Comparative proteome analysis of two Streptococcus agalactiae strains from cultured tilapia with different virulence

Veterinary Microbiology 170 (2014) 135–143

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Comparative proteome analysis of two Streptococcus agalactiae strains from cultured tilapia with different virulence Wei Li a,1, You-Lu Su a,b,1, Yong-Zhan Mai a, Yan-Wei Li a, Ze-Quan Mo c, An-Xing Li a,* a

Key Laboratory for Aquatic Products Safety of Ministry of Education/State Key Laboratory of Biocontrol, The School of Life Sciences, Sun Yat-sen University, 135 Xingang West Street, Haizhu District, Guangzhou 510275, Guangdong Province, PR China Key Laboratory of South China Sea Fishery Resources Exploitation & Utilization, Ministry of Agriculture, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, Guangdong Province, PR China c College of Animal Science, South China Agricultural University, Guangzhou, Guangdong Province, PR China b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 29 November 2013 Received in revised form 26 January 2014 Accepted 27 January 2014

Streptococcus agalactiae is a major piscine pathogen, which causes significant morbidity and mortality among numerous fish species, and results in huge economic losses to aquaculture. Many S. agalactiae strains showing different virulence characteristics have been isolated from infected tilapia in different geographical regions throughout South China in the recent years, including natural attenuated S. agalactiae strain TFJ0901 and virulent S. agalactiae strain THN0901. In the present study, survival of tilapia challenged with S. agalactiae strain TFJ0901 and THN0901 (107 CFU/fish) were 93.3% and 13.3%, respectively. Moreover, there are severe lesions of the examined tissues in tilapia infected with strain THN0901, but no significant histopathological changes were observed in tilapia infected with the strain TFJ0901. In order to elucidate the factors responsible for the invasive potential of S. agalactiae between two strains TFJ0901 and THN0901, a comparative proteome analysis was applied to identify the different protein expression profiles between the two strains. 506 and 508 cellular protein spots of S. agalactiae TFJ0901 and THN0901 were separated by two dimensional electrophoresis, respectively. And 34 strain-specific spots, corresponding to 27 proteins, were identified successfully by MALDITOF mass spectrometry. Among them, 23 proteins presented exclusively in S. agalactiae TFJ0901 or THN0901, and the other 4 proteins presented in different isomeric forms between TFJ0901 and THN0901. Most of the strain-specific proteins were just involved in metabolic pathways, while 7 of them were presumed to be responsible for the virulence differences of S. agalactiae strain TFJ0901 and THN0901, including molecular chaperone DnaJ, dihydrolipoamide dehydrogenase, thioredoxin, manganese-dependent inorganic pyrophosphatase, elongation factor Tu, bleomycin resistance protein and cell division protein DivIVA. These virulence-associated proteins may contribute to identify new diagnostic markers and help to understand the pathogenesis of S. agalactiae. ß 2014 Elsevier B.V. All rights reserved.

Keywords: Streptococcus agalactiae Comparative proteome Virulence Tilapia

1. Introduction * Corresponding author. Tel.: +86 20 84115113/+86 13 725330810; fax: +86 20 84115113. E-mail address: [email protected] (A.-X. Li). 1 Wei Li and You-Lu Su contributed equally to this work. 0378-1135/$ – see front matter ß 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2014.01.033

Streptococcus agalactiae is a major piscine pathogen responsible for huge economic losses to aquaculture. It causes significant morbidity and mortality among numerous fish species of freshwater, estuarine and marine,

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W. Li et al. / Veterinary Microbiology 170 (2014) 135–143

