></tr><tr><td>I3QWR9_CORPS</td><td>D9Q7N2_CORP1</td><td>Alpha acetolactate decarboxylase</td><td>1839,59</td><td>-1,73</td><td>0</td></tr><tr><td>I3QUU3_CORPS</td><td>D9Q5Q0_CORP1</td><td>UPF0145 protein Cp258 0101</td><td>3922,57</td><td>-1,77</td><td>0</td></tr><tr><td>I3QV73_CORPS</td><td>D9Q639_CORP1</td><td>Hydrolase domain containing protein</td><td>5759,37</td><td>-2,23</td><td>0</td></tr><tr><td>I3QZM7_CORPS</td><td>D9Q4L2_CORP1</td><td>Rhodanese related sulfurtransferase</td><td>7428,74</td><td>-2,46</td><td>0</td></tr><tr><td>I3QYY9_CORPS</td><td>D9Q3Y7_CORP1</td><td>Ankyrin domain containing protein</td><td>11,948,84</td><td>-2,84</td><td>0</td></tr><tr><td>I3QW24_CORPS</td><td>D9Q6Y4_CORP1</td><td>Hydrolase domain containing protein</td><td>15,589,93</td><td>-3,04</td><td>0</td></tr><tr><td colspan="6">Unknown function</td></tr><tr><td>I3R0H3_CORPS</td><td>D9Q5F1_CORP1</td><td>Unknown function</td><td>7862,73</td><td>3,59</td><td>1</td></tr><tr><td>I3QX01_CORPS</td><td>D9Q7W0_CORP1</td><td>Unknown function</td><td>276,68</td><td>3,43</td><td>1</td></tr><tr><td>I3QYP3_CORPS</td><td>D9Q3P0_CORP1</td><td>Unknown function</td><td>1609,29</td><td>2,84</td><td>1</td></tr><tr><td>I3QWZ8_CORPS</td><td>D9Q7V7_CORP1</td><td>Unknown function</td><td>1476,56</td><td>2,58</td><td>1</td></tr><tr><td>I3R0H2_CORPS</td><td>D9Q5F0_CORP1</td><td>Unknown function</td><td>572,19</td><td>1,90</td><td>1</td></tr><tr><td>I3QY71_CORPS</td><td>D9Q919_CORP1</td><td>Unknown function</td><td>1466,6</td><td>1,78</td><td>1</td></tr><tr><td>I3QWL3_CORPS</td><td>D9Q7H7_CORP1</td><td>Unknown function</td><td>304,96</td><td>1,65</td><td>0,98</td></tr><tr><td>I3QUW1_CORPS</td><td>D9Q5R7_CORP1</td><td>Unknown function</td><td>504,13</td><td>1,60</td><td>1</td></tr><tr><td>I3QYF2_CORPS</td><td>D9Q3D7_CORP1</td><td>Unknown function</td><td>985,97</td><td>1,47</td><td>1</td></tr><tr><td>I3QWR5_CORPS</td><td>D9Q7M8_CORP1</td><td>Unknown function</td><td>103,03</td><td>1,45</td><td>1</td></tr><tr><td>I3QVG0_CORPS</td><td>D9Q6C8_CORP1</td><td>Unknown function</td><td>4085,83</td><td>1,39</td><td>1</td></tr><tr><td>I3QXI8_CORPS</td><td>D9Q8D8_CORP1</td><td>Unknown function</td><td>87,929,25</td><td>-1,35</td><td>0</td></tr><tr><td>I3QVZ1_CORPS</td><td>D9Q6U7_CORP1</td><td>Unknown function</td><td>64,404,7</td><td>-1,38</td><td>0</td></tr><tr><td>I3R094_CORPS</td><td>D9Q572_CORP1</td><td>Unknown function</td><td>4367,05</td><td>-1,68</td><td>0</td></tr></tbody></table>
<table><caption>**Table 1** Differentially regulated proteins between 258_equi and 1002_voris (Continued)</caption><tr><td>I3QYW4_CORPS</td><td>D9Q3V8_CORP1</td><td>Unknown function</td><td>15,928,9</td><td>-1,96</td><td>0</td></tr><tr><td>I3QUX9_CORPS</td><td>D9Q5T7_CORP1</td><td>Unknown function</td><td>8100,51</td><td>-2,00</td><td>0</td></tr><tr><td>I3QYG8_CORPS</td><td>D9Q3F3_CORP1</td><td>Unknown function</td><td>78,035,52</td><td>-2,09</td><td>0</td></tr><tr><td>I3QUZ5_CORPS</td><td>D9Q5V4_CORP1</td><td>Unknown function</td><td>77,763,68</td><td>-2,09</td><td>0</td></tr><tr><td>I3QUR3_CORPS</td><td>D9Q5M1_CORP1</td><td>Unknown function</td><td>12,731,48</td><td>-2,27</td><td>0</td></tr><tr><td>I3QWV7_CORPS</td><td>D9Q6R6_CORP1</td><td>Unknown function</td><td>8564,11</td><td>-3,47</td><td>0</td></tr><tr><td>I3QXX0_CORPS</td><td>D9Q8S0_CORP1</td><td>Unknown function</td><td>19,485,3</td><td>-3,50</td><td>0</td></tr><tr><td>I3QW02_CORPS</td><td>D9Q6W1_CORP1</td><td>Unknown function</td><td>49,581,23</td><td>-3,76</td><td>0</td></tr><tr><td>I3QVS0_CORPS</td><td>D9Q6N1_CORP1</td><td>Unknown function</td><td>66,162,63</td><td>-4,87</td><td>0</td></tr><tr><td>I3R0G5_CORPS</td><td>D9Q5E4_CORP1</td><td>Unknown function</td><td>39,265,48</td><td>-5,65</td><td>0</td></tr></table>

