# A shift in the virulence potential of Corynebacterium pseudotuberculosis biovar ovis after passage in a murine host demonstrated through comparative proteomics.

Wanderson Silva, Fernanda A Dorella, Siomar C Soares, Gustavo H M F

Souza, Thiago L P Castro, Núbia Seyffert, Henrique Figueiredo, Anderson

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

► To cite this version:

Wanderson Silva, Fernanda A Dorella, Siomar C Soares, Gustavo H M F Souza, Thiago L P Castro, et al. A shift in the virulence potential of Corynebacterium pseudotuberculosis biovar ovis after passage in a murine host demonstrated through comparative proteomics. BMC Microbiology, 2017, 17 (1), pp.55. 10.1186/s12866-017-0925-6. hal-01495399

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# A shift in the virulence potential of *Corynebacterium pseudotuberculosis* biovar *ovis* after passage in a murine host demonstrated through comparative proteomics

Wanderson M. Silva<fnref n="1" />,⁴,⁵, Fernanda A. Dorella<fnref n="1" />, Siomar C. Soares<fnref n="1" />, Gustavo H. M. F. Souza³, Thiago L. P. Castro<fnref n="1" />, Núbia Seyffert<fnref n="1" />, Henrique Figueiredo⁶, Anderson Miyoshi<fnref n="1" />, Yves Le Loir⁴,⁵, Artur Silva² and Vasco Azevedo<fnref n="1" />*

**Abstract**

**Background:** *Corynebacterium pseudotuberculosis* biovar *ovis*, a facultative intracellular pathogen, is the etiologic agent of caseous lymphadenitis in small ruminants. During the infection process, *C. pseudotuberculosis* changes its gene expression to resist different types of stresses and to evade the immune system of the host. However, factors contributing to the infectious process of this pathogen are still poorly documented. To better understand the *C. pseudotuberculosis* infection process and to identify potential factors which could be involved in its virulence, experimental infection was carried out in a murine model using the strain 1002_ovis and followed by a comparative proteomic analysis of the strain before and after passage.

**Results:** The experimental infection assays revealed that strain 1002_ovis exhibits low virulence potential. However, the strain recovered from the spleen of infected mice and used in a new infection challenge showed a dramatic change in its virulence potential. Label-free proteomic analysis of the culture supernatants of strain 1002_ovis before and after passage in mice revealed that 118 proteins were differentially expressed. The proteome exclusive to the recovered strain contained important virulence factors such as CP40 proteinase and phospholipase D exotoxin, the major virulence factor of *C. pseudotuberculosis*. Also, the proteome from recovered condition revealed different classes of proteins involved in detoxification processes, pathogenesis and export pathways, indicating the presence of distinct mechanisms that could contribute in the infectious process of this pathogen.

**Conclusions:** This study shows that *C. pseudotuberculosis* modifies its proteomic profile in the laboratory versus infection conditions and adapts to the host context during the infection process. The screening proteomic performed us enable identify known virulence factors, as well as potential proteins that could be related to virulence this pathogen. These results enhance our understanding of the factors that might influence in the virulence of *C. pseudotuberculosis*.

**Keywords:** *Corynebacterium pseudotuberculosis*, Bacterial label-free proteomic, Caseous lymphadenitis, Bacterial virulence, Serial passage, Extracellular proteins

<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, Brazil
Full list of author information is available at the end of the article</footnote>
## Background

*Corynebacterium pseudotuberculosis* biovar *ovis* is a Gram-positive facultative intracellular pathogen. It is the etiologic agent of Caseous Lymphadenitis (CLA) in small ruminants, a disease characterized by abscess formation in lymph nodes and internal organs [1]. Cases of human infection caused by *C. pseudotuberculosis* have been reported and are associated with occupational exposure [1]. CLA is globally distributed and causes significant economic losses in goats, and sheep herds [2]. The pathogenic process of *C. pseudotuberculosis* in the host comprises two phases: (i) initial colonization and replication in lymph nodes that drain the site of infection, which is associated with pyogranuloma formation, and (ii) a secondary cycle of replication and dissemination via the lymphatic or circulatory systems. This dissemination is promoted by the action of phospholipase D (PLD) exotoxin, the major virulence factor of *C. pseudotuberculosis*, which allows this pathogen to contaminate visceral organs and lymph nodes, where it ultimately induces lesion formation [3–5].

Exported proteins reportedly favor the infection process in pathogenic bacteria; this class of proteins is involved in adhesion and invasion of host cells, nutrient acquisition, toxicity, and in the evasion of the host immune system [6]. Different strategies like the transposon mutagenesis have been adopted to identify *C. pseudotuberculosis* biovar *ovis* exported proteins [7]. Additionally, comparative proteomics has been applied to characterize the extracellular proteome of *C. pseudotuberculosis* biovar *ovis*, as well as, the extracellular immunoproteome (strains C231_ovis and 1002_ovis) [8–11]. In these studies, some proteins of the strain 1002_ovis, suspected to be virulence factors, were not detected suggesting this strain presents a low virulence. The surface proteome of *C. pseudotuberculosis* biovar *ovis* was also characterized using bacterial strains isolated from the lymph nodes of naturally infected sheep. This proteomic analysis allowed the identification of proteins that could favor the survival of this pathogen during the chronic phase of CLA [12].

The experimental passage of bacterial pathogens through in vitro or in an in vivo model is a strategy that has been applied to evaluate the virulence potential of several pathogens. By generating a confrontation between the pathogen and the dynamic network of host factors, including the immune system components, it helps to identify bacterial factors involved in virulence [12–19]. In this study, the strain 1002_ovis was experimentally inoculated in mice [20, 21] to identify factors which could contribute to virulence in *C. pseudotuberculosis* biovar *ovis*. Comparative proteomics of the culture supernatant from this strain collected before and after the experimental passage in mice was carried out to identify factors that might contribute to virulence of 1002_ovis.

## Methods

### Bacterial strains and growth conditions

The *C. pseudotuberculosis* biovar *ovis* strain 1002 (1002_ovis) was isolated from a goat in Brazil; this strain was cultivated under standard conditions in brain-heart infusion broth (BHI-HiMedia Laboratories Pvt. Ltd., India) at 37 °C. When necessary, 1.5% of agar was added to the medium for a solid culture. For extracellular proteomic analyses, 1002_ovis was grown in a chemically defined medium (CDM) [(Na₂HPO₄·7H₂O (12.93 g/L), KH₂PO₄ (2.55 g/L), NH₄Cl (1 g/L), MgSO₄·7H₂O (0.20 g/L), CaCl₂ (0.02 g/L) and 0.05% (v/v) Tween 80], 4% (v/v) MEM Vitamins Solution (Invitrogen, Gaithersburg, MD, USA), 1% (v/v) MEM Amino Acids Solution (Invitrogen), 1% (v/v) MEM Non-Essential Amino Acids Solution (Invitrogen), and 1.2% (w/v) glucose at 37 °C [22].