including gulf killifish, tilapia, silver pomfret, barcoo grunter and grouper (Liu et al., 2013; Pridgeon and Klesius, 2013). Clinical signs of S. agalactiae disease include septicemic infection, exophthalmia, corneal opacity, anorexia, ‘C’-shaped body posturing and erratic swimming (Ye et al., 2011). Tilapia production in the southern provinces of China, such as Guangdong, Guangxi, Fujian and Hainan, accounted for almost 40% of the world. However, the large scale disease outbreaks caused by S. agalactiae has brought a great loss in economic terms. The first isolation of S. agalactiae from tilapia in China was reported in Fujian Province, where it caused 20–30% mortality of tilapia in limited areas. Afterward, disease outbreaks spread rapidly to other tilapia farms in major cultivation areas of Southern China, especially in Guangdong and Hainan Provinces, and the mortality rates of over 95% were observed in some farms (Ye et al., 2011). In our previous studies, many S. agalactiae strains were isolated from infected fish in different geographical regions throughout South China (Li et al., 2013; Liu et al., 2013), which presented different virulence characteristics. Among them, a natural attenuated strain TFJ0901, isolated from infected tilapia in Fujian, caused no significant symptom and extremely low mortality in tilapia. There must be some unknown factors associated with the attenuated virulence characteristics for us to further study. Some virulence factors of S. agalactiae were well characterized in human, such as polysaccharide capsule, b-hemolysin/cytolysin, Srr1, nuclease A and other surface proteins that mediate binding to host cells, extracellular matrix, and blood components (Derre´-Bobillot et al., 2013; Lindahl et al., 2005; Seo et al., 2012). However, bacterial pathogens have developed such a variety of strategies to infect hosts requiring the involvement of large quantities of virulence associated factors, including bacterial adherence and invasion to the host cells, and replication inside the host to lead to the final cell death (Fittipaldi et al., 2012). The previous studies on the virulence associated factors of S. agalactiae were far from enough, so it does make sense for us to explore more proteins involved in pathogenesis. Combining two-dimensional electrophoresis with mass spectrometry resulted in a powerful technology ideally suited to recognize and identify proteins of pathogenic microorganisms (Cordwell et al., 2001). The proteomics study using S. agalactiae cells grown under different conditions has been reported, and C protein ß antigen was proposed to be a putative virulence factor (Yang et al., 2010). Hence, comparative proteome analysis was applied in our study to illustrate different protein expression profiles between the natural attenuated S. agalactiae strain TFJ0901 and virulent S. agalactiae strain THN0901 and the differentially expressed proteins may contribute to the pathogenesis of S. agalactiae. 2. Materials and methods 2.1. S. agalactiae strains with different virulence Five S. agalactiae strains used in this research were originally isolated from infected tilapia during the

outbreaks of S. agalactiae throughout South China, and they were named TFJ0901, TMM1101, TZH1201, TZC1101, and THN0901, respectively. Infection chronology, sources of isolation, hosts and serotypes of the S. agalactiae strains were summarized in Table S1. S. agalactiae strains were grown aerobically overnight at 28 8C in a shaker bath, and then overnight cultured cells were diluted into 1:100 in BHI medium. Tilapia (Oreochromis niloticus) for this experiment were purchased from a breeding farm in Guangdong province, with mean weights of 76  5 g. They were acclimated for two weeks before grouping (28–30 8C). A total of 30 fish were used in each treatment group (15 fish per tank, duplicates), and two replicate tanks of tilapia served as controls. The same amount (107 CFU/fish in a total volume of 0.1 mL) (Pridgeon and Klesius, 2013) of each S. agalactiae strain was exposed to the tilapia via intraperitoneal injection. Survivals of the 6 groups were recorded daily for 14 days. Meanwhile, brains, kidneys and spleens of tilapia infected by S. agalactiae strains were sampled after 7 days post inoculation (dpi). Following standard fixation in 10% neutral buffered formalin and processing into paraffin wax blocks, paraffin sections (5 mm thick) were stained with hematoxylin and eosin (H&E) for light microscopy observations. Approval was obtained from the animal ethics committee of the life science institute prior to using the animals for research. 2.2. Preparation of cellular proteins S. agalactiae strains THN0901 and TFJ0901 were grown aerobically overnight at 28 8C in a shaker bath, and then overnight cultured cells were diluted into 1:100 in BHI medium. Optical densities at 600 nm were measured and the growth curve of bacterial cultures was presented as Fig. S1. The cultures were collected at early stationary (12 h) by centrifugation at 10,000  g for 10 min at 4 8C and were washed by ddH2O for 3 times. Then the pellets were resuspended in lysis buffer (7 M urea, 2 M thiourea, 4% (w/ v) CHAPS), and sonicated on ice with the ultrasound power of 75 W for 30 min, worked for 4 s and stopped for 6 s in a cycle. The supernatant was collected by centrifugation at 12,000  g for 20 min at 4 8C and 100% trichloroacetic acid was added to a final concentration of 10%, kept at 4 8C for 4 h. The pellets were collected, and ice–cold acetone was added to wash it. After the pellets were centrifuged at 12,000  g for 20 min at 4 8C and dissolved in lysis buffer, the concentration of the soluble protein in the final preparation was determined by the Bradford method and BSA was used as standard protein. 2.3. Two-dimensional electrophoresis Two-dimensional gel electrophoresis (2-DE) was performed as previously described (Boguth et al., 2000). 800 mg cellular proteins were diluted to a total volume of 350 mL with rehydration buffer (7 M urea, 2 M thiourea, 4% (w/v) CHAPS and 0.005% (w/v) bromophenolblue, 1% (v/v) IPG buffer 3–10), and the proteins were separated on 17 cm pH 4–7 NL ReadyStripsTM IPG Strips (Bio-rad). IEF was performed with a PROTEAN1 IEF cell using the program: 50 V active rehydration for 12 h; 150 V (rapid ramp), 150 Vh; 300 V (rapid ramp), 300 Vh; 500 V (rapid ramp),