vascular permeability through the exchange of polar groups attached to membrane-bound lipids and helps the bacteria in spread inside the host [34, 35]. In addition, this exotoxin is able to reduce the viability of both macrophages and neutrophils [34, 36]. In comparative proteomic studies between 1002_voris and C231_voris exoproteome, Pld was detected only in the C231_voris supernatant [13, 15, 16]. A study performed with pld mutant strains presented decreased virulence [37]. Thus, in relation to 258_equi, 1002_voris could present a low potential of virulence.

The 120 differential proteins were organized by cluster of orthologous groups, and when evaluated the different biological processes that comprise each category listed above, we observed that 19 process were differentials between 258_equi and 1002_voris (Fig. 2c, Additional file 7: Figure S2 and Additional file 8: Figure S3). The majority of the more abundant proteins (258_equi:1002_voris) are related to cellular metabolism. On other hand, the majority of the less abundant proteins (258_equi:1002_voris) are classified as poorly characterized or of unknown function. However, when proteins of known or predicted function are evaluated the majority of the less abundant proteins are related to cellular processes and signaling.

## Difference among the major functional classes identified from the core-proteome analysis of 1002_voris and 258_equi

### Metabolism

During the infection process, pathogens need to adjust their metabolism in response to nutrient availability inside and outside the host. In our proteomic study, we identified several proteins related to different metabolic pathways. To determine the metabolic network of each strain, the proteins identified in this study were analyzed using Kyoto Encyclopedia of Genes and Genomes (KEGG) [38]. A total of 321 and 320 proteins, corresponding to 1002_voris and 258_equi respectively, were mapped onto different metabolic pathways (Additional file 9: Figure S4 and Additional file 10:

Figure S5). We observed differences in the metabolism of the biovars, related to Amino acid transport and metabolism, Carbohydrate transport and metabolism, Coenzyme metabolism, Energy metabolism, Lipid transport and metabolism, Nucleotide metabolism and Secondary metabolites biosynthesis, transport and catabolism. Difference in the metabolism cellular, also already were observed in others comparative proteomic study of C. pseudotuberculosis [13, 16, 17, 19], as well as in the Mycobacterium tuberculosis pathogen [39].