### Experimental infection of strain 1002_ovis in a murine model (in vivo assay)

The standardization of the parameters for infection was performed according to Moraes et al. [20] and Ribeiro et al. [21]. Female BALB/c mice between six and eight weeks old were used in all experiments. They were provided by the Animal Care Facility of the Biological Sciences Institute from the Federal University of Minas Gerais and were handled by the guidelines of the UFMG Ethics Committee on Animal Testing (Permit Number: CETEA 103/2011). For the bacterial passage assay using the murine model, two groups of three mice each was infected via intraperitoneal injection with 10⁶ colony forming units (CFU) of strain 1002_ovis. Thirty-six hours after infection, all animals were sacrificed. Their spleens were aseptically removed to recover the bacterial strain, as described below: the spleen removed from each animal was then, individually macerated in sterile saline solution (0.9% NaCl₂), seeded onto BHI agar plates and incubated for 48 h at 37 °C. Subsequently, one recovered bacterial colony was cultured in BHI broth. The recovered bacteria were then referred to as Recovered (Rc). For the bacterial virulence assay, we used the freshly recovered bacteria and bacteria that did not contact the murine host as a control, which is referred to as Control (Ct). Groups of five mice were infected with Rc and Ct, via intraperitoneal injection of a suspension containing 10⁶ CFU or 10⁵ CFU. The animals' survival rates were calculated and represented in GraphPad Prism v.5.0 (GraphPad Software, San Diego, CA, USA) using the Kaplan-Meier survival function. The results of 1002_ovis CFU count in the organs were calculated using the two-way ANOVA test.

### Preparation of proteins from culture filtrates for proteome analysis

For proteomic analysis, the Ct and Rc (three independently recovered colonies) that was obtained from infected
mice spleens as described above were grown in CDM at OD₆₀₀ = 0.8. The cultures were then centrifuged for 20 min at 2700 × g. The supernatants were then filtered using 0.22-µm filters, 30% (w/v) ammonium sulfate was added to the samples, and the pH of the mixtures was adjusted to 4.0. Next, 20 mL N-butanol was added to each sample. The samples were centrifuged for 10 min at 1350 xg and 4 °C. The interfacial precipitate was collected and resuspended in 1 mL of 20 mM Tris–HCl pH 7.2 [23]. Finally the concentration protein was determined by Bradford method [24].

## 2D-PAGE electrophoresis and Mass Spectrometry

The 2-DE procedure and in-gel protein digestion were performed as described previously [9, 10]. Approximately 300 µg of the protein extract from each condition was dissolved in rehydration buffer (Urea 7 M, thiourea 2 M, CHAPS 2%, Tris–HCl 40 mM, bromophenol blue 0.002%, DTT 75 mM, IPG Buffer 1%). Samples were applied to 18 cm pH 3–10 N.L strips (GE Healthcare, Pittsburgh, USA). Isoelectric focusing (IEF) was performed using the apparatus IPGphor 2 (GE Healthcare) under the following voltages: 100 V 1 h, 500 V 2 h, 1000 V 2 h, 10,000 V 3 h, 10,000 V 6 h, 500 V 4 h. The IPG strips were placed on 12% acrylamide/bis acrylamide gels in an Ettan DaltSix II system (GE Healthcare). The gels were stained with Coomassie Blue G-250 staining solution, and 2-DE gels were scanned using an Image Scanner (GE Healthcare). The Image Master 2D Platinum 7 (GE Healthcare) software was used to analyze the generated images and all spots were matched and analyzed by gel-to-gel comparison. The quantification of the spots was calculated according percentage volume (% Vol) and spots with reproducible changes in abundance were considered to be differentially expressed. Protein spots were excised from the gels, and in-gel digestion was carried out using trypsin enzyme (Promega, Sequencing Grade Modified Trypsin, Madison, WI, USA). The peptides were then desalted and concentrated using ZIP TIP C18 tips (Eppendorf).

The samples were subsequently analyzed for MS and MS/MS modes, using an MALDI-TOF/TOF mass spectrometer Autoflex IIITM (Bruker Daltonics, Billerica USA). The equipment was controlled in a positive/reflector way using the Flex-ControlTM software (Brucker Daltonics). External calibration was performed using peptide standards samples (angiotensin II, angiotensin I, substance P, bombe-sin, ACTH clip 1–17, ACTH clip 18–39, somatostatin 28, bradykinin Fragment 1–7, Renin Substrate tetra decapep-tide porcine) (Bruker Daltonics). The peptides were added to the alpha-cyano-4-hydroxycinnamic acid matrix, applied on an Anchor-ChipTM 600 plate (Brucker Daltonics) and analyzed by Autoflex III. The search parameters were as follows: enzyme; trypsin; fixed modification, carbamido-methylation (Cys); variable modifications, oxidation (Met);

mass values, monoisotopic; maximum missed cleavages, 1; and peptide mass tolerance of 0.005% Da (50 ppm). The results obtained by MS/MS were used to identify proteins utilizing the MASCOT_ (http://www.matrixscience.com) program and compared with the genomic data of the Actinobacteria class deposited in the NCBI nr database.

## 2D nanoUPLC-HDMSE data acquisition and Data Processing

The protein extracts from three biological replicates of each condition were concentrated using spin columns with a 10 kDa threshold (Millipore, Billerica, MA, USA) to perform the label-free proteomic analysis. The protein was denatured (0.1% RapiGEST SF at 60 °C for 15 min) (Waters, Milford, CA, USA), reduced (10 mM DTT), alkylated (10 mM iodoacetamide) and enzymatically digested with trypsin (Promega). The digestion process was stopped by adding 10 µL of 5% TFA (Fluka, Buchs, Germany), and glycogen phosphorylase (Sigma, Aldrich, P00489) was added to the digested samples after digest at 20 fmol.uL⁻¹ as an internal standard for normalization. Each replicate was injected using a two-dimensional reversed phase (2D RPxRP) nanoUPLC-MS (Nano Ultra Performance Liquid Chromatography Mass Spectrometry) approach with 171 multiplexed high definition mass spectrometry (HDMSE) label-free quantitation [25]. Qualitative and quantitative experiments were performed using both a 1 h reversed phase gradient from 7% to 40% (v/v) acetonitrile (0.1% v/v formic acid) at 500 nL.min⁻¹ and a nanoACQUITY UPLC 2D RPxRP Technology system [26]. A nanoACQUITY 174 UPLC HSS T3 1.8 µm, 75 µm × 15 cm column (pH 3) was used with an RP XBridge BEH130 C18 5 µm 300 µm × 50 mm nanoflow column (pH 10). Typical on-column sample loads were 250 ng of the total protein digests for each of the 5 fractions (250 ng/fraction/load). All analyses were performed using nano electrospray ionization in the positive ion mode nanoESI (+) and a NanoLockSpray (Waters, Manchester, UK) ionization source. The mass spectrometer was calibrated using an MS/MS spectrum of [Glu1]-Fibrinopeptide B human (Glu-Fib) solution (100 fmol.uL-1) delivered through the NanoLockSpray source reference sprayer. Multiplexed data-independent (DIA) scanning with additional specificity and selectivity for non-linear ‘T-wave’ ion mobility (HDMSE) experiments were performed using a Synapt G2-S HDMS mass spectrometer (Waters, Manchester, UK).

