Profiling and Identification of Novel Immunogenic Proteins of Staphylococcus hyicus ZC-4 by Immunoproteomic Assay

Staphylococcus hyicus has caused great losses in the swine industry by inducing piglet exudative epidermitis (EE), sow mastitis, metritis, and other diseases and is a threat to human health. The pathogenesis of EE, sow mastitis, and metritis involves the interaction between the host and virulent protein factors of S. hyicus, however, the proteins that interact with the host, especially the host immune system, are unclear. In the present study, immunoproteomics was used to screen the immunogenic proteins of S. hyicus strain ZC-4. The cellular and secreted proteins of S. hyicus strain ZC-4 were obtained, separated by 2D gel electrophoresis, and further analyzed by western blot with S. hyicus strain ZC-4-infected swine serum. Finally, 28 specific immunogenic proteins including 15 cellular proteins and 13 secreted proteins, 26 of which were novel immunogenic proteins from S. hyicus, were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. To further verify their immunogenicity, two representative proteins (acetate kinase [cellular] and enolase [secreted]) were chosen for expression, and the resultant recombinant proteins could react with S. hyicus ZC-4-infected swine serum. In mice, both acetate kinase and enolase activated the immune response by increasing G-CSF and MCP-5 expression, and acetate kinase further activated the immune response by increasing IL-12 expression. Enolase can confer better protection against S.hycius than acetate kinase in mice. For the first time to our knowledge, our results provide detailed descriptions of the cellular and secreted proteins of S. hyicus strain ZC-4. These immunogenic proteins may contribute to investigation and elucidation of the pathogenesis of S. hyicus and provide new candidates for subunit vaccines in the future.


Introduction
S. hyicus is the major pathogen causing piglet exudative epidermitis (EE), sow mastitis, and metritis, among other diseases [1,2]. EE generally occurs as an acute infection in suckling and newly weaned piglets [3] and is characterized by greasy exudation, exfoliation, and vesicle

MALDI-TOF/TOF MS and bioinformatics analysis. 2-DE gels and their immunoblot
profiles were compared by PDQuest 2-D Advance (Bio-Rad). The immunoreactive spots were excised, and in-gel protein digestion was performed as described previously [18]. Tryptic peptides were solubilized in 0.5% trifluoroacetic acid and subjected to MALDI-TOF/TOF MS with a Bruker UltraReflexTM III MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Karlsruhe, Germany). Peptide mass fingerprints were analyzed and searched against the theoretical spectra of S. hyicus. Peptide mass fingerprinting (PMF) data were analyzed using MASCOT (Matrix Science, London, UK). MASCOT searches were used to determine the possibility of each peptide and used for the combined peptide scores. The extent of sequence coverage, number of matched peptides, and the score probability obtained from the PMF data were all used to identify proteins. Low-scoring proteins were either verified manually or rejected [20].
Plasmid construction, protein expression and purification. Two proteins, acetate kinase (ACK) and enolase (ENO), representing two categories of identified immunogenic proteins were chosen for prokaryotic expression. The gene fragments encoding ACK and ENO were amplified by PCR with designed primers, digested with restriction enzymes, and ligated into vector pET32a to obtain the resultant plasmids pET32a-ACK and pET32a-ENO. The constructed plasmids were transformed into E. coli strain BL21 cells, the cells were cultured at 37˚C, and protein expression was induced by adding 1 mM IPTG when the OD 600 value was 0.6-1.0. Six hours after induction, the cells were harvested, and the recombinant proteins were subjected to western Blot analysis as described above. ACK and ENO were purified with a commercial purification kit (CW Biotech, Beijing, China) according to instructions of the manufacturer, while, HIS was purified by gel electrophoresis (data not show).
Mouse experiments. To validate the immunogenicity of identified proteins, The BALB/c mice were injected at multiple sites intramuscularly and subcutaneously with the 200 μg purified proteins, blood samples were collected at 3 h and 24 h post injection [21,22], and cytokine concentrations were determined using the Ray Biotech mouse cytokine antibody array G2 (AAM-CYT-G2-4, Ray Biotech, Norcross, GA, USA).
Animal experiments were conducted in keeping with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People's Republic of China. The present animal study was approved by the Animal Experimental Ethics Committee of the Institute of Animal Health, Guangdong Academy of Agricultural Sciences (Approval number 2014-010).
Immune protection test. Experiments were performed on female BALB/c mice, 26 mice were randomly divided into four groups, A: ENO (n = 5); B: ACK (n = 5); C: HIS (n = 5); D: PBS (n = 6); E: Control (n = 5). Mice were injected with 80ug of purified in complete Freund's adjuvant, and then boosted twice, at 7 days intervals with 80ug in Freund's incomplete adjuvant. At 7 days after the final booter injection, the blood were collected from tail vein, and then the mice were challenged with 300uL 2.8×10 9 CFU/mL S.hycius via intramuscular and subcutaneous injection. Their percent of survival were monitored at 0, 6, 12, 20, 48, 60, and 72 h after challenge, the blood from dying mice infected by ZC-4 were collected, and incubated on blood agar plate for 20 h (HKM, GZ, China) to recovery S.hycius.
Animal experiments were conducted in keeping with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People's Republic of China. The present animal study was approved by the Animal Experimental Ethics Committee of the South China Agricultural University (Approval number 2016-013).
ELISA analysis. ELISA was performed to test the antibody level, 0.25 ug purified proteins including ACK, ENO and HIS, were coated in 96 well plates (JET, GZ, China). The plates were incubates for overnight at 4˚C, washed by PBST five times, and then blocked by 5% milk at 37˚C for 2 h, washed by PBST five times again. After that, 100 uL of 1:10 diluted mouse anti-ACK serum, mouse anti-ENO serum or mouse anti-His serum were added to 96-well plates and incubated at 37˚C for 30 min, washed by PBST five times, added 100 uL HRP-conjugated goat anti-swine IgG(H+L) as the secondary antibody, incubated at 37˚C for 30 min, washed by PBST five times, added 100uL TMB (Solarbio, Beijing, China), incubated at 25˚C for 10 min, added 50 uL 2M H 2 SO 4 to stop the reaction, the absorbance was measured at 450 nm in a microplate ELISA reader (Bio-Tek, Vermont, USA).