Interestingly, the PTS system fructose-specific EIIABC component (PstF) related to carbohydrate metabolism was more abundant in 258_equi, than in 1002_voris (Table 1). This protein showed increased abundance in field isolates of C. pseudotuberculosis biovar ovis grown in BHI when compared to C231_voris, a reference strain [19]. This increased abundance of PstF in 258_equi, suggests that this protein could be important to the transport of carbon source both biovar ovis and biovar equi strains. On the other hand, the Precorrin 8X methyl mutase involved in cobalamin and vitamin B12 synthesis can be required only in biovar ovis strains, this protein beside being more abundant in 1002_voris (Table 1), was also detected with greater abundance in the field isolates of C. pseudotuberculosis biovar ovis after having been grown in BHI [19]. Glutamate dehydrogenase (GDH) was detected more abundant in 258_equi (Table 1). A study performed with the M. bovis pathogen showed that GDH contributes to the survival of this pathogen during macrophage infection [40].

In C. pseudotuberculosis, it was demonstrated that genes related the iron-acquisition are involved in the virulence of this pathogen [41]. In the core-proteome of 1002_voris and 258_equi, we detected proteins involved in this process, like CiuA, FagC and FagD; however, all these proteins were not differentially regulated between the two strains (Additional file 2: Table S1). On the other hand, HmuT protein, related to hemin uptake, was more abundant in 258_equi (Table 1). Additionally, we have also detected a cell surface hemin receptor in the
exclusive proteome of this strain. Heme represents the major reservoir of iron source for many bacterial pathogens that rely on surface-associated heme-uptake receptors [42]. The HmuT is a lipoprotein that acts as a hemin receptor. The *hmuT* gene is part of the operon *hmuTUV*, an ABC transport system (haemin transport system), which is normally present in pathogenic *Corynebacterium* [43, 44]. In addition, in the pathogen *C. ulcerans*, HmuT is required for normal hemin utilization [44].

## Information storage and processing

Of the total protein of proteins identified in the category “information storage and processing” the majority of the differential proteins were less abundant in 258_equi (Table 1). Only, Metallophosphoesterase involved in DNA repair, SAM dependent methyltransferase related to transcriptional process and Ribosomal RNA small subunit methyltransferase I involved in translation process were more induced in 258_equi. In 1002_ovis the Exodeoxyribonuclease 7 important protein related to the DNA-damage pathway was more induced in this strain. In addition, we identified the TetR family regulatory protein as more abundant in 1002_ovis, this result was also observed in field isolates of *C. pseudotuberculosis* from sheep infected naturally [19]. TerR proteins are related to regulation of multidrug efflux pumps, antibiotic biosynthesis, catabolic process and cellular differentiation process [45]. Others important transcriptional regulators also were induced in 1002_ovis such as PvdS and GreA regulators.

## Cellular processes and signaling

Our proteomic analyses detected differentially regulated proteins belonging to different antioxidant systems. These could contribute to the survival of *C. pseudotuberculosis* in various stress conditions, such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), which are generally found in macrophage. The three major thiol-dependent antioxidant systems in prokaryotic pathogens are the thioredoxin system (Trx), the glutathione system (GSH-system) and the catalase system [46]. Thioredoxin TrxA and Thiol-disulfide isomerase thioredoxin were more abundant in 258_equi (Table 1). These proteins are involved in the Trx-system, which has a major role against oxidative stress [46]. However, proteins like catalase and glutaredoxin (nrdH) were less abundant in 258_equi (Table 1), being more active in 1002_ovis. Catalase plays an important role in resistance to ROS and RNS, as well as in the virulence of *M. tuberculosis* [47]. The protein NrdH has a glutaredoxin amino acid sequence and thioredoxin activity. It is present in *Escherichia coli* [48] and *C. ammoniagenes* [49], as well as in bacteria where the GSH system is absent, such as *M. tuberculosis* [50]. Thus, the presence of NrdH may

represent one more factor that contributes to the resistance of *C. pseudotuberculosis* against ROS and RNS during the infection process, as well as to the maintenance of the balance of intracellular redox potential. Proteins like NorB and Glyoxalase/Bleomycin, which play roles in the nitrosative stress response of 1002_ovis, were identified in the exclusive proteome of this strain (Additional file 3: Table S2) [14, 18]. These results shown that beside of present proteins with difference in abundance both strains present a set of proteins that could contribute to adaptive process under stress conditions.