Following the identification of proteins, the quantitative data were packaged using dedicated algorithms [27] and searching against a database with default parameters to account for ions [28]. The databases used were reversed on-the-fly during the database queries and appended to the original database to assess the false positive rate during identification. For proper spectra
<table><caption>**Table 1** List of proteins identified in 1002_ovis control and recovered by 2D-PAGE-MS/MS</caption><thead><tr><th>Spot</th><th>Description</th><th>Accession</th><th>MW(kDa)/p.I</th><th>Peptides Number</th><th>Mascot Score</th><th>Molecular function</th></tr></thead><tbody><tr><td>5, 6, 7</td><td>Hypothetical protein</td><td>ADL20032</td><td>24.30/9.24</td><td>2</td><td>189</td><td>Unknown function</td></tr><tr><td>11,29</td><td>Trypsin-like serine protease</td><td>ADL20653</td><td>25.72/6.49</td><td>2</td><td>96</td><td>Serine-type endopeptidase activity</td></tr><tr><td>15</td><td>Hypothetical protein</td><td>ADL21714</td><td>42.04/5.22</td><td>4</td><td>159</td><td>Catalytic activity</td></tr><tr><td>20,34</td><td>Corynomycolyl transferase</td><td>ADL21610</td><td>41.80/7.05</td><td>2</td><td>58</td><td>Transferase activity</td></tr><tr><td>16</td><td>Cytochrome c oxidase sub II</td><td>ADL21302</td><td>40.33/6.03</td><td>2</td><td>96</td><td>Cytochrome-c oxidase activity</td></tr><tr><td>21</td><td>Hypothetical protein</td><td>ADL21914</td><td>12.30/5.04</td><td>2</td><td>53</td><td>Unknown function</td></tr><tr><td>12</td><td>Hypothetical protein</td><td>ADL19922</td><td>19.86/4.30</td><td>2</td><td>145</td><td>Calcium ion binding</td></tr><tr><td>8</td><td>Hypothetical protein</td><td>ADL09626</td><td>24.30/9.24</td><td>3</td><td>228</td><td>Unknown</td></tr><tr><td>27</td><td>Hypothetical protein</td><td>ADL20508</td><td>31.62/9.52</td><td>2</td><td>66</td><td>Unknown</td></tr><tr><td>22</td><td>Phospholipase D</td><td>ADL19935</td><td>34.09/8.91</td><td>4</td><td>286</td><td>Sphingomyelin phosphodiesterase D activity</td></tr><tr><td>3</td><td>Enolase</td><td>ADL20605</td><td>45.17/4.68</td><td>3</td><td>271</td><td>Phosphopyruvate hydratase activity</td></tr><tr><td>17</td><td>Trehalose corynomycolyl transferase B</td><td>ADL21814</td><td>36.67/6.90</td><td>5</td><td>245</td><td>Transferase activity, transferring acyl groups other than</td></tr><tr><td>24</td><td>Hypothetical protein</td><td>ADL21714</td><td>40.90/5.05</td><td>3</td><td>190</td><td>Catalytic activity</td></tr></tbody></table>

*Staphylococcus aureus* and *Listeria monocytogenes* [36, 37] were found induced in Rc supernatant.

Discussion

To investigate the protein factors that could influence the adaptive processes of *C. pseudotuberculosis* biovar *ovis* during the infection process, we combined a unique bacterial passage experiment in mice with proteomic analyses of 1002_ovis culture supernatants, collected before and after passage. In the first analysis, we observed that strain 1002_ovis (isolated from caprine) exhibited a low virulence potential, which is consistent with

previous reports indicating the low virulence potential of this strain [38, 39]. Although a recent in silico analysis of the 1002_ovis genome predicted various genes involved in virulence [40], studies examining the exoproteome of this strain under laboratory growth conditions failed to detect many of these virulence proteins (e.g., PLD exotoxin or proteins involved in the pathway of cell invasion, detoxification) [8–10].

One explanation for this relies on the fact that after being first isolated, strains 1002_ovis have been maintained, in vitro, under laboratory conditions with extensive passages on the culture medium, which may alter the gene

