& Comparative
Immunology
Developmental and Comparative Immunology 31 (2007) 672–686
Protein expression profiling of the shrimp cellular response to
white spot syndrome virus infection
Hao-Ching Wang
a,b, Han-Ching Wang
c, Jiann-Horng Leu
c, Guang-Hsiung Kou
c,
Andrew H.-J. Wang
a,b,d,, Chu-Fang Lo
c,a
Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan, ROC bInstitute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan, ROC
cInstitute of Zoology, National Taiwan University, Taipei 106, Taiwan, ROC dCore Facilities for Proteomics Research, Academia Sinica, Taipei 115, Taiwan, ROC Received 5 October 2006; received in revised form 30 October 2006; accepted 1 November 2006
Available online 5 December 2006
Abstract
To better understand the pathogenesis of white spot syndrome virus (WSSV) and to determine which cell pathways might be affected after WSSV infection, two-dimensional gel electrophoresis (2-DE) was used to produce protein expression profiles from samples taken at 48 h post-infection (hpi) from the stomachs of Litopenaeus vannamei (also called Penaeus vannamei) that were either specific pathogen free or else infected with WSSV. Seventy-five protein spots that consistently showed either a marked change (450%) in accumulated levels or else were highly expressed throughout the course of WSSV infection were selected for further study. After in-gel trypsin digestion followed by LC-nanoESI-MS/MS, bioinformatics databases were searched for matches. A total of 53 proteins were identified, with functions that included energy production, calcium homeostasis, nucleic acid synthesis, signaling/communication, oxygen carrier/transportation, and SUMO-related modification. 2-DE results were shown to be consistent with relative EST database data from a previously developed EST database of two Penaeus monodon cDNA libraries. For seven selected genes, 2-DE and EST data were also compared with transcriptional time-course RT-PCR data. This study is the first global analysis of differentially expressed proteins in WSSV-infected shrimp, and in addition to increasing our understanding of the molecular pathogenesis of this virus-associated shrimp disease, the results presented here should be useful both for identifying potential biomarkers and for developing antiviral measures.
r2006 Elsevier Ltd. All rights reserved.
Keywords: WSSV infection; Protein expression profiling; Host response; Proteomic analysis; White spot syndrome virus
1. Introduction
White spot syndrome (WSS) is a lethal disease that affects cultured shrimp species and many other crustaceans [1–9]. In farmed shrimp, the virus can cause 100% cumulative mortality in 2–10 days. The causative agent of WSS is an enveloped, ellipsoid, large (300 kb), double stranded DNA virus known
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E-mail addresses:[email protected] (A.H.-J. Wang),
as white spot syndrome virus (WSSV) [10–12]. WSSV has many unique properties. In addition to many non-standard proteins with functions that are not yet known, WSSV was also recently shown to have at least 39 structural proteins [13,14], one of which, WSSV664 is the largest viral protein reported to date [15]. WSS has been formally recognized since 1992, but it was only recently that the virus was designated by the International Committee on the Taxonomy of Viruses as the type species of a new genus, Whispovirus, family Nimaviridae[16].
To understand the pathogenesis of any disease, knowledge of the interactions between virus and host is critical. Virus–host interactions may result in immune responses against the invader, and may also result in changes in the expression levels of host genes that favor virus replication. To date, virus– host interactions of WSSV have been studied at the transcription level using expressed sequence tags (ESTs), RT-PCR, microarray chips, suppression subtractive hybridization and differential hybridiza-tion [17–24]. However, many of these studies focused on immune cells (lymphoid organ cells and hemocytes) and although this has provided good insights into biodefense mechanisms, these immune-related cells are not primary WSSV targets
[25–27], and their cellular pathways are, therefore, not necessarily representative of the host cells in which virus replication occurs. Further, even those studies that investigated the modulation of protein expression were not global but instead focused mainly on only a few immune-related defense genes such as beta-glycan-binding protein [28–30]. In consequence, little is known about the cellular events associated with WSSV infection in permissive cells. In the present paper we, therefore, use comparative proteomics to identify proteins whose accumulated levels in stomach cells (a main target organ of WSSV) are altered significantly after WSSV infection. Identifying these proteins is an important first step toward improving our under-standing of the cellular pathways that are necessary for WSSV infection.
Our basic approach was to use two-dimensional electrophoresis (2-DE) [31] with immobilized pH gradients (IPG)[32]to produce protein profiles for stomach cells from specific pathogen free (SPF) and WSSV-infected Litopenaeus vannamei (also called Penaeus vannamei). In addition to an invariable control (beta-actin), proteins that were either markedly up- or down-regulated or else were highly
expressed throughout WSSV infection were then identified by in-gel trypsin digestion followed by LC-nanoESI-MS/MS and a search of bioinfor-matics databases. ESTs and RT-PCR were used to confirm that changes in transcription levels over time were in good agreement with the accumulated protein (i.e. translation level) results given by the proteomic analysis. Lastly, we look at which cellular pathways might be altered and discuss the physio-logical implications of these results.