## Difference proteomic observed in the exclusive proteome of 258_equi and 1002_ovis

We found respectively 105 and 96 proteins in the exclusive proteome of 1002_ovis and 258_equi (Fig. 1) (Additional file 3: Table S2 and Additional file 4: Table S3), related to different biological process (Additional file 7: Figure S2 and Additional file 8: Figure S3). Interestingly, in this exclusive proteome of 1002_ovis and 258_equi, we detected specific proteins in each strain (Table 2, Additional file 3: Table S2 and Additional file 4: Table S3). In the exclusive proteome of 258_equi, the ORFs that codify twenty proteins are annotated as pseudogene in 1002_ovis (Table 2, Additional file 3: Table S2 and Additional file 4: Table S3). On the other hand, the ORFs that encode six proteins were not detected in the genome of 1002_ovis. These proteins are two CRISPR, MoeB, and three unknown function proteins. CRISPR is an important bacterial defense system against infections by viruses or plasmids, this immunity is obtained from the integration of short sequences of invasive DNA ‘spacers’ into the CRISPR loci [51].

The distinction between the biovar *ovis* and biovar *biovar equi* strains is based on a biochemical assay, where biovar ovis strains are negative for nitrate reduction, whereas biovar equi strains are positive [52]. However, to date, there is no available information regarding the molecular basis underlying nitrate reduction in *C. pseudotuberculosis* biovar *equi*. MoeB is involved in the molybdenum cofactor (Moco) biosynthesis, which plays an important role in anaerobic respiration in bacteria and also are required to activation of nitrate reductase (NAR) [53]. In the closely related pathogen *M. tuberculosis* several studies have showed the great importance of molybdenum cofactor in its virulence and pathogenic process, mainly macrophage intracellular environmental [54]. Therefore, more studies are necessary to explore the true role of Moco both physiology and virulence of biovar *equi* strains. Other protein that also could contribute to resistance of 258_equi macrophage is NADPH dependent nitro/flavin reductase (NfrA), a pseudogene in 1002_ovis. In addition, studies performed in *Bacillus*
<table><caption>**Table 2** Exclusive proteins identified in 258_equi and 1002_voris</caption><thead><tr><th>Locus</th><th>Locus</th><th>Description</th><th>Biological Process</th></tr></thead><tbody><tr><td></td><td></td><td>1002_voris</td><td></td></tr><tr><td>Cp1002_1457</td><td>–</td><td>DNA methylase<sup>b</sup></td><td>DNA Metabolism: replication, recombination and repair</td></tr><tr><td>Cp1002_1872</td><td>Cp258_1887</td><td>Collagen binding surface protein Cna<sup>d</sup></td><td>Adhesion and motility cell</td></tr><tr><td>Cp1002_1859</td><td>Cp258_1875</td><td>Sdr family related adhesin<sup>d</sup></td><td>Adhesion and motility cell</td></tr><tr><td>Cp1002_2025</td><td>Cp258_2050</td><td>Glycoside hydrolase 15 related protein<sup>d</sup></td><td>Carbohydrate transport and metabolism</td></tr><tr><td>Cp1002_0387</td><td>Cp258_0396</td><td>Neuraminidase Sialidase<sup>d</sup></td><td>Lipid transport and metabolism</td></tr><tr><td>Cp1002_0262</td><td>Cp258_0266</td><td>Ppx/GppA phosphatase family<sup>d</sup></td><td>General function prediction only</td></tr><tr><td>Cp1002_1151</td><td>Cp258_1168</td><td>Zinc metallopeptidase<sup>d</sup></td><td>General function prediction only</td></tr><tr><td>Cp1002_0077</td><td>Cp258_0091</td><td>Unknown function<sup>d</sup></td><td>Unknown