<figure><img src="image_3.png" /><figcaption>**Fig. 3** Volcano Plot show Log(2) Fold Change of the differentially expressed proteins detected by label-free proteomics between the recovered and control condition. Green: Up-regulated proteins; Grey: unchanged proteins; Red: Down-regulated proteins</figcaption></figure>
<table><caption>**Table 2** Proteins differentially produced among the recovered and control condition</caption><thead><tr><th>Accession</th><th>Description</th><th>Score</th><th>Fold Change_Log<sup>a</sup>(2)</th><th>SecretomeP</th></tr></thead><tbody><tr><td colspan="5"><strong>Transport</strong></td></tr><tr><td>D9Q5H9_CORP1</td><td>Periplasmic binding protein Lacl</td><td>5601,78</td><td>3,26</td><td>0.612642</td></tr><tr><td>D9Q6G4_CORP1</td><td>Oligopeptide binding protein oppA<sup>b</sup></td><td>4120,1</td><td>3,00</td><td>0.892226</td></tr><tr><td>D9Q4T5_CORP1</td><td>ABC transporter domain containing ATP</td><td>1264,05</td><td>2,57</td><td>0.084974</td></tr><tr><td>D9Q7K5_CORP1</td><td>Oligopeptide binding protein oppA<sup>b</sup></td><td>33697,17</td><td>2,11</td><td>0.873687</td></tr><tr><td>D9Q5B8_CORP1</td><td>Oligopeptide binding protein oppA<sup>b</sup></td><td>852,88</td><td>1,88</td><td>0.849217</td></tr><tr><td>D9Q6C3_CORP1</td><td>ABC type metal ion transport system permease</td><td>650,43</td><td>1,59</td><td>0.078043</td></tr><tr><td>D9Q796_CORP1</td><td>Glutamate binding protein GluB</td><td>6254,68</td><td>-1,46</td><td>0.840325</td></tr><tr><td>D9Q7W9_CORP1</td><td>Iron(3+)-hydroxamate-binding protein fhuD</td><td>2774,62</td><td>-1,62</td><td>0.824030</td></tr><tr><td colspan="5"><strong>Cell division</strong></td></tr><tr><td>D9Q7G1_CORP1</td><td>Septum formation initiator protein</td><td>2071,46</td><td>1,38</td><td>0.551153</td></tr><tr><td colspan="5"><strong>Cell adhesion</strong></td></tr><tr><td>D9Q5H7_CORP1</td><td>Hypothetical protein</td><td>115906,3</td><td>1,51</td><td>0.840443</td></tr><tr><td colspan="5"><strong>DNA synthesis and repair</strong></td></tr><tr><td>D9Q7J1_CORP1</td><td>GTP binding protein YchF</td><td>3487,98</td><td>2,68</td><td>0.042575</td></tr><tr><td>D9Q5F7_CORP1</td><td>Chromosome partitioning protein ParB<sup>b</sup></td><td>2467,24</td><td>2,44</td><td>0.052395</td></tr><tr><td>D9Q5G6_CORP1</td><td>DNA polymerase III subunit beta</td><td>1907,74</td><td>1,80</td><td>0.071008</td></tr><tr><td>D9Q5V6_CORP1</td><td>Nucleoid associated protein<sup>c</sup></td><td>68097,59</td><td>1,59</td><td>0.070074</td></tr><tr><td colspan="5"><strong>Transcription</strong></td></tr><tr><td>D9Q6J8_CORP1</td><td>DNA directed RNA polymerase subunit</td><td>29671,46</td><td>1,38</td><td>0.094910</td></tr><tr><td>D9Q748_CORP1</td><td>tRNA rRNA methyltransferase</td><td>2467,24</td><td>1,27</td><td>0.060356</td></tr><tr><td>D9Q8L3_CORP1</td><td>DNA directed RNA polymerase subunit omega</td><td>3784,13</td><td>-1,21</td><td>0.700214</td></tr><tr><td>D9Q6D1_CORP1</td><td>DNA directed RNA polymerase subunit beta</td><td>2611,89</td><td>-1,27</td><td>0.067182</td></tr><tr><td>D9Q8A5_CORP1</td><td>RNA polymerase-binding protein RbpA</td><td>10787,51</td><td>-1,75</td><td>0.103548</td></tr><tr><td colspan="5"><strong>Translation</strong></td></tr><tr><td>D9Q584_CORP1</td><td>30S ribosomal protein S6</td><td>20750,74</td><td>4,82</td><td>0.047667</td></tr><tr><td>D9Q6E4_CORP1</td><td>Elongation factor G<sup>b</sup></td><td>16882,71</td><td>3,25</td><td>0.082321</td></tr><tr><td>D9Q5I3_CORP1</td><td>Peptidyl prolyl cis trans isomerase<sup>b</sup></td><td>61648,39</td><td>2,91</td><td>0.142641</td></tr><tr><td>D9Q835_CORP1</td><td>Phenylalanine tRNA ligase beta subunit</td><td>1269,7</td><td>2,74</td><td>0.064869</td></tr><tr><td>D9Q6L0_CORP1</td><td>50S ribosomal protein L13</td><td>5689,37</td><td>2,64</td><td>0.101816</td></tr><tr><td>D9Q6H2_CORP1</td><td>50S ribosomal protein L5<sup>b</sup></td><td>3269,32</td><td>2,12</td><td>0.076250</td></tr><tr><td>D9Q918_CORP1</td><td>Proline tRNA ligase<sup>b</sup></td><td>932,79</td><td>2,12</td><td>0.072151</td></tr><tr><td>D9Q6C0_CORP1</td><td>50S ribosomal protein L10<sup>b</sup></td><td>27143,51</td><td>1,86</td><td>0.031374</td></tr><tr><td>D9Q6F6_CORP1</td><td>50S ribosomal protein L23<sup>b</sup></td><td>6947,79</td><td>1,85</td><td>0.060878</td></tr><tr><td>D9Q6H1_CORP1</td><td>50S ribosomal protein L24</td><td>27887,33</td><td>1,75</td><td>0.078408</td></tr><tr><td>F9Y2W9_CORP1</td><td>Hypothetical protein</td><td>3152,39</td><td>1,75</td><td>0.591013</td></tr><tr><td>D9Q6H6_CORP1</td><td>30S ribosomal protein S8<sup>c,b</sup></td><td>4941,19</td><td>1,56</td><td>0.088407</td></tr><tr><td>D9Q6F3_CORP1</td><td>30S ribosomal protein S10<sup>b</sup></td><td>25117,55</td><td>1,54</td><td>0.048124</td></tr><tr><td>D9Q6G2_CORP1</td><td>50S ribosomal protein L29</td><td>2467,24</td><td>1,44</td><td>0.050948</td></tr><tr><td>D9Q401_CORP1</td><td>50S ribosomal protein L27<sup>b</sup></td><td>2467,24</td><td>1,38</td><td>0.081399</td></tr><tr><td>D9Q7E8_CORP1</td><td>50S ribosomal protein L25</td><td>1358,05</td><td>-1,28</td><td>0.037225</td></tr><tr><td>D9Q6H8_CORP1</td><td>50S ribosomal protein L18</td><td>8920,94</td><td>-1,31</td><td>0.049024</td></tr><tr><td>D9Q7S4_CORP1</td><td>Homoserine dehydrogenase</td><td>698,17</td><td>-1,40</td><td>0.035138</td></tr></tbody></table>
<table><caption>**Table 2** Proteins differentially produced among the recovered and control condition (Continued)</caption><tbody><tr><td>D9Q6B7_CORP1</td><td>50S ribosomal protein L1</td><td>10218.08</td><td>-1,63</td><td>0.633387</td></tr><tr><td>D9Q4T4_CORP1</td><td>ATP dependent chaperone protein ClpB</td><td>1883,16</td><td>-1,80</td><td>0.045308</td></tr><tr><td>D9Q8N9_CORP1</td><td>Aspartate tRNA ligase</td><td>1004,33</td><td>-2,18</td><td>0.092415</td></tr><tr><td>D9Q7S2_CORP1</td><td>Arginine tRNA ligase</td><td>2679,11</td><td>-2,44</td><td>0.051908</td></tr><tr><td colspan="5">Pathogenesis</td></tr><tr><td>D9Q8M7_CORP1</td><td>Metallopeptidase family M24</td><td>3213,83</td><td>5,55</td><td>0.050024</td></tr><tr><td>D9Q608_CORP1</td><td>Penicillin binding protein transpeptidase<sup>b</sup></td><td>1215,32</td><td>3,68</td><td>0.859830</td></tr><tr><td>D9Q827_CORP1</td><td>Metallo beta lactamase superfamily protein<sup>c</sup></td><td>629,38</td><td>2,64</td><td>0.144158</td></tr><tr><td>D9Q721_CORP1</td><td>Hypothetical protein<sup>c</sup></td><td>112025</td><td>2,24</td><td>0.260801</td></tr><tr><td>D9Q7K8_CORP1</td><td>Trypsin like serine protease</td><td>35041,27</td><td>1,96</td><td>0.648370</td></tr><tr><td>D9Q416_CORP1</td><td>ATP dependent Clp protease proteolytic<sup>b</sup></td><td>2467,24</td><td>1,77</td><td>0.087255</td></tr><tr><td>D9Q639_CORP1</td><td>Secreted hydrolase<sup>b</sup></td><td>22798,13</td><td>1,75</td><td>0.072385</td></tr><tr><td>D9Q588_CORP1</td><td>Penicillin binding protein<sup>b</sup></td><td>9951,61</td><td>1,26</td><td>0.916125</td></tr><tr><td colspan="5">Energy metabolism</td></tr><tr><td>D9Q787_CORP1</td><td>Glucose-6-phosphate isomerase</td><td>1025,89</td><td>4,50</td><td>0.058841</td></tr><tr><td>D9Q7G0_CORP1</td><td>Enolase<sup>b</sup></td><td>53290,95</td><td>2,18</td><td>0.068928</td></tr><tr><td>D9Q651_CORP1</td><td>Succinate dehydrogenase flavoprotein</td><td>797,48</td><td>2,02</td><td>0.159059</td></tr><tr><td>D9Q4P2_CORP1</td><td>Acetate kinase<sup>b</sup></td><td>10828,79</td><td>1,96</td><td>0.063340</td></tr><tr><td>D9Q8G5_CORP1</td><td>Aconitate hydratase<sup>b</sup></td><td>4250,81</td><td>1,85</td><td>0.217637</td></tr><tr><td>D9Q4Z7_CORP1</td><td>Phosphoenolpyruvate carboxykinase GTP<sup>b</sup></td><td>8764,35</td><td>1,66</td><td>0.147167</td></tr><tr><td>D9Q7X0_CORP1</td><td>6 