2. Materials and methods
2.1. Virus, virus inoculum and experimental animals The virus used in this study, WSSV T-1 isolate (GenBank accession numberAF440570)[8,33], was prepared from a batch of WSSV-infected Penaeus monodon collected in Taiwan in 1994. To prepare the WSSV inoculum, we first selected one of the original, frozen (80 1C) 1994 P. monodon speci-mens that tested PCR positive for WSSV, but tested negative for other shrimp viruses (infectious hypo-dermal and haematopoietic necrosis virus [IHHNV], Taura syndrome virus [TSV], yellowhead disease virus/gill associated virus [YHV/GAV], P. monodon-type baculovirus [PMBV], mourilyan virus) using commercial PCR detection kits (Farm-ing IntelliGene Tech. Corp., Taiwan). Carapace and integument tissues (0.5 g) from this frozen specimen (body weight 30 g) were minced and then homo-genized in 4.5 ml of sterile PBS buffer. After centrifugation (400g, 10 min, 4 1C), the supernatant was filtered through a 0.45 mm membrane and used immediately to infect an adult, SPF L. vannamei (body weight 45 g; High Health Aquaculture. Inc, Hawaii) by injection (200 ml) as described previously
[26]. At 24 hours post-infection (hpi), hemolymph was extracted from this moribund shrimp, diluted 4 with phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM
KH2PO4), and frozen at 80 1C for use as virus
stock. The experimental inoculum was then pre-pared from the supernatant of this stock after centrifugation at 400g for 10 min at 4 1C and further dilution (102) with PBS [34].
Since it is still very difficult to acquire P. monodon specimens that are SPF, the experimental shrimp for the 2-DE/MS studies were the SPF offspring (mean body weight 2.6 g) of SPF L. vannamei brooders purchased from High Health Aquaculture, Inc., Hawaii. These shrimp were bred and cultured at the
Marine Research Station, Academia Sinica, Tai-wan. The disease-free status of randomly selected samples of the experimental shrimp was confirmed using the same commercial PCR detection kits for WSSV, IHHNV, TSV, YHV/GAV, PMBV and mourilyan virus (see above), and the shrimp were then challenged with WSSV (100 ml/shrimp) by intramuscular injection following Tsai et al. [26]. Shrimp injected with PBS vehicle only were used as controls. The experimental shrimp (n ¼ 480) were kept in 2.5 l tanks (30 shrimp/tank) containing filtered, aerated seawater (33% salinity; 1.8 l sea-water/tank) at constant temperature (2871 1C). Every 8 h, the tanks were checked, and dead and moribund shrimp were removed. A parallel study (data not shown) found that cumulative mortality reached 40–50% at 48 hpi, and at this time, the stomachs (and other tissues) of the active (as opposed to moribund) surviving infected and control shrimps in all but 4 of the tanks were collected and frozen using liquid nitrogen. For the time-course study, SPF L. vannamei offspring (n ¼ 40; mean body weight 9 g) were experimentally infected using the method described above, and sample specimens were collected at 0, 6, 12 and 36 hpi.
2.2. Two-dimensional electrophoresis
For each 2-DE, the frozen stomachs from three shrimp were ground to a fine powder at 80 1C. The powder was then suspended in a three-fold dilution of PBS buffer containing protease inhibitor cocktail (applied according to the manufacturer’s protocol, Roche Diagnostics, Mannheim, Germany). After centrifugation at 3000g (30 min, 4 1C), the super-natant was collected and a TCA/DTT mixture was added (final concentration: 10% w/v TCA and 0.1% DTT). After standing on ice for 30 min and another centrifugation (10,000g, 30 min, 4 1C), the supernatant was discarded and the pellet resus-pended in acetone containing 0.1% DTT. The sample was spun again (10,000g, 30 min, 4 1C), and the pellet-dried under vacuum and then solubilized in rehydration buffer (9.8 M urea, 2% CHAPS, 20 mM DTT, 0.5% IPG buffer [pH 4–7 or 3–10; Amersham Biosciences]). After a final cen-trifugation (10,000g, 30 min, 15 1C), the superna-tant, which contained the soluble protein fraction, was used as a 2-DE sample. Protein concentration of 2-DE samples was estimated using a 2-D Quant Kit (Amersham Biosciences).
The first dimension of the 2-DE, isoelectric focusing (IEF), was performed in 13 cm Immobiline DryStrip gel (Amersham Biosciences) using an integrated system, the Ettan IPGphor (Amersham Biosciences), where rehydration with the sample and IEF are performed automatically. Two pH gradient strips were used: linear pH 4–7 and 3–10. Each sample (250 mg protein) was dissolved in 250 ml rehydration buffer with a trace of bromophenol blue and placed in the base well of an IPGphor stripholder. An IPG strip was then placed on the top of the sample, and after rehydration in the IPGphor (16 h at 50 V), automatic IEF was performed using the following step voltage focusing protocol: 1 h at 300 V, 1 h at 500 V, 2 h at 1000 V, 2 h at 4000 V and 10 h at 8000 V. All the above procedures were carried out at 20 1C. After the first dimensional IEF, the IPG strips were equilibrated in a sodium dodecyl sulfate (SDS) equilibration buffer
(6 M urea, 2% SDS, 30% glycerol, 50 mM
Tris–HCl, pH 8.8) containing 1% DTT for 15 min. The IPG gel strips were then removed to another equilibration buffer containing 2.5% iodoacetamide and equilibrated for a further 15 min. The equili-brated IPG strips were then placed onto a poly-acrylamide gel that consisted of 14% poly-acrylamide, pH 8.8, for the separating gel, and 4% acrylamide, pH 6.8, for the stacking gel. The second dimensional separation was run at 20 mA per gel at 15 1C for 5–6 h. At the end of each run, the gels were stained with sypro ruby, and the protein patterns of the gels were scanned using a Typhoon 9400 scanner (Amersham Biosciences). Gel image matching was done using PDQuest software (Bio-Rad).
