iTRAQ analysis of gill proteins from the zebrafish (Danio rerio) infected with Aeromonas hydrophila

Fish & Shellfish Immunology 36 (2014) 229e239

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iTRAQ analysis of gill proteins from the zebrafish (Danio rerio) infected with Aeromonas hydrophila Aijun Lü*,1, Xiucai Hu 1, Yi Wang, Xiaojing Shen, Xue Li, Aihua Zhu, Jun Tian, Qinglei Ming, Zhaojun Feng School of Life Sciences, Key Laboratory for Biotechnology on Medicinal Plants of Jiangsu Province, Jiangsu Normal University, Xuzhou 221116, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 January 2013 Received in revised form 4 November 2013 Accepted 5 November 2013 Available online 21 November 2013

The gills are large mucosal surfaces and very important portals for pathogen entry in fish. The aim of this study was to determine the gill immune response at the protein levels, the differential proteomes of the zebrafish gill response to Aeromonas hydrophila infection were identified with isobaric tags for relative and absolute quantitation (iTRAQ) labeling followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 1338 proteins were identified and classified into the categories primarily related to cellular process (15.36%), metabolic process (11.95%) and biological regulation (8.29%). Of these, 82 differentially expressed proteins were reliably quantified by iTRAQ analysis, 57 proteins were upregulated and 25 proteins were downregulated upon bacterial infection. Gene ontology (GO) enrichment analysis showed that approximately 33 (8.8%) of the differential proteins in gills were involved in the stress and immune responses. Several upregulated proteins were observed such as complement component 5, serpin peptidase inhibitor clade A member 7, annexin A3a, histone H4, glyceraldehyde 3-phosphate dehydrogenase, creatine kinase, and peroxiredoxin. These protein expression changes were further validated at the transcript level using microarray analysis. Moreover, complement and coagulation cascades, pathogenic Escherichia coli infection and phagosome were the significant pathways identified by KEGG enrichment analysis. This is first report on proteome of fish gills against A. hydrophila infection, which contribute to understanding the defense mechanisms of the gills in fish. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Zebrafish Gill Differential proteome Aeromonas hydrophila

1. Introduction In fish, the gills are large mucosal surfaces and very important portals for pathogen entry [1]. Gills are multifunctional and play primary roles in respiration, osmoregulation and detoxification. Recently, the transcriptional studies have revealed that fish gill plays an important role in immune response [2,3], but gill immune mechanism at the proteome level remains unclear. Fish gills are the first target organ for environmental stress, and thus it is most commonly recognized in gill proteomic responses to abiotic stresses (e.g., salinity, metal ion, and irradiation). The effect of salinity on gill proteome in ayu (Plecoglossus altivelis) was investigated by Lu et al. [4], who found the altered proteins involved in osmoregulation, cytoskeleton, energy metabolism and stress responses, suggesting that gill cell volume-regulatory

* Corresponding author. Fax: þ86 516 83403173. E-mail address: [email protected] (A. Lü). 1 These authors contributed equally to this work. 1050-4648/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsi.2013.11.007

response might play an important role in fish acclimation to salinity change. Furthermore, the differential protein profiles in anterior gills of crab (Eriocheir sinensis) after cadmium exposure were characterized using a proteomic approach, and results demonstrated that acute and chronic treatment induced different responses [5]. Proteomic changes in response to acute cadmium in the gills of flounder (Paralichthys olivaceus) were also evaluated by Ling et al. [6]. Moreover, the differential proteins were identified using liquid chromatography-tandem mass spectrometry (LC-MS/ MS) in the gills of European bullhead (Cottus gobio) following cadmium treatment, which were categorized into several functional classes related to stress response, cytoskeleton, protein repair and proteolytic pathways [7]. Recently, the effects of copper exposure on the proteome of gills from rainbow trout (Oncorhynchus mykiss), common carp (Cyprinus carpio) and gibel carp (Carassius auratus gibelio) were studied using a two-dimensional differential gel electrophoresis (2D-DIGE) and an isobaric tags for relative and absolute quantitation (iTRAQ) approaches [8], and indicating that the iTRAQ labeling is more reliable and accurate, especially when used with a well annotated related species.