phosphofructokinase</td><td>1806,65</td><td>1,60</td><td>0.052885</td></tr><tr><td>D9Q648_CORP1</td><td>Dihydrolipoyl dehydrogenase</td><td>4110,08</td><td>1,57</td><td>0.047180</td></tr><tr><td>D9Q7T8_CORP1</td><td>ATP synthase subunit alpha</td><td>2467,24</td><td>1,49</td><td>0.070875</td></tr><tr><td>D9Q752_CORP1</td><td>Citrate synthase</td><td>6299,21</td><td>-1,21</td><td>0.116042</td></tr><tr><td>D9Q895_CORP1</td><td>6-Phosphogluconate dehydrogenase</td><td>4246,26</td><td>-1,89</td><td>0.050906</td></tr><tr><td colspan="5">Lipid metabolism</td></tr><tr><td>D9Q520_CORP1</td><td>Glycerophosphoryl diester phosphodieste<sup>c</sup></td><td>2494,25</td><td>4,03</td><td>0.802154</td></tr><tr><td>D9Q718_CORP1</td><td>Methylmalonyl CoA carboxyltransferase 1<sup>b</sup></td><td>2467,24</td><td>2,16</td><td>0.049504</td></tr><tr><td colspan="5">Amino acid metabolism</td></tr><tr><td>D9Q5X8_CORP1</td><td>Aspartokinase<sup>b</sup></td><td>1944,81</td><td>2,86</td><td>0.043575</td></tr><tr><td>D9Q4C2_CORP1</td><td>Succinyl CoA Coenzyme A transferase</td><td>10894,63</td><td>1,63</td><td>0.061344</td></tr><tr><td>D9Q3L8_CORP1</td><td>Glutamine synthetase</td><td>320,71</td><td>-1,23</td><td>0.263700</td></tr><tr><td>D9Q8H7_CORP1</td><td>Cysteine desulfurase</td><td>1689,36</td><td>-1,70</td><td>0.067087</td></tr><tr><td colspan="5">Stress response</td></tr><tr><td>D9Q929_CORP1</td><td>Mycothione glutathione reductase</td><td>490,36</td><td>2,67</td><td>0.085017</td></tr><tr><td>D9Q5T5_CORP1</td><td>Glyoxalase Bleomycin resistance protein<sup>c</sup></td><td>8420,32</td><td>2,21</td><td>0.226764</td></tr><tr><td>D9Q424_CORP1</td><td>DSBA oxidoreductase</td><td>12179,8</td><td>2,09</td><td>0.061566</td></tr><tr><td>D9Q566_CORP1</td><td>Universal stress protein A<sup>b</sup></td><td>2498,69</td><td>1,70</td><td>0.034684</td></tr><tr><td>D9Q4P4_CORP1</td><td>Ferredoxin ferredoxin NADP reductase<sup>b</sup></td><td>1086,71</td><td>1,69</td><td>0.083585</td></tr><tr><td>D9Q824_CORP1</td><td>Stress related protein<sup>b</sup></td><td>2467,24</td><td>1,54</td><td>0.035291</td></tr><tr><td>D9Q692_CORP1</td><td>Thiol disulfide isomerase thioredoxin</td><td>3721,88</td><td>-2,25</td><td>0.438415</td></tr><tr><td colspan="5">Metabolism of nucleotides and nucleic acids</td></tr><tr><td>D9Q4Y6_CORP1</td><td>Deoxycytidine triphosphate deaminase</td><td>887,26</td><td>2,39</td><td>0.216897</td></tr></tbody></table>
<table><caption>**Table 2** Proteins differentially produced among the recovered and control condition (Continued)</caption><tbody><tr><td>D9Q6J1_CORP1</td><td>Adenylate kinase</td><td>15629,86</td><td>2,21</td><td>0.059568</td></tr><tr><td>D9Q8L4_CORP1</td><td>Guanylate kinase</td><td>2467,24</td><td>1,34</td><td>0.050095</td></tr><tr><td>D9Q6T2_CORP1</td><td>Ribokinase</td><td>890,09</td><td>-1,23</td><td>0.032324</td></tr><tr><td>D9Q4E9_CORP1</td><td>Adenylosuccinate lyase</td><td>1441,99</td><td>-1,54</td><td>0.035597</td></tr><tr><td>D9Q6P0_CORP1</td><td>D methionine binding lipoprotein metQ</td><td>11519,67</td><td>-1,93</td><td>0.817217</td></tr><tr><td colspan="5">Carbohydrate metabolism</td></tr><tr><td>D9Q8V2_CORP1</td><td>UDP glucose 4 epimerase<sup>b</sup></td><td>2001,76</td><td>3,13</td><td>0.094403</td></tr><tr><td>D9Q6V6_CORP1</td><td>Phosphomannomutase ManB</td><td>1730,63</td><td>2,05</td><td>0.053146</td></tr><tr><td>D9Q659_CORP1</td><td>Formate acetyltransferase</td><td>5456,95</td><td>1,54</td><td>0.539548</td></tr><tr><td>D9Q423_CORP1</td><td>Ribose-5-phosphate isomerase B</td><td>2467,24</td><td>1,38</td><td>0.064467</td></tr><tr><td>D9Q6V1_CORP1</td><td>Mannose-1-phosphate guanylyltransferase</td><td>1612,45</td><td>-1,21</td><td>0.068085</td></tr><tr><td colspan="5">Nitrogen metabolism</td></tr><tr><td>D9Q4Q8_CORP1</td><td>Cytochrome c nitrate reductase small</td><td>1118,33</td><td>2,68</td><td>0.901856</td></tr><tr><td colspan="5">Unknown function</td></tr><tr><td>D9Q6T0_CORP1</td><td>Hypothetical protein</td><td>2277,6</td><td>3,62</td><td>0.050552</td></tr><tr><td>D9Q4R2_CORP1</td><td>Hypothetical protein</td><td>442,07</td><td>3,35</td><td>0.866986</td></tr><tr><td>D9Q6N1_CORP1</td><td>Hypothetical protein</td><td>561,84</td><td>3,02</td><td>0.062141</td></tr><tr><td>D9Q8Q4_CORP1</td><td>Hypothetical protein<sup>c</sup></td><td>72711,5</td><td>2,96</td><td>0.974016</td></tr><tr><td>D9Q832_CORP1</td><td>Hypothetical protein</td><td>1774,59</td><td>2,90</td><td>0.752478</td></tr><tr><td>D9Q3S8_CORP1</td><td>Hypothetical protein<sup>d</sup></td><td>837,6</td><td>2,78</td><td>0.231421</td></tr><tr><td>D9Q7M9_CORP1</td><td>Hypothetical protein</td><td>3246,28</td><td>2,60</td><td>0.147602</td></tr><tr><td>D9Q7I6_CORP1</td><td>Hypothetical protein</td><td>3751,96</td><td>2,42</td><td>0.707595</td></tr><tr><td>D9Q739_CORP1</td><td>Hypothetical protein</td><td>2845,77</td><td>2,28</td><td>0.836229</td></tr><tr><td>D9Q4C5_CORP1</td><td>Hypothetical protein</td><td>1339,3</td><td>1,83</td><td>0.023133</td></tr><tr><td>D9Q5C3_CORP1</td><td>Hypothetical protein</td><td>111234,6</td><td>1,49</td><td>0.946918</td></tr><tr><td>D9Q700_CORP1</td><td>Hypothetical protein</td><td>2467,24</td><td>1,49</td><td>0.072810</td></tr><tr><td>D9Q657_CORP1</td><td>Hypothetical protein</td><td>1172,66</td><td>1,41</td><td>0.830926</td></tr><tr><td>D9Q6F2_CORP1</td><td>Hypothetical protein</td><td>2467,24</td><td>1,34</td><td>0.061860</td></tr><tr><td>D9Q7X5_CORP1</td><td>Hypothetical protein</td><td>38716,45</td><td>-1,21</td><td>0.825761</td></tr><tr><td>D9Q4T9_CORP1</td><td>Hypothetical protein</td><td>553,76</td><td>-1,28</td><td>0.934591</td></tr><tr><td>D9Q6R6_CORP1</td><td>Hypothetical protein</td><td>1457,62</td><td>-1,40</td><td>0.206908</td></tr><tr><td>D9Q890_CORP1</td><td>Hypothetical protein</td><td>1948,52</td><td>-1,51</td><td>0.847549</td></tr><tr><td>D9Q6M6_CORP1</td><td>Hypothetical protein<sup>c</sup></td><td>1935,68</td><td>-1,90</td><td>0.823541</td></tr><tr><td colspan="5">Others</td></tr><tr><td>D9Q6I3_CORP1</td><td>Maltotriose binding protein</td><td>5210,9</td><td>5,22</td><td>0.864851</td></tr><tr><td>D9Q4A3_CORP1</td><td>DsbG protein</td><td>3101,13</td><td>2,06</td><td>0.814366</td></tr><tr><td>D9Q6N9_CORP1</td><td>D methionine binding lipoprotein metQ</td><td>2665,58</td><td>1,79</td><td>0.764416</td></tr><tr><td>D9Q732_CORP1</td><td>Carbonic anhydrase<sup>b</sup></td><td>689,15</td><td>1,66</td><td>0.130559</td></tr><tr><td>D9Q6W6_CORP1</td><td>Lipoprotein LpqB</td><td>1484,31</td><td>1,63</td><td>0.670057</td></tr><tr><td>D9Q556_CORP1</td><td>LSR2 like protein</td><td>2714,21</td><td>1,49</td><td>0.096802</td></tr><tr><td>D9Q5Q0_CORP1</td><td>UPF0145 protein</td><td>2467,24</td><td>1,37</td><td>0.025009</td></tr><tr><td>D9Q7W0_CORP1</td><td>Hypothetical protein</td><td>2467,24</td><td>1,26</td><td>0.039678</td></tr><tr><td>D9Q701_CORP1</td><td>UPF0182 protein</td><td>1682,98</td><td>1,26</td><td>0.869411</td></tr><tr><td>D9Q8A3_CORP1</td><td>Protein ycel<sup>b</sup></td><td>16885,01</td><td>1,21</td><td>0.901679</td></tr></tbody></table>
<table><caption>**Table 2** Proteins differentially produced among the recovered and control condition (Continued)</caption><tr><td>D9Q5X4_CORP1</td><td>Serine aspartate repeat containing protein</td><td>528,36</td><td>-1,82</td><td>0.892317</td></tr><tr><td>D9Q826_CORP1</td><td>DoxX family protein</td><td>697,26</td><td>-2,08</td><td>0.614317</td></tr><tr><td>D9Q7W3_CORP1</td><td>Mycothiol acetyltransferase</td><td>947,33</td><td>-2,11</td><td>0.214833</td></tr><tr><td>D9Q407_CORP1</td><td>Ornithine cyclodeaminase</td><td>2566,18</td><td>-2,58</td><td>0.048247</td></tr></table>