W. Li et al. / Veterinary Microbiology 170 (2014) 135–143

500 Vh; 1000 V (rapid ramp), 1000 Vh; 5000 V (rapid ramp), 5000 Vh; 10,000 V (rapid ramp), 1 h; 10,000 V (line ramp), 55,000 Vh at 20 8C. After IEF, IPG strips were first soaked with equilibration buffer (375 mM Tris–HCl, pH 8.8, 6 M urea, 20% (v/v) glycerol, 2% (w/v) SDS) supplemented with 20 mg/mL DTT for 15 min and then in another equilibration buffer supplemented with 25 mg/mL iodoacetamide for additional 15 min. The equilibrated IPG strips were transferred to the top of 12% polyacrylamide gels for the secondary dimensional electrophoresis at 15 mA for 12 h at 25 8C. Proteins were visualized by staining with Coomassie Brilliant Blue G-250, and then the wet gels were scanned by GSC-8000 (Bio-rad) and analyzed by PDQuestTM v. 8.0 analysis software. Protein spots were automatically detected, matched, and normalized to a master gel, and then manually checked to guarantee correct matching across the gels. Unique spots were marked with different symbols. 2.4. Mass spectrometry analysis Selected spots were excised and washed with 120 mL 50% methanol for 30 min. Then the liquid was removed and 100 mL acetonitrile was added. After the gel particles became white and shrunk, the acetonitrile was removed and gel pieces were rehydrodrated in 0.1 M NH4HCO3 and 50% acetonitrile. All the liquid was removed and 110 mL acetonitrile was added to make the particles dried. In-gel tryptic degradation was performed 12 h at 37 8C with 20 ng/mL trypsin (Promega). The pooled extracts were lyophilized and reconstituted in 1.5 mL 0.1% TFA prior to MALDI-TOF mass spectrometer analysis. MALDI-TOF spectra were obtained using an Ultraflex III MALDI-TOF/ TOF mass spectrometer (Bruker Dalton), MALDI-TOF MS analysis was according to a previously described procedure (Su¨ss et al., 2009). Peptide masses were searched against the NCBI database using the MASCOT program. 2.5. Gene cloning and real time quantitative PCR analysis of differentially expressed proteins Genomic DNA was extracted from the TFJ0901 and THN0901 using Wizard genomic DNA purification kit (Promega) following the manufacturer’s protocol. Primers (Table S2) specific for the genes were designed. PCR condition was one cycle of 94 8C for 2 min, 30 cycles of 94 8C for 30 s, 60 8C for 30 s, 72 8C for 20 s. PCR products were separated using electrophoresis in a 3% agarose gel containing ethidium bromide and visualized under UV light (Tanon 2500 Gel Image System). The cultured cells of TFJ0901 and THN0901 were collected at late logarithmic phase (8 h) by centrifugation at 10,000  g for 10 min at 4 8C. Total RNA extraction, DNase I treatment and first-strand cDNA synthesis were performed as previously described (Li et al., 2011). The primers used in real time quantitative PCR were designed using Beacon Designer 7.80 software and listed in Table S1, rRNA 16 s was used as the reference gene. RT-PCR was carried out with Roche Light Cycler 480 Real-time PCR Detection System using SYBR Green qPCR Master Mix (Thermo). The cycling protocol was pre-denaturation at