2.3. In-gel protein digestion and protein identification Protein spots of interest were manually excised from the gels, washed twice with 25 mM ammonium bicarbonate buffer (pH 8.5) in 50% acetonitrile, for 15 min each time, dehydrated with 100% acetoni-trile for 5 min, vacuum dried, and rehydrated with 100 ng of sequencing-grade, modified trypsin (Pro-mega) in 25 mM ammonium bicarbonate, pH 8.5, at 37 1C for 16 h. Following digestion, tryptic peptides were extracted twice with 5% formic acid in 50% acetonitrile for 15 min each time with sonication. The extracted solutions were pooled and evaporated to dryness under vacuum. Samples were dissolved in 0.1% formic acid in 50% acetonitrile and analyzed by LC-nanoESI-MS/MS. Proteins were identified by MS/MS ion search using the search program
MASCOT and the NCBI protein and EST sequence databases. For MS/MS ion search, the mass tolerance parameter was 0.25 Da, MS/MS ion mass tolerance was 0.25 Da, and up to one missed cleavage was allowed. Variable modifications con-sidered were methionine oxidation and cysteine carboxyamidomethylation. Significant hits (as de-fined by Mascot probability analysis) were regarded as positive identification.
2.4. EST-based transcription analysis of identified protein genes
This analysis used two cDNA libraries, PmTwI (WSSV-infected) and PmTwN (non-infected). To construct these libraries, RNA was extracted from P. monodon postlarvae (PL20) that had either been exposed to WSSV by immersion 66 h previously (for PmTwI), or else were left unchallenged (for PmTwN). From the extracted RNA, cDNA was synthesized by RT-PCR, and the two libraries were then constructed using a l-Zap II vector construc-tion kit (Stratagene), followed by conversion to the pBluescript plasmid by mass excision according to the manufacturer’s instructions. From these libraries, a total of 14152 ESTs (7335 from PmTwI; 6817 from PmTwN) were generated by subjecting randomly selected clones to 30sequencing. The raw traces were base-called by running Phred (Q413), and the resultant sequences were then masked for pBluescript vector and WSSV (AF440570) using the ‘‘Cross_match’’ package with default parameters (minimatch 12, penalty 2, minscore 20). The Prap assembly program produced a total of 2183 and 2075 unique sequences from PmTwI and PmTwN, respectively, and these were then checked for matches in the GeneBank nr (non-redundant) peptide sequence databases (http://www.ncbi. nlm.nih.gov) and SWISS PROT (http://www.ebi. ac.uk/swissprot/) using BlastX and InterPro Scan with default parameters. After checking, there were still 2155 unique sequences that remained un-matched, but these were further subjected to 50 sequencing and analysis as above, and ultimately all but 1056 unique sequences were successfully identi-fied. After the genes had been identified, the gene ontology (GO) database (http://www.geneontology. org/) was used to classify the ESTs by biological process, cellular component, and molecular func-tion. In addition, the Kyoto Encyclopedia of Genes and Genomes (KEGG http://bioinfo.weizmann. ac.il:3456/kegg/kegg.html)[35] was used to classify
the genes according to their biochemical roles. To identify genes that might be differentially expressed, the number of EST clones that matched the gene in each library was expressed as a percentage relative to the total number of ESTs in the same library. 2.5. Time-course RT-PCR
WSSV-challenged L. vannamei were sampled at 0 (i.e., immediately before infection), 6, 12 and 36 hpi. Total RNA was extracted from the stomachs of the L. vannamei harvested at each time point. The stomachs (n ¼ 3) from each time point were pooled, purified with TRIzol Reagent (Invitrogen) and then treated with RNase-free DNase I (Roche) to remove any residual DNA. First strand cDNA synthesis was performed using the oligo-dT primer, and 2 ml (1 mg) of the cDNA was subjected to PCR in a 50-ml reaction mixture containing an appropriate primer pair (Table 1). For comparison, an ICP11 gene fragment was also amplified from the same tem-plates by the primer pairs ICP11-F/ICP11-R. A shrimp beta-actin primer set, actinF1/actinR1, was used as an internal control for RNA quality and amplification efficiency. To confirm there was no WSSV DNA contamination of the RNA samples, a WSSV genomic DNA-specific primer pair, IC-F2/ IC-R3, derived from an intergenic region of the WSSV genome, was also used as a quality control. 3. Results
3.1. 2-DE analysis and protein identification of protein spots in stomach cells of WSSV-infected shrimp
Mortality among the WSSV-infected shrimp approached 50% at 48 hpi and at this time, the stomachs of surviving PBS control (mock-infected) and WSSV-infected shrimp were collected and subjected to 2-DE. After staining with sypro ruby, automatic detection of the protein profiles revealed 500 protein spots (Fig. 1). A total of 75 eligible spots (i.e. spots that were present in three replicates) were subjected to LC-nanoESI-MS/MS and submitted to database searches for peptide matching and protein identification.Table 2 showed the 53 protein spots that were successfully identified. Some proteins were identified in more than one spot (e.g. glyceralde-hydes-3-phosphate dehydrogenase in spots 37, 38 and 43). For the known proteins, the experimental
and theoretical MW and pI were mostly in close agreement (Table 2).