<table><caption>**Table 3** List of proteins identified in the exclusive proteome of recovered-condition</caption><thead><tr><td>Accession</td><td>Description</td><td>Score</td><td>Biological process</td><td>SecretomeP</td></tr></thead><tbody><tr><td>D9Q869_CORP1</td><td>Esterase<sup>a</sup></td><td>251.44</td><td>Others</td><td>0.862935</td></tr><tr><td>D9Q575_CORP1</td><td>Cation transport protein</td><td>1961.29</td><td>Transport</td><td>0.062276</td></tr><tr><td>D9Q5N5_CORP1</td><td>Uncharacterized iron regulated membrane<sup>a</sup></td><td>46.77</td><td>Transport</td><td>0.855681</td></tr><tr><td>D9Q3T9_CORP1</td><td>Pyridoxamine kinase</td><td>216.2</td><td>Cofactor metabolism</td><td>0.083313</td></tr><tr><td>D9Q751_CORP1</td><td>Phosphoserine aminotransferase</td><td>639.64</td><td>Amino acid metabolism</td><td>0.151778</td></tr><tr><td>D9Q537_CORP1</td><td>LytR family transcriptional regulator<sup>a</sup></td><td>375.8</td><td>Transcription</td><td>0.766483</td></tr><tr><td>D9Q7F2_CORP1</td><td>Multicopper oxidase</td><td>74.63</td><td>Stress response</td><td>0.278840</td></tr><tr><td>D9Q525_CORP1</td><td>ABC transporter substrate binding lipoprotein</td><td>283.38</td><td>Transport</td><td>0.452814</td></tr><tr><td>D9Q6P2_CORP1</td><td>Manganese ABC transporter substrate binding<sup>a</sup></td><td>236.6</td><td>Transport</td><td>0.774461</td></tr><tr><td>D9Q4C8_CORP1</td><td>Phosphate ABC transporter phosphate binding<sup>a</sup></td><td>125.4</td><td>Transport</td><td>0.840195</td></tr><tr><td>D9Q4L0_CORP1</td><td>D alanyl D alanine carboxypeptidase OS</td><td>426.74</td><td>Others</td><td>0.232261</td></tr><tr><td>D9Q4T7_CORP1</td><td>Hyphotetical protein</td><td>157.52</td><td>Unknow function</td><td>0.349026</td></tr><tr><td>D9Q5A9_CORP1</td><td>Hyphotetical protein</td><td>218.02</td><td>Unknow function</td><td>0.907333</td></tr><tr><td>D9Q476_CORP1</td><td>Hyphotetical protein</td><td>510.32</td><td>Unknow function</td><td>0.066368</td></tr><tr><td>D9Q5B3_CORP1</td><td>Glucosamine-6-phosphate deaminase<sup>b</sup></td><td>524,55</td><td>Carbohydrate metabolism</td><td>0.079507</td></tr><tr><td>D9Q474_CORP1</td><td>Glutamate racemase</td><td>343,98</td><td>Cell wall organization</td><td>0.040278</td></tr><tr><td>D9Q7N5_CORP1</td><td>O-methyltransferase</td><td>619,11</td><td>DNA process</td><td>0.032455</td></tr><tr><td>D9Q5N3_CORP1</td><td>Gamma type carbonic anhydratase</td><td>577,75</td><td>Others</td><td>0.035357</td></tr><tr><td>D9Q4X0_CORP1</td><td>Urease accessory protein UreD</td><td>333,12</td><td>Others</td><td>0.055896</td></tr><tr><td>D9Q5J0_CORP1</td><td>Phospholipase D<sup>b</sup></td><td>40,25</td><td>Pathogenesis</td><td>0.409585</td></tr><tr><td>D9Q8S8_CORP1</td><td>Copper resistance protein CopC</td><td>4315,26</td><td>Stress response</td><td>0.964015</td></tr><tr><td>D9Q493_CORP1</td><td>Glutaredoxin like protein nrdH</td><td>725,98</td><td>Stress response</td><td>0.033036</td></tr><tr><td>D9Q6Y6_CORP1</td><td>ATP dependent RNA helicase rhLE</td><td>1438,25</td><td>Transcription</td><td>0.060627</td></tr><tr><td>D9Q4M0_CORP1</td><td>Cell wall channel</td><td>4008,59</td><td>Transport</td><td>0.025882</td></tr><tr><td>D9Q4V1_CORP1</td><td>CP40</td><td>558,79</td><td>Pathogenesis</td><td>0.926013</td></tr><tr><td>D9Q6V9_CORP1</td><td>Hyphotetical protein</td><td>1278,45</td><td>Unknow function</td><td>0.953803</td></tr><tr><td>D9Q6A8_CORP1</td><td>Hyphotetical protein</td><td>326,47</td><td>Unknow function</td><td>0.918886</td></tr><tr><td>D9Q485_CORP1</td><td>Hyphotetical protein</td><td>2795,11</td><td>Unknow function</td><td>0.890081</td></tr><tr><td>D9Q4N2_CORP1</td><td>Hypothetical protein<sup>a</sup></td><td>708,75</td><td>Unknow function</td><td>0.857050</td></tr><tr><td>D9Q559_CORP1</td><td>Hypothetical protein<sup>a</sup></td><td>475,62</td><td>Unknow function</td><td>0.472378</td></tr><tr><td>D9Q4L8_CORP1</td><td>Hyphotetical protein</td><td>5324,08</td><td>Unknow function</td><td>0.038893</td></tr><tr><td>D9Q4T0_CORP1</td><td>Hyphotetical protein</td><td>732,37</td><td>Unknow function</td><td>0.037132</td></tr></tbody></table>