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95 8C for 10 min followed by 40 cycles of 95 8C for 15 s, 60 8C for 30 s, and 72 8C for 30 s. Specificity of PCR products were detected by melting curve analysis. The relative mRNA expression of target genes to reference genes were DDCt calculated using the 2 (Livak and Schmittgen, 2001). All data were analyzed using SPSS (version19.0) software. Transcription levels were considered significantly different between TFJ0901 and THN0901 using the Duncan test (p < 0.05), and the expression ratio must be more than 2fold or less than 0.5. 3. Results 3.1. The attenuated virulence characteristics of the S. agalactiae strain TFJ0901 were tested on tilapia The survival and clinical symptoms of tilapia were recorded daily for a period of 14 dpi (Fig. S2). Tilapia mortalities occurred in large quantities from 1 to 6 dpi and the survival of each group challenged with TFJ0901, TMM1101, TZH1201, TZC1101, and THN0901 were 93.3%, 46.7%, 30.0%, 20.0%, and 13.3%, respectively. There was no significant symptom appeared in the tilapia challenged with S. agalactiae strain TFJ0901, while the clinical signs appeared in the tilapia from the third day post challenge with S. agalactiae strain TMM1101, TZH1201, TZC1101, and THN0901, including exophthalmia, corneal opacity, and anorexia. The survival assay revealed that the virulence of S. agalactiae TFJ0901 is extremely lower than the other S. agalactiae strains, especially the S. agalactiae strain THN0901. Histopathological examination showed that the most severe lesions of the examined tissues were appeared in tilapia infected by S. agalactiae strain THN0901. The brain capillaries were expanded and congested associated with proliferation of glial cells. The meninges were thickened by the infiltration of inflammatory cells (Fig. 1b). Similar inflammatory cell infiltrations were observed in the renal tubules of kidney and some of the renal capsules were narrowing. There was marked hemorrhage in the renal interstitial tissue (Fig. 1d). Severe hemorrhage was observed in the white pulp of spleen accompanied by lymphocytes decreasing and melano-macrophage center increasing. Another finding was the presence of severe necrosis in lymphocytes and reticular endothelial cells (Fig. 1f). In contrast, no obvious histopathological changes were observed in fish injected with S. agalactiae TFJ0901 (Fig. 1a, c, and e). The histopathological examination was the further evidence of the attenuated virulence characteristics of the S. agalactiae strains TFJ0901. 3.2. Comparative proteome analysis The soluble proteins extracted from the S. agalactiae virulent strain THN0901 and the natural attenuated strain TFJ0901, were separated on high-resolution 2D electrophoresis gels (pH 4–7). Triple replicated analytical gels were analyzed and compared by the PDQuest 8.0 (Bio-rad), which generated standardized synthetic images. The two resulting synthetic gels allowed detection of 506 and 508 spots for S. agalactiae TFJ0901 and THN0901, respectively

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Fig. 1. Histological sections in the brain, kidney and spleen of tilapia infected with Streptococcus agalactiae strain TFJ0901 (a), (c) and (e) and strain THN0901 (b), (d) and (f). (a) No significant histopathological changes in the brain. (b) Expansion and congestion of brain capillaries (arrowhead); proliferation of glial cells; brain meninges inflammatory oedema, thickening, with inflammatory cells infiltration (arrow). (c) No significant histopathological changes in the kidney. (d) The inflammatory oedema and inflammatory cell infiltration among the renal tubules; renal capsule narrow or disappear (arrow); renal interstitial hemorrhage (arrowhead). (e) No significant histopathological changes in the spleen. (f) Severe hemorrhage in splenic white pulp; lymphocytes and reticular endothelial cell necrosis (arrow); decreased the number of lymphocytes in the white pulp (arrowhead) and increased of melano-macrophage center. Hematoxylin and eosin (H.E.) stain, bar = 50 mm.