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Evidence is accumulating for a protective response by the gill proteome of rainbow trout (O. mykiss) exposed to X-ray induced bystander signals [9]. Proteomic changes in the gills of medaka (Oryzias latipes) following direct irradiation and bystander signals were further examined and indicated the both immediate protection and longer term adaptation in gills [10]. Additionally, proteomic modification in gills of marine medaka (O. melastigma) after brevetoxin-1 exposure were analyzed by Tian et al. [11], the altered proteins were identified and categorized into three main functional classes such as cell structure, metabolism and signal transduction. It is noted that the proteomic changes in shrimp gill have been focused on responses to pathogen infections (e.g., virus, bacterium). In the proteomic analysis of gills from yellow head virus (YHV)infected Penaeus vannamei, the findings indicated that the upregulated proteins included enzymes in the glycolytic pathway, the tricarboxylic acid cycle (TAC) and amino acid metabolism [12]. Analysis of protein expression of thioredoxin in the gills from Litopenaeus vannamei infected with two different viruses demonstrated that white spot syndrome virus (WSSV), or infectious hypodermal and hematopoietic necrosis virus (IHHNV) may induce a differential response of the thioredoxin protein [13]. Recently, fish gill and bacterial pull-down approaches were applied to isolate both bacterial outer membrane proteins that bind to gills and fish gill proteins that interact with bacterial cells, indicating that the twelve gill proteins are known to be involved in host immunity [14]. Beyond these studies, little is known about the gill response to bacterial infection at the protein level in fish. Zebrafish (Danio rerio) has been extensively used to study genetics, development, disease and immunology [15]. Recently, experimental infections using zebrafish were conducted by our group with two different bacteria [16,17]. The immune response of zebrafish host against the bacterium Aeromonas hydrophila was demonstrated at mRNA expression level by Rodríguez et al. [18]. The proteome profile of zebrafish gill was firstly established using the two-dimensional liquid chromatography-electrospray ionization tandem mass spectrometry (2D LC-ESI MS/MS), and the gill proteome exhibited important physiological functions relevant to respiration, homeostasis and energy metabolism [19]. In the present study, the differential protein expressions of zebrafish gill tissues after A. hydrophila infection were identified by iTRAQ analysis, with the aim of clarifying the mechanism of gill immune response against bacterial pathogen in fish. 2. Materials and methods 2.1. A. hydrophila infection in zebrafish Adult zebrafish were infected by immersion with A. hydrophila as previously described with some modifications [18]. A. hydrophila bacteria were cultured from a single isolate (ZF-1201), and reisolated from a single symptomatic zebrafish and biochemically confirmed to be A. hydrophila, before being inoculated into tryptic soy broth (TSB) and incubated in a shaker incubator at 28  C for overnight. The concentration of the bacteria was determined using colony-forming unit (cfu) per mL by the agar plat count method. Zebrafish wild type adults (w0.5 g in weight) were bought from a pet shop in Xuzhou, China. Fish were maintained at 26  C in 60 L tanks containing 40 L of aerated water and acclimatized for 15 d prior to the experiment, a 12/12 h lightedark period was maintained and supplemental aeration was supplied by an air stone. Fish were fed with commercial dry feed distributed manually twice a day. Experimental fish were confirmed to be culture negative for bacterial infection by culturing liver and kidney tissues from representative groups of fish on tryptic soy agar (TSA) plates. For the challenge, a total of 180 fish were randomly divided into six

groups with 30 fish in each aquarium. Three infected fish groups were immersed in a suspension of A. hydrophila at a density of 1.0  108 cfu/mL in 0.65% PBS for 1 h and then were distributed to 10 L glass aquaria. Three groups of control fish were treated with sterile PBS alone. At 72 h post-infection, 20 fish from each group of three replicates were randomly selected, and the gill tissues were aseptically excised and placed in a microcentrifuge tube. The pooled gill samples were flash frozen in liquid nitrogen, and then stored at 80  C for total proteins extraction. 2.2. Protein extraction Gill tissues from three control and three infected groups were respectively homogenized to powder in liquid nitrogen. The powder was added to 4-fold volume of ice-cold acetone containing 10 mM dithiothreitol (DTT) and precipitated at 20  C overnight, the supernatant was discarded following centrifugation at 20,000 g for 20 min. After repeating this step twice, the pellets were then resuspended in a lysis buffer (7 M Urea, 2 M Thiourea, 4% CHAPS, 40 mM TriseHCl), with a final concentration of 1 mM PMSF and 2 mM EDTA. After mix, the samples were sonicated with 200 W for 5 min and the supernatant was reduced with 10 mM DTT and blocked with 55 mM IAM, and then added to 5-fold volume of icecold acetone for 2 h at 20  C. After centrifugation as describe above, the pellets were dissolved in 400 mL 0.5 M TEAB and resuspended with sonication. The homogenates were centrifuged and the resulting supernatant protein samples were stored at 80  C. The protein concentration was quantified by 2-D Quant Kit (GE Healthcare). 2.3. iTRAQ analysis The iTRAQ assays were performed as described previously with minor modification [20]. Protocol-iTRAQ chemistry labeling reagents were obtained from Applied Biosystems. Control and infected tissue samples were treated in parallel throughout the labeling procedure. Samples were dissolved with 0.5 M triethylammonium bicarbonate. Each sample was incubated with 10 mL of 1 mg/mL trypsin (Promega, Madison, WI, USA) overnight at 37  C. The proteins from the infected and non-infected samples were labeled with iTRAQ reagents 115 and 116, respectively. The labeled samples were pooled and purified using a strong cation exchange choematography (SCX) column (Phenomenex, Torrance, CA, USA), and separated by liquid chromatography (LC) using a LC-20AB HPLC system (SHIMADZU). The LC fractions were analyzed by using a Triple TOF 5600 mass spectrometer (AB SCIEX). Briefly, the peptides were separated using nanobored C18 column with a picofrit nanospray tip and performed at a constant flow rate of 20 mL/min with a splitter achieving an effective flow rate of 0.2 mL/min. The mass spectrometer data acquired in the positive ion mode, with a selected mass range of 300e2000 m/z. Peptides with þ2 to þ4 charge states were selected for MS/MS. The three most abundantly charged peptides above a 5 count threshold were selected for MS/ MS and dynamically excluded for 30 s with 30 mDa mass tolerance. The fragment intensity multiplier was set to 20 and maximum accumulation time was 2 s. For peptide identification, it also has high mass accuracy (<2 ppm), fragment mass tolerance of 0.1 Da and max missed cleavages of 1. Mass values were monoisotopic and peptide mass tolerances were 0.05 Da. Other parameters used included: Gln / pyro-Glu (N-term Q), oxidation (M), iTRAQ8plex (Y), carbamidomethyl (C), iTRAQ8plex (N-term), iTRAQ8plex (K). iTRAQ data from three biological replicates were analyzed by MASCOT 2.3.02 software, then protein identification was performed using the most recently updated zebrafish SwissProt