function</td></tr><tr><td></td><td></td><td>258_equi</td><td></td></tr><tr><td>Cp258_0374</td><td>–</td><td>MoeB protein<sup>a</sup></td><td>Coenzyme metabolism</td></tr><tr><td>Cp258_1675</td><td>–</td><td>CRISPR associated protein<sup>a</sup></td><td>DNA Metabolism: replication, recombination and repair</td></tr><tr><td>Cp258_0028</td><td>–</td><td>CRISPR-associated protein<sup>a</sup></td><td>DNA Metabolism: replication, recombination and repair</td></tr><tr><td>Cp258_0076</td><td>–</td><td>Unknown function<sup>a</sup></td><td>Unknown function</td></tr><tr><td>Cp258_0585</td><td>–</td><td>Unknown function<sup>a</sup></td><td>Unknown function</td></tr><tr><td>Cp258_0586</td><td>–</td><td>Unknown function<sup>a</sup></td><td>Unknown function</td></tr><tr><td>Cp258_0896</td><td>Cp1002_0888</td><td>Acetolactate synthase<sup>c</sup></td><td>Amino acid transport and metabolism</td></tr><tr><td>Cp258_0465</td><td>Cp1002_0455</td><td>Cystathionine gamma synthase<sup>c</sup></td><td>Amino acid transport and metabolism</td></tr><tr><td>Cp258_0313</td><td>Cp1002_0310</td><td>Aminopeptidase G<sup>c</sup></td><td>Amino acid transport and metabolism</td></tr><tr><td>Cp258_0893</td><td>Cp1002_0884</td><td>Dihydroxy acid dehydratase<sup>c</sup></td><td>Amino acid transport and metabolism</td></tr><tr><td>Cp258_1223</td><td>Cp1002_1203</td><td>Inositol 1 monophosphatase<sup>c</sup></td><td>Carbohydrate transport and metabolism</td></tr><tr><td>Cp258_1360</td><td>Cp1002_1337</td><td>Unknown function<sup>c</sup></td><td>Coenzyme metabolism</td></tr><tr><td>Cp258_1909</td><td>Cp1002_1892</td><td>Aldehyde dehydrogenase<sup>c</sup></td><td>Energy metabolism</td></tr><tr><td>Cp258_0123</td><td>Cp1002_0109</td><td>ABC type metal ion transport system<sup>c</sup></td><td>Inorganic Ion Transport and Metabolism</td></tr><tr><td>Cp258_1854</td><td>Cp1002_1838</td><td>Disulfide bond formation protein DsbB<sup>c</sup></td><td>Post-translational modification, protein turnover, chaperones</td></tr><tr><td>Cp258_0395</td><td>Cp1002_0386</td><td>Methionine aminopeptidase<sup>c</sup></td><td>Post-translational modification, protein turnover, chaperones</td></tr><tr><td>Cp258_1923</td><td>Cp1002_1906</td><td>Oligopeptide binding protein oppA<sup>c</sup></td><td>Intracellular trafficking secretion and vesicular transport</td></tr><tr><td>Cp258_1549</td><td>Cp1002_1541</td><td>ABC transporter ATP binding protein<sup>c</sup></td><td>Intracellular trafficking secretion and vesicular transport</td></tr><tr><td>Cp258_1566</td><td>Cp1002_1561</td><td>ABC transporter<sup>c</sup></td><td>Intracellular trafficking secretion and vesicular transport</td></tr><tr><td>Cp258_0693</td><td>Cp1002_0689</td><td>Phosphatase YbjL<sup>c</sup></td><td>General function prediction only</td></tr><tr><td>Cp258_1503</td><td>Cp1002_1497</td><td>Alpha beta hydrolase<sup>c</sup></td><td>General function prediction only</td></tr><tr><td>Cp258_1265</td><td>Cp1002_1243</td><td>Unknown function<sup>c</sup></td><td>General function prediction only</td></tr><tr><td>Cp258_0169</td><td>Cp1002_0157</td><td>NADPH dependent nitro flavin reductase<sup>c</sup></td><td>General function prediction only</td></tr><tr><td>Cp258_1351</td><td>Cp1002_1329</td><td>Unknown function<sup>c</sup></td><td>Unknown function</td></tr><tr><td>Cp258_1916</td><td>Cp1002_1899</td><td>Unknown function<sup>c</sup></td><td>Unknown function</td></tr><tr><td>Cp258_2099</td><td>Cp1002_2077</td><td>Unknown function<sup>c</sup></td><td>Unknown function</td></tr></tbody></table>

