S. suis and the ccpA-mutant strains from our laboratory (9) were cultured in Todd-Hewitt broth (Oxoid Ltd., Basingstoke, UK) at 37°C under 5% CO₂. Cells in the exponential growth phase were used.

After 200 µg cells were collected by centrifugation, SDT lysis buffer [4% SDS, 100 mM Tris-HCl pH 8.0, 100 mM dithiothreitol (DTT)] purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA) was added and samples were then mixed by vortexing and placed in a boiling water bath for 10 min. The samples were then ultrasonically disrupted and heated again for 5 min in a boiling water bath. The supernatant was removed and protein quantification was performed using the bicinchoninic acid method.

From each sample, 200 µg protein was taken and DTT was added to a final concentration of 100 mM. The mixtures were heated in a boiling water bath for 3 min and then cooled to room temperature. The products were combined with 200 µl UA buffer (Bio-Rad Laboratories, Inc.; 8 M Urea and 150 mM Tris HCl, pH 8.0) and mixed. The mixtures were transferred into ultrafiltration centrifuge tubes with a 10-kDa cutoff point and centrifuged at 14,000 xg for 15 min. The precipitates were re-suspended in 200 µl UA buffer and centrifuged at 14,000 xg for 15 min. The filtrates were discarded and the precipitates were re-suspended in 100 µl Iodoacetamide (Bio-Rad Laboratories, Inc.; 50 mM in UA) in the dark for 30 min and then centrifuged at 14,000 xg for 10 min. The precipitates were re-suspended in 100 µl UA buffer and centrifuged at 14,000 xg for 10 min. This process was repeated twice. The precipitates were re-suspended in 100 µl dissolution buffer and centrifuged at 14,000 x g for 10 min, which was also repeated twice. The precipitates were re-suspended in 40 µl trypsin buffer (Promega, Madison, WI, USA; 5 µg trypsin in 40 µl dissolution buffer), agitated at 600 rpm for 1 min and incubated at 37°C for 16-18 h. The products were centrifuged at 14,000 xg for 10 min using fresh collection tubes, the filtrates were collected, and peptide quantification was performed by measuring the optical density at 280 nm. After enzymolysis, peptide labeling was performed using an iTRAQ 8-plex Multiplex kit (Ab Sciex, Framingham, MA, USA) according to the manufacturer's instructions. For native S. suis, iTRAQ reagents 113, 114 and 115 were used, and for mutant strains, reagents 116, 117 and 118 were used.

SCX chromatography was performed using an AKTA Purifier 100 (GE Healthcare, Little Chalfont, UK) with a 4.6×100 mm polysulfoethyl column (5 µm; 200 Å) (PolyLC Inc., Columbia, MD, USA). Buffer A contained 10 mM KH₂PO₄ and 25% acetonitrile (ACN; pH 3.0; Sigma-Aldrich, St. Louis, MO, USA). Buffer B contained 10 mM KH₂PO₄ pH 3.0, 500 mM KCl and 25% ACN. Samples were collected and lyophilized prior to desalination in a C18 Cartridge (Sigma-Aldrich).

An Easy nLC system (Thermo Fisher Scientific, Waltham, MA, USA) run at a nanoliter flow rate was used for liquid separation of each sample. Buffer A was 0.1% formic acid in water and Buffer B was 0.1% formic acid in 84% ACN. Chromatographic columns were equilibrated with 95% Buffer A. Samples were loaded into a Thermo scientific EASY-Spray column (2 cmx100 µm 5 µm-C18) using an auto sampler and then separated on a Thermo scientific EASY column (75 µmx100 mm 3 µm-C18) at a flow rate of 250 nl/min. MS was performed using a Q-Ex active mass spectrometer (Thermo Fisher Scientific) with an analysis time of 120 min, a positive ion detection method, a precursor ion scanning range of 300–1,800 m/z, a first-order mass spectrum resolution of 70,000 at m/z 200, an AGC target of 3e6, a first order maximum injection time (IT) of 10 msec, one scan range and a dynamic exclusion of 40.0 sec. The mass-charge ratios of the peptides and peptide fragments were obtained as follows: Ten-fragments spectra (MS2 scan) were collected after each full scan, the MS2 Activation Type was HCD, the isolation window was 2 m/z, the second-order MS resolution was 17,500 at m/z 200 with one microscan. The second-order Maximum IT was 60 msec, the normalized collision energy was 30 eV and the under fill ratio was 0.1%.