<footnote>ªFold change - Ratio values to: 1002Rc:11002Ct_Log(2)Ratio ≥ 1.2 proteins with *p* < 0.05</footnote>

<footnote>ᵇIdentified in an isolated of *C. pseudotuberculosis* from ovine lymph nodes [Rees et al. [12]</footnote>

<footnote>ᶜInduced in 1002_ovis during to stress nitrosative [Pacheco et al. [57], Silva et al. [58]</footnote>

<footnote>ᵈPredicted LPXTG cell wall-anchoring motif</footnote>

<footnote>ªInduced in 1002_ovis during to stress nitrosative [Pacheco et al. [57], Silva et al. [58]</footnote>

<footnote>ᵇIdentified in an isolated of *C. pseudotuberculosis* from ovine lymph nodes [Rees et al. [12]</footnote>
<figure><img src="image_4.png" /><figcaption>**Fig. 4** Biological processes differentially regulated in 1002_ovis after passage in mice. Analysis of the differentially expressed proteins grouped into biological processes for strain 1002_ovis after passage in mice</figcaption></figure>

expression profile of the strain, especially for effectors related to bacterial virulence. This phenomenon has also been reported in other pathogens such as *Mycobacterium bovis*, *Helicobacter pylori*, *S. aureus*, and *L. monocytogenes*. In vitro passages of these bacteria on culture medium altered both bacterial physiology and virulence profile [41–44]. However, we showed that the bacterial passage process in a murine model changed the virulence potential of strain 1002_ovis. Previous reports on experimental serial passages showed that pathogens such as *H. pylori*, *Escherichia coli*, *Xenorhabdus nematophila*, *Arcobacter butzleri*, and *Salmonella enterica* also exhibited altered virulence profiles after in vivo passage in a host, which helped identifying factors that contribute to infectious process [14–19]. Thus, as observed in these pathogens, the recovered condition also showed increased capacity to persist into host, when compared with control condition. The altered physiology and virulence status observed in 1002_ovis is supported by our proteomic analyses, where several proteins involved in processes favoring infection and host adaptation were differentially expressed after passage in mice.

Although our study focused on the *C. pseudotuberculosis* extracellular proteins, cytoplasmic proteins were also detected in the proteomic analyses. The presence of cytoplasmic proteins in the extracellular fraction is reported in several other proteomic studies [8–10, 12, 45]. It may be partially due to cell lysis and thus, be considered artifacts. However, cytoplasmic proteins in the culture supernatant may act as *moonlighting* proteins and be

exported via a non-classical secretion pathway [30, 46]. The *moonlighting* proteins are described both Gram-positive and Gram-negative bacteria, and can be detected in different subcellular locations (cytoplasm, membrane, cell surface, and extracellular environment) and exhibit distinct functional behavior depending on the host cell type [46, 47]. Interestingly, some proteins, such as Chromosome partitioning protein ParB, Phosphoenolpyruvate carboxykinase GTP, Methylmalonyl CoA carboxyltransferase 12S subunit, Acetate kinase, and Enolase, induced in the Rc supernatants were identified only in the membrane shaving of *C. pseudotuberculosis* harvested directly from ovine lymph nodes [12].