3.2. Differential analysis of WSSV-infected and mock-infected stomach cells
For normalization purposes, we regarded beta actin as an invariable protein in response to WSSV infection because its mean intensity (spot 20) in the four replicates for the PBS controls (96,497; 15,246,292; 18,453,858; 15,785,132; mean ¼ 12,395,445) was always very close (within 5%) to the four corresponding replicates for the WSSV-infected samples (99,023; 16,145,483; 19,094,090; 17,171,348; mean ¼ 13,127,486). To normalize the spot intensities before they were compared, the intensity of each protein spot as measured in the gels was corrected by reference to the beta-actin protein intensity in the same gel. The changes in accumu-lated protein levels for the 53 spots were presented as ratios (7standard deviation) that represent the normalized, accumulated protein expression levels at 48 hpi relative to the mock-infected PBS controls. As shown in Table 2, WSSV infection for 48 h caused no change (defined aso50% change, ratios between 0.67 and 1.5) for 24 spots, while the other 27 spots showed at least a 50% increase (ratios41.5) or decrease (ratioso0.67) in intensity. The differentially expressed genes in the stomach included only one WSSV gene, the non-structural
protein ICP11 (spot1), which was strikingly in-creased. Differentially expressed host genes had functions that included: chitin and chitodextrin hydrolysis (up-regulation of chitinase [spot 3]); carbohydrate metabolism and/or energy production (up-regulation of cytochrome oxidase polypeptide VIb [spot 16], enolase [spots 32 and 33], the alpha and beta subunits of mitochondrial ATP synthase [spots 7 and 47], triosephosphate isomerase [spot 24; but spot 31 was unchanged], glyceraldehydes-3-phosphate dehydrogenase [spots 37 and 38; but spot 43 was unchanged] and fructose biphosphate aldolase [spot 41; but spots 42 and 48 were unchanged]); nucleic acid synthesis (up-regulation of nucleoside diphosphate kinase [spot 46]); the major pathway for metabolite flux through the mitochondrial outer membrane (up-regulation of voltage-dependent anion-selective channel protein 2 [spot 51]); signaling/communication (up-regulation of a 14-3-3 like protein [spot 11] and a putative activated protein kinase C receptor [spot 50]); oxygen carrier/transportation (up-regulation of hemocyanin [spot 28]); structure/mobility (up-reg-ulation of Rab GDP dissociation inhibitor [spot 18] and down-regulation of calponin [spot 45]); tricar-boxylic-acid (TCA) cycle (up-regulation of aconi-tase [spot 27] and isocitrate dehydogenase [spot 30]); calcium homeostasis (up-regulation of sarco/endo-plasmic reticulum (ER)-type calcium-transporting ATPase [spot 6], and down-regulation of a calcium Table 1
Primers used in temporal RT-PCR analysis
Target gene Primer name Primer sequence (50–30)
Glycoaldehyde-3-phosphate dehydrogenase GAPDH-F 50-CGAGTGCTCCTACGATGATCAAGG-30 GAPDH-R 50
-GTACTTAGCGTCGAAGATGGAGGACC-30 Fructose-bisphosphate aldolase Aldolase-F 50-CAACGTTGAGAACACCGAGGAGAACC-30
Aldolase-R 50
-GTTCTTGCCGATCTTCAGGACACAGC-3 Enolase Enolase-F 50-CAACCAGATTGGCAGTGTGACAGAGTC-30
Enolase-R 50-CAAACTTAGCATTGCCTCCAAGCTCCTCC-30 Cytochrome C oxidase polypeptide VIb COX6b-F 50-GCATATGTCTGAGGAAGCTAAAATGGAAACTG-30
COX6b-R 50-CCTCGAGTCCTGGGAAGATTCCATTATC-30 Calcium-binding protein alpha-B and -A chains, SCP alpha chain CBP-F 50
-CCAGGGCAAGAAATACGGCGAATTCC-30 CBP-R 50-CGCTGCAAGACTCATCAGGGTTGG-30
ICP11 ICP11-F 50
-CCATATGGCCACCTTCCAGACTGAC-30 ICP11-R 50-CCTCGAGTTCTGTTGTTGGCACAATC-30 Beta actin Actin-F 50-GAYGAYATGGAGAAGATCTGG-30
binding protein of the invertebrate sarcoplasmic calcium-binding protein (SCP) family [spot 13]); calcium binding chaperone (up-regulation of calreticulin [spot 2]); SUMO related modification (down-regulation of a small ubiquitin-like modifier [spot 14]); carotenoid astaxanthin binding (down-regulation of crustacyanin A2 subunit [spot 25]); and digestion related activity (down-regulation of trypsin [spot 4], carboxypeptidase B [spot 8] and chymotrypsin [spot 12]).
WSSV infection did not affect the accumulated levels of several other important proteins with functions that include cellular components (tropo-myosin isoforms [spot 9], beta-actin [spot 20] and actin-depolymerizing factor [spot 26]), the ATP buffering system/resistance to environmental stres-ses (cytosolic malate dehydrogenase [spots 21 and 23] and arginine kinase [spots 35 and 36]), protein folding activity (protein disulfide isomerase [spot 29]), transportation (intracellular fatty acid-binding Fig. 1. 2-DE protein profiles of the stomachs of experimental shrimp. (A) IPG 3-10, PBS control, (B) IPG 3-10, WSSV infected, (C) IPG 4-7, PBS control, (D) IPG 4-7, WSSV infected. Eligible protein spots that showed consistent expression change or were constant during WSSV infection are circled. Numbers correspond to the entries inTable 2.