subtilis showed that NfrA is involved in both oxidative stress [55] and heat shock resistance [56].

In 1002_voris, only the ORF that encodes a DNA methylase was not found in the 258_equi genome (Table 2, Additional file 3: Table S2 and Additional file 4: Table S3). In addition, the ORFs that codifies seven proteins

identified in the exclusive proteome of the strain 1002_voris are annotated like pseudogene in 258_equi (Table 2, Additional file 3: Table S2 and Additional file 4: Table S3). Inside this group, we have identified important proteins involved in the process of adhesion and invasion cellular, which might contribute in the

<footnote>(a) Strain-specific protein, ORF detected only in the genome of 258_equi</footnote>

<footnote>(b) Strain-specific protein, ORF detected only in the genome of 1002_voris</footnote>

<footnote>(c) ORF predicted like pseudogene in 1002_voris</footnote>

<footnote>(d) ORF predicted like pseudogene in 258_equi</footnote>
pathogenesis of 1002_ovis. Adhesion to host cells is a
crucial step that favors the bacterial colonization; this
process is mediated by different adhesins [57]. We identi-
fied proteins such as: collagen binding surface protein Cna-
like and Sdr family related adhesin, which are members of
the collagen-binding microbial surface components recog-
nizing adhesive matrix molecules (MSCRAMMs) (Table 2).
This class of proteins is present in several Gram positive
pathogens and plays an important role in bacterial virulence
by acting mainly in the cellular adhesion process [58–61].

Another detected protein that might contribute to the virulence of 1002_ovis is Neuraminidase (NanH) (Table 2). This protein belongs to a class of glycosyl hydrolases that contributes to the recognition of sialic acids exposed on host cell surfaces [62]. In C. diphtheriae, it was demonstrated that a protein with trans-sialidase activity promotes cellular invasion [63, 64]. In addition, NanH was reported to be immunoreactive in the immunoprotoeme of 1002_ovis, showing the antigenicity of this protein [65]. Interestingly, genomic difference in relation to gene involved in the adhesion and invasion process, also already were observed between biovar ovis strain and biovar equi strains, mainly in genes related to pilus [10, 12]. According to pathogenic process of each biovar, unlike biovar equi strains, which rarely causes visceral lesions [4], biovar ovis strains, are responsible mainly by visceral lesions [2, 35], what requires a high ability to adhere and invade the host cell, thus these protein could be responsible by this ability of biovar ovis strain in attacks visceral organs.

Proteogenomic analysis

In our proteomic analysis, the measured MS/MS spectra
from the proteomic datasets of 1002_ovis and 258_equi
were searched against a concatenated database com-
posed by genome annotation of 1002_ovis CP001809.2
version and 258_equi CP003540.2 version for identify
possible errors or unannotated genes. Thus, by adopting
more stringent criteria of considering only proteins with
a minimum representative of two peptides and a
FDR < 1%, we identified five proteins in 1002_ovis and
seven proteins in 258_equi, which were not previously
annotated. All parameters, as well as, the peptides se-
quence which were used for identification of these pro-
teins are shown in Additional file 11: Table S6 and
Additional file 12: Table S7. The proteins identified in
this proteogenomic analysis are associated to different
biological processes. For instance, the Aminopeptidase
N involved in the amino acid metabolism was detected
in 1002_ovis, whereas the Cobaltochelatase (cobN), associ-
ated to cobalt metabolism, glutamate dehydrogenase (gdh)
involved in the L-glutamate metabolism, the PTS system
fructose specific EIIABC related to fructose metabolism
and the Phosphoribosylglycinamide formyltransferase