A. Lü et al. / Fish & Shellfish Immunology 36 (2014) 229e239

database (IPI_DANRE_v3.86 database, 40470 sequences). To calculate the relative protein levels, proteins with a statistically significant label ratio of 1.2 (p < 0.05) were considered to be differentially expressed proteins. A fold change analysis was calculated as the ratio of 115e116 intensities from both experiments. 2.4. GO and pathway enrichment analysis Gene ontology (GO) enrichment analysis showed the rich GO terms of differential proteins at the background of identified proteins, which connected the differential proteins to GO databases (http://www.geneontology.org), and computed the protein number of each corresponding GO terms. Then the GO enrichment terms of differential proteins at the background of identified proteins were found out by the means of hypergeometric test (p < 0.05). As GO function enrichment analysis, pathway enrichment analysis of significant proteins was carried out using Kyoto encyclopedia of genes and genomes (KEGG) database (http://www.genome.jp/ kegg/). The pathway of significant enrichment compared with all proteins that have been tested by hypergeometric test, p < 0.05 and FDR <0.05 were used as a threshold to select significant KEGG pathways. 2.5. Microarray analysis Microarray analysis was performed according to the previous report [16]. Total RNA was extracted from the gill samples using Trizol reagent (Invitrogen), and was further purified using an RNeasy Mini kit (Qiagen) according to the manufactures’ instructions. The RNA groups were hybridized separately with the same Affymetrix GeneChip probe arrays, and compared the gene expression profiles between the infected and the control groups. The hybridization data were analyzed using GeneChip Operating Software (GCOS) version 1.4 (Affymetrix). The differentially expressed genes were identified using significant analysis of microarray (SAM) software (CapitalBio), and thus were selected on the basis of 1.2-fold changes as compared with the control groups. All experiments were repeated three times. 3. Result

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metabolic process (11.95%), biological regulation (8.29%), multicellular organismal process (7.27%), and cellular component organization or biogenesis (5.65%). In total, 82 proteins were further quantified as showing differential expression by iTRAQ analysis, including 57 upregulated proteins and 25 downregulated proteins upon A. hydrophila infection. In order to validate the results obtained from iTRAQ, the gene expression changes were further performed at the transcript level by using zebrafish cDNA microarray analysis. The genes encoding the 82 identified proteins were searched in the microarray data, and results showed that the expression trends of the corresponding genes were generally consistent with the iTRAQ data (Table 1). The functional enrichment analysis of GO annotations for biological process showed that the differentially expressed proteins identified were classified into 19 categories (Fig. 2), which included the response to stimulus (GO:0050896), immune system process (GO:0002376), and death (GO:0016265). Among them, approximately 33 (8.8%) of proteins related to stress and immune responses were identified. The upregulated proteins that were involved in gill immunity included complement component 5 (C5), serpin peptidase inhibitor clade A member 7 (SERPINA7), annexin A3a, histone H4, troponin I, parvalbumin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), creatine kinase (CK) and peroxiredoxins (PRDXs), etc. As shown in Table 2, these proteins were identified by peptide sequence analysis. The MS/MS spectrum and iTRAQ reporter ions for two representative peptides are shown in Fig. 3. 3.2. Pathway analysis The KEGG pathway analysis for the identified proteins showed that these pathways were mostly related to metabolic pathways, regulation of actin cytoskeleton, cardiac muscle contraction, oxidative phosphorylation, calcium signaling pathway, phagosome, insulin signaling pathway, peroxisome proliferator-activated receptor (PPAR) signaling pathway, vascular smooth muscle contraction, dilated cardiomyopathy, complement and coagulation cascades, and pathogenic Escherichia coli infection. Moreover, complement and coagulation cascades, pathogenic E. coli infection, and phagosome pathway were the significant pathways identified by the zebrafish gill immunity association based on KEGG enrichment analysis of 82 differential proteins. The three proposed pathways are shown schematically in Fig. 4.