Ta ble 2 Pr otein identifi cation by LC-nan oESI -MS/MS Sp ot Prote in name (specie s) Acc. No. MS/m ps a Predicte d MW( kDa)/ PI Obs erved MW (kDa)/ PI After infe ction b Up-re gulate d prote in spots 1 ICP 11/WS S V 285 (Wh ite Spot Synd rome Virus ) AAL89 153 147/3 9.21/4.20 13.70 /3.40 4 1000 fold 2 Calre ticuli n (Litopen aeus van namei ESTs) CK57249 2 365/1 0 — 78.34 /3.90 1.52 7 0.23 3 Chitin ase (Litopen aeu s vannam ei ) AAN74647 387/9 51.93/ 4.87 69.37 /4.26 2.55 7 0.56 6 Sar co/endo plasmic reticulu m-type calcium-tran sportin g ATP ase (Artemia sp) CAA3 5980 63/1 110.27/ 5.00 103.8 8/4.97 3.34 7 0.55 7 A T P synthas e beta subunit (Bombyx mori ) ABF5 1410 696/1 6 54.95/ 5.26 58.22 /4.68 1.61 7 0.24 11 14-3-3b protein (Geod ia cydoniu m ) CAA7 5860 151/4 28.36/ 4.88 30.05 /4.73 2.10 7 0.47 16 Cytoc hrom e c oxidas e polyp eptide VIb (Litop enaeus vannam ei EST s) BF02 4205 82/2 — 13.06 /4.80 4.52 7 0.53 18 GDP dissoc iation inh ibitor (Aed es aeg ypti) EAT3 4894 203/4 49.85/ 5.38 51.11 /5.74 1.81 7 0.35 24 Triose phos phate isomerase (Arch aeopotam obius sibiriensis ) CAD 29196 133/2 24.36/ 5.33 29.19 /5.69 1.81 7 0.47 27 Acon itase (Daph nia pulex ) CAB723 17 126/4 85.83/ 7.03 90.95 /6.12 1.56 7 0.15 28 Hemo cyanin (Litopen aeu s vannam ei ) CAA5 7880 247/6 74.93/ 5.27 76.51 /5.80 2.63 7 0.54 30 Isocit rate dehydro genase (Tribolium casta neum ) XP_97 0446 316/9 48.95/ 8.72 50.00 /6.22 1.51 7 0.49 32 Enol ase (Penaeus monodon ) AAC7 8141 934/3 4 47.24/ 6.18 53.35 /6.56 1.64 7 0.25 33 Enol ase (Penaeus monodon ) AAC7 8141 549/1 3 47.24/ 6.18 49.14 /7.10 1.58 7 0.31 37 Gly ceraldeh yde-3-p hosp hate de hydroge nase (Homaru s ame ricanus ) 1GPD_R 185/3 35.80/ 6.29 37.99 /6.58 1.53 7 0.33 38 Gly ceraldeh yde-3-p hosp hate de hydroge nase (Procam barus clar kii ) BAC770 82 184/3 35.69/ 6.54 39.55 /7.00 1.59 7 0.52 41 Fruc tose-bisphosp hate aldolas e (Trib olium casta neum ) XP_97 5842 238/6 39.72/ 7.6 45.33 /7.10 1.83 7 0.18 46 Nucle oside diphosp hate kin ase (Litopen aeus van namei ESTs) CX535 995 322/1 3 — 18.09 /7.49 1.53 7 0.23 47 Mit ochondria l A T P syn thase alph a subunit precurso r (Bombyx mori ) ABD3 6284 1021 /33 59.61/ 9.21 60.70 /8.43 2.16 7 0.27 50 Put.ac tivated protein kinase C recep tor (Litopen aeu s styliros tris EST s) CD52667 3 263/6 — 38.63 /8.15 2.33 7 0.43 51 Voltag e-dep endent anion-selective channe l protein 2 (C arcinus mae nas EST s) CX994 499 390/1 1 — 33.27 /9.14 1.63 7 0.43 Dow n-regulat ed prote in spots 4 Trypsi n (Litop enaeus vannam ei ) CAA6 0129 247/1 0 28.23/ 4.37 37.20 /3.68 0.50 7 0.04 8 Carb oxypeptid ase B (Penaeus mono don EST s) EB39 0086 345/6 — 28.54 /7.71 0.63 7 0.19 12 Chymo tryps in BII precu rsor (Litopen aeus vannam ei ) P3617 8 115/2 28.70/ 4.98 26.73 /4.79 0.48 7 0.05 13 Calciu m-bindin g protein alpha-B and -A chains, SCP alpha chain (Pe naeus sp) P0263 6 513/1 5 21.97/ 4.58 22.03 /4.59 0.54 7 0.27 14 Small ub iquitin-lik e modifie r (Litop enaeus van namei EST s) BQ1081 64 201/4 — 17.20 /4.84 0.65 7 0.06 22 Actin -1 (Penae us mono don ) AAC7 8681 140/3 41.77/ 5.23 32.77 /5.33 0.47 7 0.11 25 Crus tacyanin A2 subu nit (Litop enaeus vannam ei EST s) CV4681 94 139/2 — 23.51 /5.75 0.63 7 