Statistical analysis
The significance of different groups was analyzed statistically with the Student's t-test. The data were expressed as the mean ± standard deviation (SD), p values < 0.05 were considered to be significant.

Identification of immunogenic proteins of S. hyicus ZC-4
To identify immunogenic proteins, samples of cellular and secreted proteins were subjected to immunoproteomics analysis, and the proteins that reacted with swine immune sera against S. hyicus ZC-4 were selected as the immunogenic proteins. We identified 24 spots from the bacterial cellular protein samples and 21 and 12 spots from bacterial secreted protein samples with normal broth and peptide-free medium, respectively, by 2-DE immunoblotting. Further analysis with MALDI-TOF/TOF MS, identified 15 cellular immunogenic proteins, seven secreted immunogenic proteins from normal broth, and nine secreted immunogenic proteins from peptide-free medium. S. hyicus lipase and phosphopyruvate hydratase (enolase) were identified from both media (Fig 1). Thus, 28 different immunogenic proteins were isolated from the S. hyicus cellular and secreted fractions; lipase and metalloprotease were identified previously [26,27], and the remaining 26 proteins were novel in this study (Tables 1 and 2).

Functional analyses of identified immunogenic proteins
The functions of the identified immunogenic proteins of S. hyicus are summarized in Fig 2. The data demonstrated that most of the proteins were involved in amino acid transport and metabolism or energy production and conversion, and some were involved in translation, post-translational modification, protein turnover, and as chaperones.

Immunogenicity validation of identified proteins
To verify the immunogenicity of identified proteins obtained by immunoproteomic assay, ACK from cellular proteins and ENO from secreted proteins were chosen for biological function validation. The ACK and ENO proteins were expressed in E. coli and analyzed by SDS-PAGE, which confirmed that the proteins were correctly expressed with high abundance ( Fig  3A). Western blot analysis of recombinant ACK and ENO demonstrated that both proteins could react with pig sera against ZC-4 ( Fig 3A), suggesting that the two proteins exhibit immunogenicity. The recombinant proteins were purified on a Ni 2+ Sepharose column for further use (Fig 3B).