3.1. Protein profiling and iTRAQ quantification 4. Discussion The zebrafish is an excellent candidate for proteomic studies as it is a well-characterized vertebrate model widely used in genetics, physiology and immunology [15]. To date, the genome of the zebrafish is fully sequenced and partially annotated, which

Number

All MS/MS spectra were processed using Mascot software. As shown in Fig. 1, iTRAQ analysis of zebrafish gill proteome showed 9260 queries in IPI_DANRE_v3.86 database (40470 sequences) resulted in 1338 protein hits in Mascot, which can be categorized into diverse functional classes related to cellular process (15.36%),

Total spectra

Spectra

Unique specra

Peptide

Unique peptide

Protein

Fig. 1. Analysis of zebrafish gill proteome profile by iTRAQ. Total spectra are the secondary mass spectrums, and spectra are the secondary mass spectrums after quality control. Unique Peptide is the identified peptides which belong only to a group of proteins, and protein is identified by Mascot 2.3.02 software.

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Table 1 Differential expression of proteins in the gills of zebrafish infected with A. hydrophila by using iTRAQ. Accession number

Protein name

Scorea

Coverageb

Peptidec

Fold changed

Microarraye

IPI00499674 IPI00864674 IPI00616127 IPI00616566 IPI00508152 IPI00511139 IPI00493852 IPI00490915 IPI00971885 IPI00482295 IPI00500040 IPI00503192 IPI00487122 IPI00487665 IPI00851874 IPI00871032 IPI00494738 IPI00627705 IPI00501227 IPI00494456 IPI00638824 IPI00488652 IPI00613480 IPI00484287 IPI00481812 IPI00504921 IPI00496071 IPI00504420 IPI00493602 IPI00508438 IPI00809369 IPI00492110 IPI00497388 IPI00501009 IPI00490850 IPI00487455 IPI00487534 IPI00500416 IPI00495240 IPI00495855 IPI00508462 IPI00508226 IPI00487033

Serpin peptidase inhibitor clade A (Alpha-1 antiproteinase) member 7 Complement component 5 Ventricular myosin heavy chain-like Cardiac myosin light chain-1 Tropomyosin alpha-1 chain Troponin I, skeletal, fast 2b.2 Troponin I, skeletal, fast 2a.3 Myoglobin Microtubule-associated protein Actin, cytoplasmic 1 Tubulin beta 2c Tubulin alpha-1C chain T-complex protein 1 subunit delta Chaperonin containing TCP1 subunit 3 (gamma) Acyl-CoA-binding domain-containing protein 7 Histone H4 Ribosomal protein S13 Visinin-like 1b Parvalbumin 6 Parvalbumin 1 Isoform 1 of Synaptosomal-associated protein 25-A Syntaxin 1B Glial fibrillary acidic protein Gefiltin Ependymin Annexin A3a Fatty acid-binding protein 10-A Fatty acid binding protein 7a ATPase, Hþ transporting, V1 subunit G isoform 1 ATPase, Caþþ transporting, cardiac muscle, slow twitch 2a ATP synthase subunit beta Solute carrier family 25 alpha, member 5 Creatine kinase-B Aspartate aminotransferase Fructose-bisphosphate aldolase C-B Glyceraldehyde 3-phosphate dehydrogenase Hexosaminidase B (beta polypeptide) Isocitrate dehydrogenase Malate dehydrogenase L-lactate dehydrogenase B-A chain Nicotinamide nucleotide transhydrogenase Peptidyl-prolyl cis-trans isomerase Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide 2 Ubiquinol-cytochrome c reductase iron-sulfur subunit Ubiquinol-cytochrome c reductase core protein I Ubiquinol-cytochrome c reductase binding protein Protein phosphatase 1, catalytic subunit, alpha Phosphorylase Peroxiredoxin 5 Peroxiredoxin 3 Zgc:85717 Zgc:65851 Zgc:152830 Si:ch211-15d5.5 Uncharacterized protein Uncharacterized protein Uncharacterized protein Fetuin B Ribosomal protein L23a Apolipoprotein B-like Apolipoprotein C-II Fibrinogen alpha chain Myozenin-2 Elongation factor-1 delta, a Heat shock protein HSP 90-beta NSFL1 cofactor p47-like isoform 2 Histone H1-like H1 histone family, member X Pdlim7 protein Hemoglobin subunit beta-2 Novel alpha-globin Myosin, heavy polypeptide 9, non-muscle Beta thymosin-like protein 2

339 81 6376 561 2434 148 183 331 97 2225 1031 898 91 67 79 752 74 151 80 422 209 156 238 135 156 79 101 257 127 700 1173 498 729 112 236 367 169 439 379 486 135 222 196