(Fig. 2). In the present study, 38 strain-specific spots were taken into account, which presented exclusively on the gel of S. agalactiae TFJ0901 or THN0901. Three representative unique spots of strain THN0901 were showed in Fig. 3. Moreover, 34 spots, corresponding to 27 proteins, were identified successfully by MALDI-TOF mass spectrometry. Among these proteins, 23 of them were unique proteins of S. agalactiae TFJ0901 or THN0901 and functions were assigned to these proteins (Tables 1 and 2). As expected, most of the proteins originated from the cytoplasm, and the proteins were assigned into 6 classes of cellular function, with almost half proteins being assigned to

carbohydrate metabolism (26.1%) and translation (26.1%), followed by groups of proteins involved in transport (21.7%), folding, sorting and degradation (8.7%), cellular process (8.7%) and nucleotide metabolism (8.7%). Carbohydrate metabolism and cellular translation significantly decreased in S. agalactiae TFJ0901, whereas translation and transport improved in vivo. However, the other four proteins presented in different isomeric forms between S. agalactiae TFJ0901 and THN0901 (Table S2), including the ATP-dependent Clp protease, tRNA (uracil-5-)-methyltransferase, glucose-1-phosphate adenylyltransferase and phenylalanyl-tRNA synthetase.

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Fig. 2. Cellular proteins extracted from S. agalactiae strains THN0901 (a) and TFJ0901 (b) were separated by 2-D electrophoresis, the gels were stained by Coomassie Brilliant Blue G-250. The proteins expressed exclusively in THN0901 were numbered H1 to H20, and the proteins expressed exclusively in TFJ0901 were numbered F1 to F18. The numbered spots were identified by MALDI-TOF MS. Positions of the protein molecular weight markers are indicated at the left hand side of gel (a).

3.3. Gene cloning and real time quantitative PCR analysis of differentially expressed proteins As shown in Fig. S3, all the genes of differentially expressed proteins were amplified by PCR successfully, which means those genes were present in both S. agalactiae THN0901 and TFJ0901. Meanwhile, the transcription level of all these genes were analyzed by RT-PCR (Fig. S4). Among the genes of 23 differentially expressed proteins, 16 genes transcript significantly different. From the analysis of RT-PCR, most transcription level of genes were in correlation with the protein expression aside from accD SAK_0427, and dnaJ SAK_0148, which further demonstrated the authenticity of our comparative proteomic study.

4. Discussion There are some limitations in studying pathogenesis of pathogens, one of them is that there is no consensus on the animal model to be used to evaluate the virulence of a specific strain, including animal species, routes of inoculation and bacterial infective doses (Fittipaldi et al., 2012). Tilapia were selected as our experimental subject and survival assays were conducted according to previous studies (Pridgeon and Klesius, 2013). The survival assay revealed that the morbidity and mortality of tilapia challenged with S. agalactiae strain TFJ0901 is extremely lower than the other S. agalactiae strains, especially the S. agalactiae strain THN0901. Histopathology lesions of the tilapia infected with S. agalactiae strain THN0901 were

Fig. 3. Three representative unique spots of S. agalactiae strain THN0901 were showed, which only presented on the gel of THN0901. (A) Molecular chaperone DnaJ; (B) Elongation factor Tu; (C) Bleomycin resistance protein.

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140 Table 1 Unique spots in the gel of THN0901. Spot numbera Protein name

Gene name Accession numberb

Theoretical MW/pIc

Carbohydrate metabolism H3 Acetyl-coenzyme A carboxylase carboxyl transferase subunit beta accD

Scored Sequence coveragee (%)

Matched peptidesf

Putative functionsg

ATP binding; acetyl-CoA carboxylase activity; acetyl-CoA carboxylase complex; fatty acid biosynthetic process; malonyl-CoA biosynthetic process; transferase activity; zinc ion binding Cell redox homeostasis; cytoplasm; dihydrolipoyl dehydrogenase activity; flavin adenine dinucleotide binding Cytoplasm; inorganic diphosphatase activity; manganese ion binding Cell redox homeostasis; electron carrier activity; glycerol ether metabolic process; protein disulfide oxidoreductase activity

accD SAK_0427

527904912

32,346/5.94 247

36

9

H7 H9

Acetoin dehydrogenase, TPP-dependent, E3 component

SAK_1004

494703062

62,288/4.82 137

28

10

H10 H18

ppaC SAK_1430

494703399

33,576/4.47 190

30

7

H19

Manganese-dependent inorganic pyrophosphatase Thioredoxin

trx SAK_1725

527814307

11,847/4.39 76

25

4

Translation H13

Elongation factor Tu

tuf SAK_0887

494702959

43,954/4.83 226

35

14

GTP binding; GTP catabolic process; GTPase activity; cytoplasm; translation elongation factor activity