The passage process in mice was also able to induce other proteins identified in Rc supernatants, and which contribute to the adhesion process. Proteins with an LPTXG domain, which characterizes the cell-wall anchored proteins, were identified and included monomers of membrane pilus. This latter class of proteins is described in pathogenic *Corynebacterium* species and may contribute especially in the process of cellular adhesion [48]. In *Campylobacter jejuni*, serial passages in mice induce the expression of invasiveness and increase the capacity of cell invasion [13]. Components of the Opp system were induced by the passage process, too. The Opp system facilitates the uptake of extracellular peptides, which are further used as carbon and nitrogen sources for bacterial nutrition [49]. Proteins that comprise the Opp system also were induced in a field isolated of *C. pseudotuberculosis* biovar *ovis*, when
compared with the strain C231_ovis a laboratory reference strain [12, 50]. In the pathogen *Mycobacterium avium* the OppA gene was highly expressed during the infection in a mouse model [51]. We have identified known secreted virulence factors as CP40 serine protease, which previously shown to be necessary for *C. pseudotuberculosis* virulence potential and to induce an immune response [52, 53].

An important factor that precedes the chronic stage of infection by *C. pseudotuberculosis* is the capacity of this pathogen to disseminate within the host, which consequently favors the establishment of the disease [3]. In *C. pseudotuberculosis*, this process is mediated by the action of PLD exotoxin, a major virulence factor of this pathogen [54, 55] that catalyzes the dissociation of sphingomyelin and increases vascular permeability, which contributes to the dissemination process of *C. pseudotuberculosis* in the host. Here, PLD was only detected in the proteome of the Rc condition. This result is noteworthy because, a previous proteomic study performed by our research group, PLD was not identified in the extracellular proteome of 1002_ovis [8–10]. McKean et al. [5] showed that *pld* expression is expressed by different environmental factors, thus during the infection and recuperation process 1002_ovis was exposed to different environmental and stimulus, which may have affected the *pld* expression. A study showed that a *pld* mutant strain is indeed unable to disseminate and yields reduced virulence [55]. Here, we observed the presence of caseous lesions in different organs only at the end of experimental infection, only in the group of mice infected with the Rc condition. Altogether, the observations suggest that the expression of PLD can be modified by the passage in the host and can thus change the virulence potential of 1002_ovis.

Another attribute of PLD is its capacity to alter the viability of macrophage cells during the infection [5]. However, before promoting macrophages lysis, *C. pseudotuberculosis* has to be able to resist the hostile environment inside macrophages mainly against reactive oxygen species (ROS) and reactive nitrogen species (RNS). Thus, the induction of proteins involved in detoxification processes in Rc could be contributed for its resistance against ROS and RNS. The inductions of proteins related to oxidative stress also were observed in *Shigella flexneri*, after recuperation process in an in vivo infection model. We detected the mycothione glutathione reductase, a component of the mycothiol system, which is present in *Mycobacterium and Rhodococcus* genera. This system is used as an alternative mechanism of disulphide reduction and contributes to the cytosolic redox homeostasis and the resistance to ROS [35]. Glutaredoxin-like protein, NrdH, which plays an important role in the resistance to ROS, and is present in *C. glutamicum* [34] and *M. tuberculosis* [56] was also detected.

On the other hand, some proteins like dihydroxybiphenyl dioxygenase, Metallo beta lactamase superfamily protein, Formamidopyrimidine DNA glycosylase, MerR family transcriptional regulator, which were induced by 1002_ovis during the exposition to nitric oxide [57, 58] were also found induced in this study in the recovered condition. These proteins are related to different processes of resistance to nitrosative stress, DNA repair, antibiotic resistance, and transcription, these results show a set of proteins involved in the adaptation process of 1002_ovis to nitric oxide, which could contribute to the pathogenic process of this pathogen. Another type of defense of the host immune system against bacterial infection is the utilization of copper [59]. Here, CopC, a protein related to copper resistance, was detected in recovered 1002_ovis. In *M. tuberculosis*, proteins involved in copper resistance are essential to virulence [60, 61]. Thus, the association of this factor related to an antioxidant system with PLD could promote an effective pathway of defense against the action of the innate immune system and consequently contributes to virulence process of *C. pseudotuberculosis*.

## Conclusion

In conclusion, the virulence potential and proteomic profiles of strain 1002_ovis undergo dramatic changes after recovery from experimentally infected mice. The proteomic screening outlined, after the serial passage in murine model showed a set of proteins that were induced in the recovered condition. Into this group were detected known secreted virulence factors, as well as some proteins which could contribute in its virulence. Therefore, more study is necessary to show the true role of these proteins in the virulence of *C. pseudotuberculosis*. Altogether, our results demonstrate that in vitro passages alter the expression of *C. pseudotuberculosis* exoproteome leading to a reduced virulence and that a single passage in vivo, in a murine model, can induce significant changes in the *C. pseudotuberculosis* extracellular proteome, contributing to the increase in virulence of this pathogen.

## Additional files

**Additional file 1: Table S1.** Complete list of proteins differentially produced between the recovered and control condition of strain 1002_ovis. (XLSX 44 kb)

**Additional file 2: Table S2.** List of proteins identified in the exclusive proteome of control condition. (XLSX 12 kb)

**Additional file 3: Table S3.** Total list of peptide and proteins identified by LC-MS<sup>E</sup>. (XLSX 3 mb)

**Additional file 4: Table S4.** Proteins identified in the recovered condition detected in pathogenicity island. (XLSX 10 kb)

## Acknowledgements

The authors would like to thank to Pará State Genomics and Proteomics Network and Waters Corporation, Brazil.
## Funding

The work was supported by the Brazilian Federal Agency for the Support and Evaluation of Graduate Education (CAPES), Pará Research Foundation (FAPESPA), Minas Gerais Research Foundation (FAPEMIG) and the National Council for Scientific and Technological Development (CNPq). Yves Le Loir is the recipient of a PVE grant (71/2013) from Programa Ciências sem Fronteiras.

## Availability of data and materials

The datasets supporting the results of this article were then concatenated into a *.xlxs file at peptide and protein level to fulfill the requirements and is available at supplemental material including sequence coverage and a number of identified peptides for each protein sequence identified. It also includes the native peptide information. In addition other data are included within the article.

## Authors' contributions

VA, WMS, and FAD designed the experiments. WMS and FAD performed in vivo experiments. WMS, TLPC, and NS performed microbiological analyses and sample preparation for proteomic analysis. GHMFS and WMS conducted the proteomic analysis. WMS and SCS performed bioinformatics analysis of the data. YLL, 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

All animals used in this study were provided by the Animal Care Facility of the Biological Sciences Institute from the Federal University of Minas Gerais and were handled by the guidelines of the UFMG Ethics Committee on Animal Testing (Permit Number: CETEA 103/2011).

## Author details

Received: 15 January 2016 Accepted: 4 January 2017

Published online: 22 March 2017

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