23.9 1.6 32.5 35.2 43.3 15.9 22.7 43.5 7.8 40.8 40.9 39.4 7.9 7.6 40.9 36.9 23.8 28.8 18.3 45.9 30.4 10.1 11.9 11.7 28.1 7.1 21.4 45.5 16.9 17.9 28.6 21.8 36.7 14.3 19 21.8 12.6 31 23.7 18 7.2 23.2 18

7 2 57 6 13 3 4 5 1 12 13 13 3 3 3 4 3 5 2 4 5 2 6 6 3 2 2 4 2 14 11 6 10 5 4 4 5 11 7 5 5 3 5

1.365 1.670 1.644 1.836 1.300 2.130 1.779 1.665 1.945 1.264 1.306 1.826 1.421 1.232 1.647 1.345 1.226 1.650 1.890 1.556 1.988 1.565 1.680 2.820 1.756 1.389 1.459 1.869 1.292 1.286 1.286 1.252 1.817 1.320 1.371 1.437 1.472 1.259 1.269 1.427 1.394 1.307 1.201

2.076 3.567f 1.305 4.753 1.646 2.476 1.944 1.613 1.469 1.293 1.252 1.841 1.971 1.963 e 2.034 e e 1.645 2.145 e 1.743 1.520 2.956 1.311 2.236 e 1.320 1.455 1.570 e 1.946 1.528 1.307 1.259 2.877 1.827 1.207 1.370 1.501 1.341 1.839 1.889

70 130 206 77 330 316 66 138 145 146 92 216 65 414 114 112 640 66 520 160 99 285 152 520 106 147 2716 2427 2475 55

8.1 7.8 28.8 9.4 17.6 38.4 5.8 31.9 14.2 6.2 8.4 14.2 3.6 13.7 6.6 25.8 4.8 29 29.2 20.6 9.4 18.9 13.2 27 15.6 37.2 81.8 60.1 27.1 17.4

2 4 3 2 10 4 1 5 7 2 1 3 1 5 2 4 13 2 11 4 2 9 4 5 2 5 11 7 43 1

1.221 1.413 1.321 1.254 1.261 1.239 1.794 1.317 1.936 1.237 1.244 1.294 1.272 1.747 0.827 0.813 0.576 0.458 0.814 0.687 0.675 0.830 0.742 0.826 0.744 0.786 0.452 0.412 0.822 0.788

1.907 1.268 1.802 1.988 1.229 2.842 3.877 1.615 2.502 e 1.748 e e e 0.405 0.810 0.313 0.197 0.549 e 0.327 0.496 0.515 e 0.527 0.825 e e 0.813 0.514

IPI00491457 IPI00496979 IPI00484752 IPI00492152 IPI00491623 IPI00501058 IPI00613816 IPI00507931 IPI00482009 IPI00801939 IPI00500941 IPI00499224 IPI00854046 IPI00499018 IPI00773000 IPI00491568 IPI00503447 IPI00636975 IPI00998738 IPI00931163 IPI00817230 IPI00491598 IPI00504292 IPI00851936 IPI00774304 IPI00511792 IPI00554412 IPI00631256 IPI00570082 IPI00809846

A. Lü et al. / Fish & Shellfish Immunology 36 (2014) 229e239

233

Table 1 (continued ) Scorea

Accession number

Protein name

IPI00868406

Hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Coenzyme A thiolase/enoyl-Coenzyme A hydratase, alpha subunit a Cytosolic phospholipase A2 gamma-like Zgc:112425 Zgc:92061 Zgc:194131 Zgc:77592 Uncharacterized protein Uncharacterized protein Uncharacterized protein

IPI00901276 IPI00570286 IPI00503915 IPI00483607 IPI00506010 IPI00837174 IPI00998364 IPI00501333 a b c d e f

Coverageb

Peptidec

Fold changed

Microarraye

169

6

3

0.772

0.576

210 94 524 540 3042 896 1420 148

14.6 7.6 19.4 58.8 45.8 26.7 50.1 2.2

5 1 8 13 14 4 23 8

0.775 0.823 0.778 0.457 0.796 0.715 0.717 0.788

0.569 0.752 0.589 0.285 0.418 e e e

Score indicates identification score of proteins. Peptide indicates peptide sequence number matching a protein. Coverage indicates the coverage of protein sequence. The values are calculated as the ratio of 115 (infected) to 116 (non-infected) label. Changes in corresponding gene expression are evaluated by microarrays. Proteins either with p < 0.05 and fold change >1.5 are in bold letters. Refer to complement component 6.

facilitates proteomic identification and characterization of proteins by using existing databases [19]. It is noteworthy that the gill proteome of zebrafish were recently established, and identified 5716 proteins closely related to respiration, homeostasis and energy metabolism [19]. However, little is known about the molecular mechanism at proteomic level in zebrafish gill against the infection. In the present study, the differential expressions of the gill proteome in the zebrafish were identified by using the iTRAQ approach. To obtain valuable evidence of gill proteomic changes in the zebrafish infection model, we discuss the possible relationships between these proteins and immunological effects caused by A. hydrophila infection. To our best knowledge, this is the first report on proteome of fish gill against A. hydrophila infection.