Function validation of ACK and ENO in mice
To determine the biological function of ACK and ENO, BALB/c mice were treated with purified ACK and ENO, and the levels of 32 serum cytokines including interleukin (IL)-6, IL-8, IL- 12, and INF-γ were determined by protein chip at 3 h and 24 h. The levels of both G-CSF and MCP-5 increased significantly (p < 0.001) at 3 h and 24 h in ACK and ENO treated groups, and that the level of IL-12p40p70 increased significantly (p < 0.001) at 3 h and 24 h in the ACK-treated group (Fig 4).
Epitope prediction of identified proteins B-cell epitopes play a vital role in the antibacterial immune response and are widely used to develop peptide vaccines and diagnose diseases. We analyzed the B-cell epitopes of identified proteins, including ACK and ENO, using ABCPred and BCPreds. We identified 100 B-cell epitope antigen sequences for the cellular immunogenic proteins and 136 B-cell epitope antigen sequences for the secretory immunogenic proteins (Tables 1 and 2).
Immunoprotection of ENO and ACK as a subunit vaccine against S. hycius in mice. Blood was collected from the tail vein of immune and control mice at 0, 7, 14, 21 and 28 days after the first immunization, and antibodies in the serum were assessed by ELISA. We wanted to screen the antibody level at 0, 7, 14, 21 and 28 days, to observe the curves of antibody, but unfortunately, the blood were too little to measure the level of antibody at these time, by ELISA(data not show), we only got the value at 28 days after first immunization ( Fig 5A). The results showed that the level of antibody of treated groups were higher than that of PBS, while there was no significance between HIS treated group and PBS group, this might be because of the blood were too little, and were diluted much.
In order to evaluate the efficacy of the ENO and ACK proteins vaccine against S.hycius ZC-4 infection, the ACK and ENO immuned mice were challenged with 300uL 2.8×10 9 CFU/mL S.hycius. The PBS and His groups mice began to die at 12 h after the challenge, after 24h, the two groups had 83.3%(5/6) and 80%(4/5) mortality. Whereas the mice in the ACK and ENO immunized groups began to die after 12 or 24 hours after the challenge, and after 24 or 36h, they had 60% (3/5) and 40% (2/5) mortality (Fig 5B), these results confirmed that the ACK and ENO immuned mice protected mice against infection by S. hycius ZC-4( Fig 5B). : Mascot standard score. 3 : Theoretical mass determined from the predicted protein sequence. 4 : Theoretical isoelectric point determined from the predicted protein sequence. 5 : Predicted protein localization determined using PSORTb version 3.0.0 and GposmPLoc. C, cytoplasmic; U, unknown. 6 : Number of peptides determined using BCPreds and ABCPred.