Transport H16

CutC family protein

SAK_1587

494703532

23,500/5.15

275

39

10

Copper ion binding; copper ion homeostasis

dnaJ SAK_0148

527809703

29,970/8.57

96

29

6

ATP binding; DNA replication; cytoplasm; protein folding; response to heat; zinc ion H2

SAK_0586

527899577 29,581/4.38 227

48

12

SAK_1311

76562355

15,469/4.40

50

11

1

Cell cycle; cell division; cytoplasm Bind to bleomycin

upp SAK_1601

494703542

22,778/5.38 86

35

6

Folding, sorting and degradation H2 Chaperone protein DnaJ

Cellular process H12 Cell division protein DivIVA H20 Bleomycin resistance protein Nucleotide metabolism H15 Uracil phosphoribosyltransferase

a b c d e f g

GTP binding; UMP salvage; magnesium ion binding; uracil phosphoribosyltransferase activity; uracil salvage

Refers to the proteins labeled in Fig. 2. Accession ID of each protein is the GenInfo number in the NCBI protein database. pI values and molecular weights represent reported values for the full-length protein. Mascot score. Percent amino acid coverage of entire protein. Number of non-redundant peptides identified for each protein. As given in the SWissprot database for S. agalactiae.

consistent with previous studies, including meningitis, inflammatory cell infiltration, severe necrosis in lymphocytes and reticular endothelial cells (Suanyuk et al., 2008), while S. agalactiae TFJ0901 caused no significant pathological damage. Attenuated virulence characteristics of S. agalactiae strain TFJ0901 were demonstrated which may attribute to the different protein expression profiles, especially the expression of virulence associated proteins. For the comparative proteome analysis, the isoforms expressed differentially in charge or mass probably arose as a result of posttranslational modifications. This charge

or mass heterogeneity probably reflected specific amino acid substitutions or post-translational protein modification. A striking characteristic of Streptococcus pneumoniae phosphoproteome was the large number of specific phosphorylated sites, indicating that high level of protein phosphorylation might play an important role in regulating many metabolic pathways and bacterial virulence (Sun et al., 2009). Of the 23 differentially expressed proteins, 13 and 10 proteins were detected exclusively in the cellular proteins of S. agalactiae strains TFJ0901 and THN0901, respectively. Most of the strain-specific proteins were just

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Table 2 Unique spots in the gel of TFJ0901. Spot numbera Protein name

Carbohydrate metabolism F12 2,3-Bisphosphoglyceratedependent phosphoglycerate mutase F15 Phosphoglycerate kinase

Gene name

Accession numberb

gpm SAK_0889 76787711

pgk SAK_1788

Theoretical MW/pIc

Scored Sequence coveragee (%)

Matched Putative functionsg peptidesf

25,944/5.13

87

22

5

255

63

9

527916853 14,302/5.91

2,3-Bisphosphoglyceratedependent phosphoglycerate mutase activity; glycolysis ATP binding; cytoplasm; glycolysis; phosphoglycerate kinase activity

Translation F1

Arginyl-tRNA synthetase

argS SAK_2042 76787802

63,304/5.18

209

25

16

F3

Glutamyl-tRNA synthetase gltX SAK_0165 76787836

54,881/4.93

95

22

8

F4

GTP-binding protein EngA

258

37

16

F8

50S ribosomal protein L4

der engA 76788647 48,950/5.15 SAK_1634 rplD SAK_0092 494702590 22,126/9.84

173

22

3

F11

50S ribosomal protein L3

rplC SAK_0091 527912148 21,301/10.03 96

23

6

Transport F9

ABC transporter

SAK_1033

78

18

4

494703502 26,298/5.76

253

49

8

76788663

11,477/5.01

192

66

5

SAK_0435

76788411

36,115/5.23

168

25

9

SAK_0393

494702476 51,928/5.01

112

13

5

Hydrolase activity; peptidyl-prolyl cis-trans isomerase activity; protein folding; protein peptidyl-prolyl isomerization