0.12 45 Calp onin (Litop enaeus vannam ei EST s) CK59206 4 629/1 2 — 22.81 /7.09 0.64 7 0.03 C onstant prote in spots 5 Trypsi n (Litop enaeus vannam ei ) CAA6 0129 234/9 28.23/ 4.37 34.70 /3.82 0.83 7 0.24 9 Slow tropomyo sin isoform (Hom arus america nus ) AAC4 8287 779/1 6 32.89/ 4.47 38.76 /4.67 1.02 7 0.19 10 Pream ylase 1 (Litop ena eus van namei ) CAA5 4524 366/1 6 56.96/ 5.21 32.83 /4.98 1.01 7 0.24 15 Cyclic AMP-regulate d pro tein like protein (Marsu penaeu s japon icus ) BAB8 5575 149/3 17.05/ 5.39 15.22 /5.01 0.87 7 0.13 17 Valo sin containin g protein -1 (Eise nia fetida ) BAD9 1024 464/8 89.58/ 5.23 96.06 /5.46 1.38 7 0.12 19 Actin 1 (Penae us monodon ) AAC7 8681 693/2 4 41.71/ 5.23 45.51 /5.37 1.14 7 0.11
20 Bet a-actin (Homaru s gamm arus ) CAE467 25 490/2 1 41.84/ 5.30 46.38 /5.47 1.00 7 0.00 21 Cytoso lic malate dehy droge nase (C aenorha bditis brigg sae ) CAE718 99 264/7 35.86/ 6.46 40.74 /6.06 1.09 7 0.11 23 Cytoso lic manga nese superox ide dism utase (Penaeus mono don ) AAW5 0395 402/8 31.36/ 5.46 32.25 /5.86 0.86 7 0.08 26 Actin -depolym erizing facto r (Anoph eles gamb iae ) EAA03 029 237/1 0 17.79/ 8.57 18.42 /6.25 1.20 7 0.19 29 Prote in disulfi de-isomerase (Penae us monodon EST s) EB39 0086 395/8 — 65.80 /5.87 0.92 7 0.14 31 Triose phos phate isomerase (Arch aeopotam obius sibiriensis ) CAD 29196 249/8 24.36/ 5.33 28.47 /6.19 0.98 7 0.12 34 Isocit rate dehydro genase (Anoph eles gamb iae ) XP_97 0446 278/4 48.95/ 8.72 51.02 /6.89 0.91 7 0.05 35 Argini ne kinase (Penae us mono don ) AAO 15713 1003 /39 40.09/ 6.05 42.05 /6.75 1.06 7 0.05 36 Argini ne kinase (Penae us mono don ) AAO 15713 793/2 5 40.09/ 6.05 42.54 /6.50 1.03 7 0.44 39 Catalas e (Litop ena eus van namei ) AAR 99908 251/9 57.63/ 6.71 64.42 /7.35 1.34 7 0.57 40 Acet ylcholine stera se C (Nipp ostron gylus brasili ensis ) AAF04 599 56/2 64.63/ 4.78 58.37 /7.58 1.02 7 0.06 42 Fruc tose 1,6-bisphosp hate aldolas e (Trib olium casta neum ) XP_97 5842 236/8 39.72/ 7.60 45.14 /7.62 1.37 7 0.24 43 Gly ceraldeh yde-3-p hosp hate de hydroge nase (Homaru s ame ricanus ) 1GPD_R 393/1 3 35.80/ 6.29 39.24 /7.25 1.44 7 0.58 44 Pho sphoglyc erate mutase (Bombyx mori ) ABA00 463 214/9 28.60/ 6.33 31.76 /7.31 1.11 7 0.08 48 Fruc tose 1,6-bisphosp hate aldolas e (Trib olium casta neum ) XP_97 5842 210/7 39.72/ 7.60 44.14 /8.41 1.32 7 0.15 49 Asp artate amino transferas e (Aede s aegypti ) AAQ 02892 127/6 47.23/ 9.14 43.95 /8.69 0.86 7 0.04 52 Cyclop hilin A (Litop enaeus vannam ei EST s) BQ1083 95 673/2 5 — 17.30 /8.54 1.01 7 0.30 53 Intra cellular fatty acid bindin g protein (Pacifastacus leniu sculus ) ABE7 7153 147/1 0 15.31/ 7.68 15.44 /8.40 1.14 7 0. aMS/m ps: M owse Sc ore/ma tched peptides. bData represen t rat ios (7 standar d deviat ion) as calculated from eith er 3 o r 4 replica tes of the normalized, accumu lated prot ein exp ression levels at 48 hpi relativ e to the nor m alized, acc umulat ed expre ssio n leve ls of the PBS contro l. Norm alizat ion of the raw data was calc ulated from the accum ulated beta-actin leve ls on the same gel .
protein [spot 53]), antioxidant activity (catalase [spot 39]) and amino acid catabolism (aspartate aminotransferase [spot 49]).