Immune system process, 1.60%

In this study, several upregulated proteins (e.g., complement, serine peptidase inhibitor, annexin, histone) were found to be related to bacterial infection and host immunity. The complement system plays a critical role in the innate defense against the pathogens. Just as in mammals, the classical, alternative and lectin mediated pathways have been characterized in fish [21]. Complement C5 is the central component in the terminal stage of the three complement activation pathways, which plays an important role in inflammatory responses and complement mediated cytolysis. Complement C5 protein was purified from the rainbow trout (O. mykiss) [22], the gene sequences encoding C5 were reported for common carp (C. carpio) and shark (Ginglymostoma cirratum) [23,24], the RNA expression level of C5 was significantly increased by stimulating carp with lipopolysaccharide (LPS) [23]. A significant Cell proliferation, 0.80%

Death, 1.60%

Positive regulation of biological process, 1.60%

Viral reproduction, 0.53% Biological adhesion, 0.53% Growth, 0.27%

Negative regulation of biological process, 1.87%

Cellular process, 16.27%

Locomotion, 3.20% Signaling, 3.47%

Metabolic process, 12.53%

Establishment of localization, 6.13% Cellular component organization, 6.40%

Multicellular organismal process, 9.33%

Response to stimulus, 8.00% Biological regulation, 8.80%

Localization, 8.27% Developmental process, 8.80%

Fig. 2. Gene ontology (GO) analysis of differentially expressed proteins in gills. A total of 82 proteins were identified as differentially expressed by iTRAQ analysis. Shown above is the classification of these proteins in different categories based on biological process.

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Table 2 Immune-related proteins identified by peptide sequence analysis in the gills of the infected zebrafish. Protein name

pI/Mwa

Serpin peptidase inhibitor clade A (Alpha-1 antiproteinase) member 7

5.16/42899.83

Complement component 5

7.95/117575.16

Annexin A3a

5.99/37305.39

Creatine kinase B

5.49/42883.59

Histone H4

11.36/11367.34

Glyceraldehyde 3-phosphate dehydrogenase

6.55/36107.40

Peroxiredoxin 5

7.61/20201.50

Peroxiredoxin 3

9.99/25875.10

a b

Peptide sequenceb

Position

(R)LIESPDYQSK(N) (K)TTVPVQMMHQYER(L) (K)VFYDVELFSK(V) (K)DLEMTVSR(Q) (K)GMGMTDMFSDK(A) (K)ADFTGVSEENIFVSK(V) (R)FDHPFMIFITDQTNDNILFVGK(V) (K)AELVADSTWIDVK(A) (R)AAIDFLMDAPLSQVR(S) (K)TLIEILTHR(S) (K)EMSGNLEELLVSIVK(C) (K)MNYSAEQEYPDLTK(H) (K)VLTPEMYANLR(D) (K)ELLDPVIEDR(H) (K)GGDDLDPNYVLSSR(V) (R)GIESLSVEALGALDGDLK(G) (R)DWPDAR(G) (K)TFLVWVNEEDHLR(V) (R)GTGGVDTAAVGGVFDISNADR(L) (R)LGFSEVELVQMVVDGVK(L) (K)RLESGQSIDDLMPEQK (R)ISGLIYEETR(G) (K)VFLENVIR(D) (R)KTVTAMDVVYALK(R) (R)TLYGFGG (R)VVVSAPSPDAPMFVMGVNQDK(Y) (K)VIHDNFGIEEALMTTVHAYTATQK(T) (R)VPVADVSVVDLTCR(L) (K)LISWYDNEFGYSHR(V) (R)LPAVEVQEEDPGNSLSMAELFSCK(R) (R)GVLFGVPGAFTPGCSK(T) (K)THLPGFIQMAGELR(A) (K)AVDLVLNNAQLIPVLGNLR(S) (R)DYGVLLEGPGIALR(L)

13e22 186e198 201e210 238e245 281e291 292e306 343e364 552e564 764e778 57e65 248e262 12e25 33e43 87e96 117e130 153e170 210e215 224e236 321e341 342e358 366e381 47e56 61e68 80e92 97e103 118e138 163e186 235e248 310e323 37e60 62e77 78e91 135e153 197e210

pI/Mw is isoelectric point and molecular weight of proteins. Peptide sequence before and after amino acids are in brackets, respectively.