SAK_1838

76787246

282

44

16

‘De novo’ AMP biosynthetic process; GTP binding; adenylosuccinate synthase activity

F10 F16

F17

Metal ABC transporter SAK_1555 mtsB PTS system, IIB component SAK_0399 lactose/cellobiose family

PTS system, IIAB component mannose/ fructose/sorbose family

Folding, sorting and degradation F2 Cof-like hydrolase/ peptidyl-prolyl cis-trans isomerase domain protein

Nucleotide metabolism F14 Adenylosuccinate synthetase purA

a b c d e f g

76787616

26,371/5.89

47,692/5.70

ATP binding; arginine-tRNA ligase activity; arginyl-tRNA aminoacylation; cytoplasm ATP binding; cytoplasm; glutamate-tRNA ligase activity; glutamyl-tRNA aminoacylation; GTP binding; ribosome biogenesis rRNA binding; ribosome; structural constituent of ribosome; translation rRNA binding; ribosome; structural constituent of ribosome; translation ATP binding; ATP catabolic process; ATPase activity ATP binding; ATP catabolic process; ATPase activity Phosphoenolpyruvatedependent sugar phosphotransferase system; protein-N(PI)phosphohistidine-sugar phosphotransferase activity Cytoplasm; integral to membrane; phosphoenolpyruvatedependent sugar phosphotransferase system; protein-N(PI)phosphohistidine-sugar phosphotransferase activity

Refers to the proteins labeled in Fig. 2. Accession ID of each protein is the GenInfo number in the NCBI protein database. pI values and molecular weights represent reported values for the full-length protein. Mascot score. Percent amino acid coverage of entire protein. Number of non-redundant peptides identified for each protein. As given in the SWissprot database for S. agalactiae.

involved in metabolic pathways, while some proteins were demonstrated to be virulence factors in other bacteria, which might be responsible for the virulence differences of S. agalactiae strain TFJ0901 and THN0901, including

molecular chaperone DnaJ, dihydrolipoamide dehydrogenase, thioredoxin, manganese-dependent inorganic pyrophosphatase, elongation factor Tu, bleomycin resistance protein and cell division protein DivIVA.

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Spot H2 matched the molecular chaperone DnaJ which belongs to the Hsp40 stress protein family. DnaJ acts synergistically with DnaK and the cochaperone GrpE, in the folding of nascent protein chains in the bacterial cytoplasm and it is essential for the selective breakdown of many unfolded proteins. Previous experiments revealed that a Campylobacter jejuni DnaJ mutant was unable to colonize newly hatched chickens, suggesting DnaJ played an important role in enabling C. jejuni to colonize chickens (Konkel et al., 1998). Edwardsiella tarda DnaJ also has been proved to be essential to overall bacterial virulence to invade into host blood and macrophages (Dang et al., 2011). The DnaJ expressed much lesser in the natural attenuated S. agalactiae strain TFJ0901, which probably contributed to the attenuated virulence characteristics of strain TFJ0901. The transcript changes of S. agalactiae in response to blood contact from culture medium occurred for genes involved in carbohydrate metabolism, including multi-functional proteins and regulators putatively involved in pathogenesis (Mereghetti et al., 2008). The same changes appeared in our experiment, dihydrolipoamide dehydrogenase, thioredoxin and manganese-dependent inorganic pyrophosphatase were all involved in the carbohydrate metabolism and also putatively involved in pathogenesis, which were highly expressed in virulent strain THN0901. Dihydrolipoamide dehydrogenase (DLDH) is a flavoenzyme which makes up the E3 component of the pyruvate dehydrogenase. Studies demonstrated that the DLDH-negative S. pneumoniae, produced only 50% of normal capsular polysaccharide, were impaired for galactose and raffinose metabolism and were avirulent in sepsis and lung infection models in mice (Tyx et al., 2011). Thioredoxin is a kind of cytosolic highly potent reductase, involving in defense against oxidative stress. Thioredoxin also possesses chaperone function that is disconnected from cysteine interactions. Previous studies illustrated that the redox potential of thioredoxin was related with intracellular replication and virulence (Bjur et al., 2006), and catalytic activity of thioredoxin was essential for Salmonella pathogenicity island 2 to infect phagocytic and epithelial cells (Negrea et al., 2009). Manganese-dependent inorganic pyrophosphatase was found to be a mediate associated with virulence of S. agalactiae which was consistent with our study. It can be reversibly phosphorylated by serine/threonine protein kinase and serine/threonine protein phosphatase (Rajagopal et al., 2003), which coupled to a novel regulating system affecting normal growth, virulence, and cell segregation of S. agalactiae (Jiang et al., 2005). Spot H13 matched the elongation factor Tu (EF-Tu), a typical multifunctional protein, which is implicated in the sorting and amplification of transmembrane signals and the direction of the synthesis and translocation of proteins. Surfaced localized elongation factor Tu (EF-Tu) of Mycoplasma pneumoniae mediate mycoplasma binding to fibronectin, facilitating interactions between mycoplasmas and extracellular matrix (Balasubramanian et al., 2009). The highly expressed elongation factor Tu may play an important role in S. agalactiae virulent strain THN0901. Bleomycin resistance protein only expressed in the S. agalactiae THN0901. It is a kind of 14-kDa acidic protein,