The raw data of the MS analysis were derived from RAW files. Database searches and quantitative analyses were performed using the software Mascot 2.2 of Proteome Discoverer 1.4 (Thermo Fisher Scientific). For protein-abundance ratios measured using iTRAQ, a 1.2-fold change was set as the threshold and a two-tailed P-value <0.05 to identify significant changes. The database was downloaded from the National Center of Biotechnology Information (NCBI) on 2013-08-09, and the NCBI_Streptococcus_suis. Fasta results contained 89,409 sequences (http://www.ncbi.nlm.nih.gov/protein?term=txid1307 [Organism]). Mascot 2.2 was used for the library search. The localized sequence alignment software NCBI Basic Local Alignment Search Tool (BLAST 2.2.28+-win32. ext; http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to perform sequence alignment between the identified proteins and protein sequences in the NCBInr database. Using the similarity principle, functional information from homologous proteins was used to create protein function annotation. The mapping function of BLAST2GO (https://www.blast2go.com/; version 2.7.0) was used to extract gene ontology (GO) function entries correlated with the aligned sequences for all differentially expressed proteins. The Go Slim was annotated for the function of the target protein by GO for generic.obo. The KEGG (Kyoto Encyclopedia of Genes and Genomes) Automatic Annotation Server (KAAS; http://www.genome.jp/tools/kaas/) was used to align target-protein and Streptococcal sequences in the KEGG GENES database. KEGG Orthology (KO) identifiers of homologous/similar proteins were assigned to the relevant KEGG pathway. Carbon metabolism pathway analysis was performed with the Pathway Builder Tool 2.0 (Protein Lounge, San Diego, CA, USA).

All statistical analyses were performed using the Student's t-test by Perseus 1.3 (http://141.61.102.17/perseus_doku/doku.php?id=start). Differences with a P-value of 0.05 or less were considered statistically significant.

A total of 1,167 proteins were identified from S. suis and ccpA mutant strains from the original mass spectrometry data, 55 of which were differentially expressed.

The mapping function of Blast2GO (Version 2.7.0) was used to extract the GO function entries correlated with the aligned sequences for all of the differentially expressed proteins. As a result, 269 GO function entries associated with the sequences of 45 differentially expressed proteins (81.8%) were extracted. In the functional annotation process, the sequences of a total of 37 differentially expressed proteins were annotated with 87 GO function entries. The final statistical results after supplementary annotation showed that a total of 47 proteins were annotated with 188 GO function entries (Fig. 1).

GO for generic.obo was used to annotate the functions of the target proteins, and a total of 47 protein sequences were annotated by 194 GO Slim function entries (Fig. 2).

KAAS was used to align target-protein and Streptococcal sequences from the KEGG GENES database, and the KO identifiers of homologous/similar proteins were assigned to the relevant KEGG pathways. A total of 34 KEGG signal/metabolic pathways associated with the sequences of 21 differential proteins were extracted (Fig. 3).

The data of the metabolic pathway analyses indicated that dihydrolipoyl transacetylase and dihydroxyacetone kinase and acetate kinase exhibited differential effects on the carbon metabolism between the native S. suis type 2 and ccpA mutant strains. These differential effects on the carbon metabolic pathways between the native and the ccpA mutant strain indicate that ccpA is directly or indirectly involved in the carbon metabolism of bacteria. In particular, ccpA was indicated to have certain effects on the cellular metabolism of bacteria (Fig. 4).