increasing expression of C5 was detected in the gills of zebrafish after infection with A. hydrophila. Published results indicated that the expression of complement molecules in the common carp (C. carpio) were significantly stimulated with the parasite Ichthyophthirius multifiliis [25], and in the zebrafish were stimulated by the bacterium Citrobacter freundii [16]. Moreover, serine peptidase inhibitors (also called a1-antiproteinase) are a dominant class of proteinase inhibitors that are involved in maintaining homeostasis through multiple regulatory functions, such as the complement cascade, blood coagulation and modulation of inflammatory responses. Recently, a novel Kazal-type serpin gene was cloned from the scallop (Chlamys farreri), and serpin mRNA expression level was upregulated in hemocytes after Vibrio anguillarum challenge [26]. It was found to possess bacteriostatic activity against the Bacillus subtilis, a five-domain Kazal-type SERPIN from the shrimp (P. monodon) is involved in innate immune response [27]. Interestingly, the expression of SERPINA7 protein was significantly upregulated in gills and indicated that SERPINA7 may play a key role in local immune responses. Since there is the co-upregulated expression of C5 and SERPINA7 in gill immune response, these associations need to be further investigated in future studies. Previous studies reported that annexins have anti-inflammatory properties for bacteria stimuli in host defense [28,29]. Six different annexins (i.e., A1, A2, A4, A5, A6, and A11) were upregulated in the gills of channel catfish (Ictalurus punctatus) following infection with Edwardsiella ictaluri [29]. Furthermore, annexin A4 was upexpressed in fish gill cells interacted with E. ictaluri [30], and annexin A13 can induce protective immune response against fish gill fluke Microcotyle sebastis infection [31]. A recent study showed

that the annexin II protein was elevated in the gills of rainbow trout (O. mykiss) exposed to X rays [9]. Smith et al. [10] reported that the two-dimensional gel location of annexin max 3 in medaka (O. latipes) is similar to that of annexin II in rainbow trout, and results showed that the annexin max 3 protein expression was increased in gills exposed to direct irradiation. In this study, the annexin A3a protein was upregulated in zebrafish infected with A. hydrophila, which may be indicative of an active immune response in gills. The histones proved to actively participate in the immune defenses of fish, in addition to their important role in chromatin structure and transcriptional regulation [32,33]. High expressions of histone proteins (i.e., H2A, H2B, H3, and H4) were found in the hemocytes of shrimp (L. vannamei), and a mixture of them demonstrated the growth inhibition against Micrococcus luteus [34]. Dorrington et al. [35] reported that oyster (Crassostrea virginica) histone H4 has antimicrobial activity against the bacteria E. coli, V. anguillarum, Staphylococcus aureus and M. luteus, and histone H4 of the snail Biomphalaria glabrata was responsive to challenge with Echinostoma caproni [36]. The histone H4 protein was upregulated in the gills of zebrafish infected with A. hydrophila, while zebrafish skin exposed to C. freundii showed significant upregulation of linker histone-like protein H1M [16], also suggesting that histones could be a potential antimicrobial agent for the treatment and prevention of bacterial infection in fish [32]. In fish, gills are vital organs for the respiratory and metabolic function, and also play a role in mucous immune system. In this study, analysis of differential proteomes in the gills with and without A. hydrophila infection showed the upregulation of several

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Fig. 3. Tandem mass spectrum (MS/MS) and corresponding iTRAQ reporter ions. (A) MS/MS spectrum identifying a peptide from SERPINA7, (B) iTRAQ reporter ions for the SERPINA7 peptide shown in A; (C) MS/MS spectrum identifying a peptide from C5, (D) iTRAQ reporter ions for the C5 peptide shown in C. The MS/MS spectrum was obtained from the precursor ions (m/z 751.067, m/z 651.014) using a Triple TOF 5600 Analyzer.

enzymes of energy metabolism (e.g., GAPDH, CK, PRDX). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was recognized as a multifunctional protein displaying diverse activities with being distinct from its original role in glycolytic functions [37,38]. Comparative proteomic analysis shown that GAPDH was associated with the high susceptibility of channel catfish (I. punctatus) after exposure to E. ictaluri [39], and GAPDH was identified as upregulated protein in the buccal cavity mucus of parental tilapia (Oreochromis spp.) [40]. It is well consistent with our findings that GAPDH protein was upregulated in the gills of zebrafish against A. hydrophila infection. Therefore, GAPDHs have activities potentially involved in immune function or cellular stress responses in fish. Creatine kinase (CK) is an enzyme catalyzing the conversion of adenosine diphosphate (ADP) to adenosine triphosphate (ATP), which plays a central role in energy homeostasis in the tissues displaying high and variable rates of energy turnover such as cardiac, skeletal muscles and brain [41]. CK was identified as an acute phase protein in the amphioxus Branchiostoma belcheri [42] and distinctly elevated in yellowtail (Seriola quinqueradiata) infected with Lactococcus garvieae [43]. Notably, An et al. [41] demonstrated that CK is a bacteriostatic factor with a lectin-like activity in inhibiting the growth of E. coli. Moreover, CK was increased in the gills of medaka (O. latipes) following exposure to direct irradiation and bystander signals [10]. It is highly likely