which reportedly protects against bleomycin-induced DNA damage in vitro by binding to bleomycin (Keyt et al., 1996). Cell division protein DivIVA is division site-selection protein present simultaneously both as a ring at the division septum and as dots at the cell poles in most Grampositive bacteria (Kang et al., 2008). Moreover, a divIVA deletion mutant reduced extracellular levels of the autolysins p60 and MurA, exhibited a pronounced chaining phenotype which were unable to swarm, severely impaired in biofilm formation on plastic surfaces, and clearly affected both invasiveness and cell-to-cell spread. (Halbedel et al., 2012). The growth curve showed slightly slow growth of S. agalactiae strain TFJ0901, which probably due to the low expression of DivIVA, suggesting that DivIVA is involved in the process of replication in the host. However, there were 4 spots, exclusively presented on the gel of S. agalactiae TFJ0901, should be taken into account. These 4 polypeptides matched ABC transporter, metal ABC transporter, PTS system (IIB component) and PTS system (IIAB component), which were all associated with the transport of S. agalactiae. In our present study, PTS system (IIB component) and PTS system (IIAB component) were only expressed in S. agalactiae TFJ0901, the same results occurred in some previous comparative proteome analysis (Yang et al., 2012, 2010). Furthermore, transcriptome analysis showed that each component gene of the PTS system were expressed differentially in response to amniotic fluid (Sitkiewicz et al., 2009), so we speculated some components of phosphotransferase system (PTS) might negatively correlate with pathogenesis of S. agalactiae. The bacterial phosphotransferase system not only catalyzes the concomitant transport and phosphorylation of its sugar substrates, such as lactose, cellobiose, mannose, sorbose, fructose, but also takes part in a variety of ramifications for metabolic regulation and the synthesis of group B carbohydrate (Sutcliffe et al., 2008). ABC transporters, contributing to acquisition of vital nutrients, stress responses and intercellular signaling, were confirmed that they had significant roles during disease pathogenesis for a range of bacteria, including S. pneumoniae (Brown et al., 2001) and Staphylococcus aureus (Remy et al., 2013). But in the present study, ABC transporters were also only expressed in the natural attenuated S. agalactiae TFJ0901, which was contradictory to these studies. The real-time quantitative PCR analysis showed that transcription level of most genes were in correlation with the protein expression, further demonstrating the authenticity of our comparative proteomic study. While transcription level of a few genes were unrelated with the protein expression, which may because the translation of individual mRNA species into their encoded proteins were regulated, producing discrepancies between mRNA and protein levels (MacKay et al., 2004). In conclusion, this is the first report using comparative proteome analysis to find the different protein expression patterns through different virulence strains of S. agalactiae. It is meaningful for us to find virulence candidates to further study the pathogenesis and diagnostic markers of S. agalactiae. However, many virulence candidates were demonstrated to be multifunctional proteins and found to be localized in the different fraction of the bacteria. For

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