involved in the purine biosynthesis were all detected in
258_equi. Proteins involved in DNA processes, such as
Uracil DNA glycosylase in 258_equi; and Exodeoxyribonu-
clease 7 small subunit in 1002_ovis were also detected in
both strains. Proteins with general function prediction
only and unknown function were also identified in both
strains.

Conclusion

In conclusion, we used a label-free quantitative approach
to compare, for the first time, the proteome of C. pseu-
dotuberculosis strains belonging to both ovis and equi
biovars. Taken together, the findings reported here show
a set of shared and exclusive factors of 1002_ovis and
258_equi at the protein level, which can contribute to
understanding both the physiology and the virulence of
these strains. In addition, the functional analysis of the
genome of 1002_ovis and 258_equi allows the in silico
validation of data of the genome of these strains. Thus,
the proteins identified here may be used as potential
new targets for the development of vaccines against ovis
and equi C. pseudotuberculosis in future investigations.

Availability of supporting data

The datasets supporting the results of this article were
then concatenated into a *.xlsex file at peptide and protein
level to fulfill the requirements and is available at supple-
mental material including sequence coverage and a num-
ber of identified peptides for each protein sequence
identified. It also includes the native peptide information.

Additional files

**Additional file 1: Figure S1.** Growth rates in BHI media of 1002_ovis (blue circles) and 258_equi (red triangles). (JPEG 278 kb)

**Additional file 2: Table S1.** Total list of proteins identified in the core-proteome of 1002_ovis and 258_equi. (XLSX 215 kb)

**Additional file 3: Table S2.** Total list of proteins identified in the exclusive proteome of 1002_ovis. (XLSX 20 kb)

**Additional file 4: Table S3.** Total list of proteins identified in the exclusive proteome of 258_equi. (XLSX 21 kb)

**Additional file 5: Table S4.** Total list of peptide and proteins identified 1002_ovis. (XLSB 31769 kb)

**Additional file 6: Table S5.** Total list of peptide and proteins identified 258_equi. (XLSB 33204 kb)

**Additional file 7: Figure S2.** The protein-protein interaction network of 1002_ovis. (A) General interactome of differentially regulated proteins, identified in the exclusive proteome of 1002_ovis. The proteins are marked with different shapes: exclusive proteome, circle; more abundant, square; less abundant, rhombus. The biological processes were marked with different colors: amino acid transport and metabolism, yellow; secondary metabolites biosynthesis, transport and catabolism, aquamarine; inorganic ion transport and metabolism, orange; coenzyme metabolism, brown; carbohydrate transport and metabolism, chartreuse green; nucleotide metabolism, cerulean; energy metabolism, olive; lipid transport and metabolism, viridian; adhesion and motility cell, crimson; iuntracellular trafficking secretion and vesicular transport, persian blue; signal transduction mechanisms, maroon; cell wall/membrane and envelope, gray; defense mechanism, red; post-translational modification, protein
turnover, chaperones, electric blue; DNA metabolism, replication, recombination and repair, violet; translation, ribosomal structure and biogenesis, amber; transcription, regulation, degradation and RNA processing, salmon; poorly characterized, white. (JPEG 3310 kb)