that CK up-expressed in the gills was capable of recognizing invading bacterial pathogens and eventually protecting the fish from infection [41,42]. Peroxiredoxins (PRDXs) are antioxidant enzymes that catalyze the reduction of hydrogen peroxide, organic hydroperoxides and peroxynitrite to less reactive products [44]. PRDXs can represent a first line of defense against oxidative stress, and are also crucial in inflammatory and immune response [45]. Li et al. [46] reported that PRDX3 knockout mice were more susceptible to LPS-induced oxidative stress, and upregulation of PRDX3 was a key event of apoptosis [47]. The expression of PRDX gene was upregulated in European flounder (Platichthys flesus) injected with cadmium [48], and level of PRDX5 gene was increased in Antarctic bivalve (Laternula elliptica) induced by thermal stress [49]. The expression of PRDX3 was significantly upregulated, whereas PRDX5 remained unaltered in the head kidney of gilthead sea bream (Sparus aurata) exposed to the parasite Enteromyxum leei. Importantly, the concomitant gene expression of PRDX3 and PRDX5 was recently demonstrated in fish [44]. In this study, PRDX3 and PRDX5 protein expressions were also upregulated in the gills of zebrafish against A. hydrophila infection, which supports a role of PRDXs on the local immune defense in fish. Besides the metabolic proteins, several cytoskeleton-related proteins including actin (cytoplasmic 1), myosin heavy chainlike, myosin light chain-1, troponin I, tropomyosin alpha-1

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Fig. 4. Network pathways for association of identified proteins in gills for (A) complement and coagulation cascades, (B) pathogenic E. coli infection and (C) phagosome. The associated proteins for different pathways are shown in red frames. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

chain, tubulin alpha-1C chain, tubulin beta 2c, and microtubuleassociated protein showed apparent upregulation in the gills of zebrafish challenged by A. hydrophila. Proteomic analysis of muscle tissue from sea bream (S. aurata) was documented by means of 2-DE/MS techniques, and the differences in expression were also observed for cytoskeleton-related proteins (e.g., actin, myosins, troponin) [50]. Accumulating studies in fish have shown that cytoskeleton-related proteins (e.g., actin, tubulin, tropomyosin) fulfilled important roles in resistance to bacterial or viral infections [51,52]. Being an important component of cytoskeletal structures, different expression levels of these proteins can be

presumed to influence the formation of new phagosomes and thus affect phagocytic activity. Identification of changes in the macrophage cellular proteome induced by calcium oxalate monohydrate (COM) was performed by 2-DE/MS analyses, results enlightened the significant role of the altered proteins, including F-actin and a-tubulin in enhanced phagocytic activity, and association of HSP90 and F-actin is important for phagosome formation [53]. The increased phagosome prevalence in the gills of Chinook salmon (O. tshawytscha) contributed to mortality during exposure to abiotic stress [54]. Whether these changes could be bacteria mediated needs to be investigated in phagosome

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Fig. 4. (continued).

pathway analysis. Atlantic cod (Gadus morhua) infected with V. anguillarum showed an increase of b-tubulin in the skin mucus, possibly due to its increased expression in the mucosal cells associated with phagocytic processes [55]. Interestingly, the differential protein expressions (e.g., actin, tubulin, HSP90) were also found in the gills of zebrafish against A. hydrophila infection, indicating that they may probably activate the phagosome pathways in gill local immune responses. Additionally, zebrafish infected with A. hydrophila may result in reddened gills and rapid breathing (our unpublished data), which agreed well with the upregulation of these cytoskeleton-related proteins involved in gill-respiratory movement. In mammals, myosins were well known for their role in muscle contraction and involvement in cell motility processes. Tropomyosin, along with the troponin complex, was associated with actin in muscle fibers and regulated muscle contraction by regulating the binding of myosin [56]. The upregulated expression of them in this study indicated that these proteins might play important roles in gill response to A. hydrophila infection.

In conclusion, we have revealed that fish gill is involved in immune response against A. hydrophila infection at the proteome level. The differential proteomes of the zebrafish gill response to A. hydrophila infection were first identified and quantified by iTRAQ analysis. When compared with the proteome profiles of zebrafish gills, obvious similarities and differences have been observed [19]. Both non-infected and infected fish gills showed the similar proteomic profiles relevant to respiration, homeostasis and energy metabolism. Nonetheless, it was found the differences in the infected-gill that the potential proteins are involved in stress and immune responses, which represents a local defense mechanism of the gills and may help to clear the bacteria. Moreover, we showed some significant pathways related to complement and coagulation cascades, pathogenic E. coli infection and phagosome pathway in zebrafish after A. hydrophila infection, which contribute to understanding the molecular mechanism of the gill immune response in fish. However, further studies are needed to determine the function of the described proteins, as well as their roles in the immune response against bacteria.

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Fig. 4. (continued).

Acknowledgments This study was supported by the National Natural Science Foundation of China (No. 31272692, No. 30800847), Jiangsu Government Scholarship for Overseas Studies (2009), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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