3.3. EST analysis of gene transcription levels Out of the 53 protein spots that were identified, 39 spots (a total of 30 genes) were cross-referenced in two P. monodon cDNA libraries and EST databases, PmTwI and PmTwN (Table 3). The relative EST abundances of 11 of these protein genes were quite consistent with the corresponding changes in accumulated protein levels in Table 2, namely: the increased transcription levels of enolase, fructose biphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), mitochon-drial ATP synthase and nucleotide diphosphate kinase; the decreased transcriptional level of cal-cium-binding protein alpha chain, SUMO proteins, carboxypeptiase B, chymotrypsin BI, trypsin and crustacyanin A2. From this we conclude that regulation of the accumulated protein levels of these genes is mediated by regulation at the transcription stage. We note, however, that for two genes, the data were inconsistent in the two tables: tropomyo-sin and intracellular fatty acid-binding protein were both down-regulated according toTable 3, but their protein levels were unchanged inFig. 1andTable 2. Other genes (including phosphoglycerate mutase and calreticulin) were not sufficiently well represented in the EST databases to derive mean-ingful data.
3.4. Time-course transcriptional and translational analysis of selected differentially expressed genes
Time-course RT-PCR assays were used to confirm the expression levels of several genes that were consistently up- or down-regulated in both the proteomic analysis and EST database analysis. Three of the selected up-regulated genes were for three enzymes in the glycolytic pathway (glyceraldehydes-3-phosphate dehydrogenase, fructose biphosphate aldolase and enolase). Also selected were the down-regulated gene for the calcium-binding protein SCP alpha chain, the energy production-related gene for cytochrome c oxidase polypeptide VIb (which had up-regulated protein levels [Table 2], but which was only represented once in the EST databases), and the WSSV gene for the non-structural protein ICP11. The differential mRNA levels of these genes in the time-course RT-PCR
(Fig. 2) showed that the transcription levels of the calcium-binding protein decreased dramatically at the late phase (12 and 36 hpi), which is in good agreement with the two-fold decrease found in the EST and proteomic analyses. The other RT-PCR results were also in broad agreement with the
EST and proteomic analyses: GAPDH and
fructose biphosphate aldolase and enolase-produced transcripts that increased at 6 hpi and remained fairly constant thereafter, and increases in the cytochrome c oxidase subunit VIb and WSSV non-structural protein ICP 11 transcripts were also observed.
The 2-DE time course results for the same 7 genes (Figs. 3A and B) are also broadly in agreement with the proteomic, EST and RT-PCR analyses. The expression levels of GAPDH (spots 37 and 38), fructose biphosphate aldolase (spot 41), enolase (spots 32 and 33), and cytochrome c oxidase subunit VIb (spot 16) were all increased after WSSV infection. However, the calcium-binding protein SCP alpha chain (spot 13), WSSV infection resulted in an increase at 12 hpi, but a decrease thereafter (Figs. 1, 3A and B). The divergence between the RT-PCR and 2-DE results for this gene may be due to the different regulation mechanisms for tran-scription and translation.
4. Discussion
The aim of this study was to characterize host protein expression changes in shrimp stomach cells after WSSV infection. To do this, we combined proteomic and EST approaches to explore these interactions at the molecular level. Both of these technologies are powerful, high through-put tools that can quickly identify differentially expressed genes associated with a disease at the transcriptional and (in effect) the translational levels, respectively. A better understanding of host response to WSSV will help to elucidate this unique pathogen’s mechanisms of virulence and pathogenesis, and here we identified 26 host proteins and 1 WSSV protein with altered abundance at the mRNA and/or protein levels after WSSV infection. Most of these proteins have important biological roles in the cell, and should be useful for identifying potential biomarkers as well as development of antiviral measures. We now attempt to interpret the possible biological significance of the observed infection-induced changes.
Table 3
Increase/decrease in relative EST abundance of protein genes in the PmTwI (WSSV infected) and PmTwN (non-infected) Penaeus monodon postlarva cDNA libraries
Spot Gene name PmTwIa
(No/ percentage%)
PmTwNb
(No Percentage%)
Relative abundance after infectionc Glycolysis 32, 33 Enolase 26/0.355 16/0.235 1.51 41, 42, 48 Fructose-bisphosphate aldolase 6/0.082 3/0.044 1.86 37, 38, 43 Glyceraldehyde-3-phosphate dehydrogenase 11/0.150 1/0.015 10.00 44 Phosphoglycerate mutase 1/0.014 0/0 N 24, 31 Triosephosphate isomerase 5/0.068 5/0.073 0.93 Calcium homeostasis 2 Calreticulin 1/0.014 0/0 N
13 Calcium-binding protein alpha chain 18/0.245 41/0.601 0.41 Signaling/communication
11 14-3-3b protein 1/0.014 0/0 N
Energy production
16 Cytochrome c oxidase polypeptide VIb 1/0.014 0/0 N 7 Mitochondrial ATP synthase beta subunit 3/0.041 0/0 N TCA cycle
21 Cytosolic malate dehydrogenase 4/0.041 1/0.015 2.73 30, 34 Isocitrate dehydrogenase 3/0.041 4/0.059 0.69 Cellular component
20 Actin 62/0.845 66/0.968 0.87
9 Slow tropomyosin isoform 3/0.041 22/0.322 0.13
ATP buffering system/resistance to environmental stress
35, 36 Arginine kinase 42/0.572 32/0.469 1.22
Protein folding activity
29 Protein disulfide-isomerase 2/0.027 1/0.015 1.80 Transportation
53 Intracellular fatty acid binding protein 0/0 6/0.088 0 Antioxidant activity
23 Superoxide dismutase like protein 2/0.027 1/0.015 1.80 Amino acid catabolism
49 Aspartate aminotransferase 1/0.014 0/0 N
SUMO related modification system
14 Small ubiquitin-like modifier 2/0.027 4/0.059 0.46 Structure/mobility
26 Actin-depolymerizing factor 0/0 1/0.015 0
Digestion related activity
8 Carboxypeptidase B 0/0 2/0.029 0
3, 39 Chitinase 3/0.041 2/0.029 1.41
12 Chymotrypsin BI 2/0.027 11/0.161 0.17
10 Preamlase 0/0 4/0.059 0
4, 5 Trypsin 4/0.041 101/1.480 0.03
Major pathway for metabolite flux through the mitochondrial outer membrane
51 Voltage-dependent anion-selective channel protein 2 0/0 1/0.015 0 Carotenoid astaxanthin binding activity
25 Crustacyanin A2 subunit 4/0.041 13/0.101 0.41
Nucleobase, nucleoside and nucleotide interconversion
46 Nucleoside diphosphate kinase 37/0.504 2/0.029 17.38 ATP binding/Caspase activation
17 Valosin containing protein-1 1/0.014 0/0 N
aTotal ESTs ¼ 7335. bTotal ESTs ¼ 6817.