**Additional file 8: Figure S3.** The protein-protein interaction network of 258\_equi. (A) General interactome of the differentially regulated proteins, identified in the exclusive proteome of 258\_equi. The proteins are marked with different shapes: exclusive proteome, circle; more abundant, square; less abundant, rhombus. The biological processes are marked with different colors: amino acid transport and metabolism, yellow; secondary metabolites biosynthesis, transport and catabolism, aquamarine; inorganic ion transport and metabolism, orange; coenzyme metabolism, brown; carbohydrate transport and metabolism, chartreuse green; nucleotide metabolism, cereulian; energy metabolism, olive; lipid transport and metabolism, viridian; adhesion and motility cell, crimson; intracellular trafficking secretion and vesicular transport, persian blue; signal transduction mechanisms, maroon; cell wall/membrane and envelope, gray; defense mechanism, red; post-translational modification, protein turnover, chaperones, electric blue; DNA metabolism, replication, recombination and repair, violet; translation, ribosomal structure and biogenesis, amber; transcription, regulation, degradation and RNA processing, salmon; poorly characterized, white. (JPEG 4178 kb)

**Additional file 9: Figure S4.** Metabolic network of 1002\_ovis. Red line, proteins identified in the proteomic analysis, other colors represent proteins not identified in this study. (JPEG 8633 kb)

**Additional file 10: Figure S5.** Metabolic network of 258\_equi. Red line, proteins identified in the proteomic analysis, other colors represent proteins not identified in this study. (JPEG 1267 kb)

**Additional file 11: Table S6.** Proteins identified in 1002\_ovis by Proteogenomics. (XLSX 216 kb)

**Additional file 12: Table S7.** Proteins identified in 258\_equi by Proteogenomics. (XLSX 266 kb)

## Abbreviations

2D-RP: Two-dimensional reversed phase; C: Citosine; FDR: False discovery rate; G: Guanine; HSS: High strength silica; LC/MS: Liquid chromatograph mass spectrometry; nanoESI-HDMS: Nano Electrospray High Definition Mass Spectrometry; nanoUPLC: Nano Ultra Performance Liquid Chromatography; nanoUPLC-MS: Nano Ultra Performance Liquid Chromatography Mass Spectrometry; PBS: Phosphate buffered saline; PLGS: Protein Lynx Global Server; RNS: Reactive nitrogen species; ROS: Reactive oxygen species

## Acknowledgment

This work involved the collaboration of various institutions, including the Genomics and Proteomics Network of the State of Pará of the Federal University of Pará, the Amazon Research Foundation (FAPESPA), the National Council for Scientific and Technological Development (CNPq), the Brazilian Federal Agency for the Support and Evaluation of Graduate Education (CAPES), the Minas Gerais Research Foundation (FAPEMIG) and the Waters Corporation, Brazil.

## Authors' contributions

WMS performed microbiological analyses and sample preparation for proteomic analysis. GHMFS and WMS conducted the proteomic analysis. SCS and ELF performed bioinformatics analysis of the data. CSS, AVS, AM and HF contributed substantially to data interpretation and revisions. VA, AS and YLL participated in all steps of the project as coordinators, and critically reviewed the manuscript. All authors read and approved the final manuscript.

## Competing interests

The authors declare that they have no competing interests.

## Consent for publication

Not applicable.

## Ethics approval and consent to participate

No ethics approval was required for any aspect of this study.

## Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

## Author details

¹Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brasil.

²Instituto de Ciências Biológicas, Universidade Federal do Pará, Belém, Pará, Brasil. ³Centro de Biotecnologia, Universidade Federal da Paraíba, João Pessoa, Paraíba, Brasil. ⁴Waters Corporation, Waters Technologies Brazil, MS Applications Laboratory, Alphaville, São Paulo, Brasil. ⁵INRA, UMR1253 STLO, 35042 Rennes, France. ⁶Agrocampus Ouest, UMR1253 STLO, 35042 Rennes, France. ⁷Escola de Veterinária, Aquavet, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brasil. ⁸Departamento de Microbiologia, Imunologia e Parasitologia, Instituto de Ciências Biológicas e Naturais, Universidade Federal do Triângulo Mineiro, Uberaba, Minas Gerais, Brasil.

Received: 26 July 2016 Accepted: 31 May 2017

Published online: 08 June 2017

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