Discussion
S. hyicus is the causative agent of EE, which mainly occurs in piglets [28] with long-term clinical indicators. In particularly, because of the high temperatures and humidity in south China, there are frequent disease outbreaks that seriously damage pig farms. However, the mechanism  Although there is no database with sufficient information about proteins in S. hyicus, we identified 28 specific immunogenic proteins from S. hyicus strain ZC-4, including 15 structure proteins and 13 secreted proteins. Of the 28 proteins, some were previously reported in other bacteria, several were subunits of multissubunitproteins, and others such as ABC transporter ATP binding protein, ENO, and ACK were novel immunogenic proteins for S. hyicus. Among the identified bacterial cellular proteins, phosphoenolpyruvate carboxykinase, 23S rRNA (uracil-5)-methyltransferase (RumA), ornithine carbamoyl transferase, lysyl-tRNA synthetase, and ACK are appealing candidates for further study. Phosphoenolpyruvate carboxykinase can effectively induce the cell-mediated immune response by increasing CD4 T cells and cytokines such as IFN-γ, IL-12, and TNF-α, thus displaying high immunogenicity [29], and might be a promising new subunit vaccine candidate. RumA has contributed to the spread of bacterial drug resistance by interfering with initiation factor IF2 and blocking antibiotic drug binding to rRNA through the site-specific methylation of 23 rRNA by RumA [30]. Ornithine carbamoyl transferase was first discovered by Winierhoff [31] and catalyzes the carbamoylation of ornithine to form citrulline in the urea cycle. Deficiency of ornithine carbamoyl transferase leads to an X-linked aminoacidopathy characterized by hyperammonemia, neurologic abnormalities, and orotic aciduria [32]. Hughes et al. demonstrated that the major outer surface phosphoenolpyruvate carboxykinase of Streptococcus agalactiae could trigger the host immune response [33], our data showed that phosphoenolpyruvate carboxykinase from S. hyicus had a similar immune regulation function in pigs.
Another important protein identified in the current study was ACK. S. hyicus predominantly accumulates acetate in the culture medium, suggesting that the phosphor-transacetylase (Pta)-ACK pathway plays a crucial role in bacterial fitness. The Pta-ACK pathway in S. aureus plays an indispensable role for maintaining energetic and metabolic homeostasis during overflow metabolism [34]. Our results indicated that the recombinant ACK exhibited good immunogenicity and could react with S. hyicus antiserum. The in vivo mouse experiments also indicated that ACK can stimulate G-CSF, MCP-5, and IL-12 expression. IL-12 comprises covalently linked p40 and p35 subunits [35] and is an important regulator of T-helper 1 (Th1) cell responses [36]. G-CSF displays a function of enhance survival and antiapoptotic activity [37]. Mindy et al suggested that the expression of MCP-5 is involved in inflammation and the host response to pathogens [38], these proteins were upregulated in our results suggested that the S. hyicus ACK might be related to disease development. The immuneprotection test suggested that ACK can confer the week protection against S.hycius ZC-4 strain The pathogeneses of bacteria are remarkably diverse; nevertheless, all mechanisms of bacterial incursion might be classified in three principal strategies: microbial adhesion, secretion of toxins into the extracellular milieu, and injection of virulence factors into host cells [15]. Thus, the identification of secreted proteins was of crucial importance to understanding the pathogenesis of S. hyicus.
In the present study, we identified several important secreted proteins with implications for future research. Among these, ENO has been identified several times [18,21]in other bacteria including Staphylococcus species, but this was the first report in S. hyicus to our knowledge. ENO has several auxiliary functions, e.g., as a cell-surface plasminogen receptor, as a secreted protein for a variety of pathogenic microorganisms, and as a factor in bacteria adhesion [39]. Our results showed that S. hyicus ENO can increase the cytokine levels of G-CSF and MCP-5 in mice; however, whether it is an important factor that induces IL-10 expression, as demonstrated for Streptococcus sobrinus ENO [21], whether or how enolase involved in S.hycius infection, all requires further study. In our study, we found that similar to enolase of Streptococcus iniae and Plasmodium spp (just enolase specific peptide sequence) [40,41], enolase of S.hycius would confer effective protection in mice against S.hycius infection. We analyzed the homology of enolase between S.hycius and S. iniae and found that their similarity was up to 84% and higher than that betwwen S.hycius and S. sobrinus (76%). These results suggested the enolase had multifunction in different species, and in S.hycius, would be a good protective antigen. S. hyicus also secreted lipase, which was reported in many bacterial species as a potential contributor to colonization and persistence on the skin and is produced during bacterial infection [42][43][44]; Thus, lipase has been suggested as an important bacterial virulence factor and might play important role in the pathogenesis of S. hyicus. Bacterial ABC transporters are essential in cell viability, virulence, and pathogenicity. In addition to their function in transport, some bacterial ABC proteins are involved in regulation of several physiological processes. Basavanna et al. and Mei et al. provided more evidence that functioning ABC transporters were required for the full virulence of bacterial pathogens Streptococcus pneumoniae and S. aureus [45,46]. To the best of our knowledge, our study was the first to identify ABC transporters as secreted proteins of S. hyicus, and their function will be explored in subsequent studies. Serine/threonine protein phosphatase is involved in the DNA damage response by regulating dephosphorylation and mediating cell apoptosis [47,48]. Whether, this protease plays a role similar to that of ENO in S. hyicus invasion of host cells remains to be studied.
In addition to those discussed above, seven structural proteins and five secreted proteins reacted with antisera. So far, studies about these proteins are very limited, and the biological functions of these proteins in immune-regulation during S. hyicus infection remain unclear and require further investigation. Additional studies are necessary to elucidate the biological functions of these unknown proteins.
Interestingly, S. hyicus lipase and phosphopyruvate hydratase (ENO) were identified in both normal broth and peptide-free medium, whereas the other proteins were only secreted in one condition, indicating lipase and ENO might be essential for bacteria growth in either condition, next step, we hoped to express lipase to explore its function.

Conclusions
In summary, we identified 28 proteins, including 15 structural and 13 secreted proteins, as specific immunogenic proteins of S. hyicus ZC-4 by immunoproteomic analysis. Two of these were identified previously, and the remaining 26 were novel for S. hyicus. Furthermore, two of these proteins, ACK and ENO from the cellular and secreted fractions, respectively, were chosen for verification by western blotting and in mouse models, which indicated that ACK and ENO had some level of immune protection against S.hycius in mice. Moreover, we analyzed the important B-cell epitopes and subcellular localizations of these proteins. This study contributes to the current understanding of the map of cellular and secreted proteins of the virulent S. hyicus ZC-4 strain and provides important information about S. hyicus proteins that can help reveal the molecular mechanisms of S. hyicus pathogenicity and develop efficient subunit vaccines.