cThe relative abundance of a gene transcript is defined as the percentage of the ESTs matched to a gene in PmTwI divided by the percentage of the ESTs matched to the same gene in PmTwN.
4.1. Energy production
Our present results show a marked or moderate increase in accumulated protein levels in key glycolytic enzymes (glyceraldehydes-3-phosphate dehydrogenase, fructose biphosphate aldolase and enolase). Up-regulation was also observed in the oxygen carrier hemocyanin, in the electron transport chain protein cytochrome c oxidase polypeptide VIb (a subunit of cytochrome oxidase, the terminal enzyme of the respiratory chain that transfers electrons to molecular oxygen, which then reacts with hydrogen ions to form water), and in both the alpha and beta subunits of mitochondrial ATP synthase. Taken together, the increased avail-ability of oxygen and the increased levels of some key glycolytic enzymes and oxidative respiration enzymes would suggest that oxidative respiration was enhanced in WSSV-infected cells. If so, this would allow the infected cells to increase their energy yield by extracting energy from glucose through aerobic respiration to yield ATP. Bearing in mind that WSSV is an extremely virulent pathogen with a very rapid onset [4], it seems probable that the up-regulation of these energy production-related proteins might not only meet the requirement of a large burst of oxygen and energy during rapid virus replication, but would also facilitate energy-dependent detoxification (which
would help the cells tolerate the environmental changes caused by WSSV infection) as well as other energy-dependent biological processes. Improving the survival capability of the infected cells also benefits WSSV because cell-survival is essential for successful virus replication.
4.2. Nucleic acid synthesis
In addition to energy production, we also note that several of the proteins that are up-regulated after WSSV infection have roles in energy-depen-dent processes. For example, when nucleotide triphosphates (NTPs) are synthesized from nucleo-tide diphosphates (NDPs), the high-energy phos-phate transfer from ATP proceeds via the phosphorylated NDP kinases, and NDP kinase is up-regulated after WSSV infection. NDP kinases provide NTPs for nucleic acid synthesis, CTP for lipid synthesis, UTP for polysaccharide synthesis and GTP for protein elongation, signal transduction and microtubule polymerization. NDP kinases are also involved in cell growth, differentiation, and tumor metastasis [36], and the control of endocy-tosis through the regulation of dynamin[37]. All of these functions are potentially important for WSSV replication.
Fig. 2. Temporal RT-PCR transcription analysis for 7 selected genes. These time-course results are quite consistent with the corresponding changes in accumulated protein levels.
4.3. Calcium homeostasis
Through the course of the virus infection cycle, the intracellular Ca2+ concentration is critical for events such as virus infection and capsid transpor-tation to the nucleus [38]. Moreover, increases in intracellular Ca2+ concentration have been asso-ciated with many host cell defense responses, such as apoptosis. The modulation of calcium related proteins during WSSV infection is, therefore, likely to be important.
Our present results show the protein expression levels of three calcium-related proteins were altered after WSSV infection. Expression of sarco/ER-type calcium pump-ER Ca2+-ATPase was up regulated
about 7-fold. This protein is located on the ER membrane and its function is to refill the ER with Ca2+. Another up-regulated protein, calreticulin, is an important molecular chaperone involved in ‘‘quality control’’ within secretory pathways. This protein is also involved in the regulation of intracellular Ca2+homeostasis and it increases the Ca2+ storage capacity of the ER [39]. The third calcium-related protein, invertebrate SCP (SCP-alpha chain) was down regulated. SCP-(SCP-alpha chain has three Ca2+-binding sites that are common to EF-hand-type Ca2+-binding proteins. The EF-hand calcium-binding proteins have remarkable sequence homology and structural similarity, and they func-tion in Ca2+ buffering. A decrease in the accumu-lated levels of a protein in this family might, therefore, disrupt normal physiological function by interfering with the Ca2+-dependent signaling path-way[40,41], but it is interesting that several WSSV proteins (e.g. the proteins encoded by WSSV ORFs 136 and 486 in GenBank Accession NoAF440570) contain an EF-hand calcium-binding motif that would allow them to take over this function. 4.4. Voltage-dependent anion-selective channel
Another up-regulated protein was the voltage-dependent anion channel (VDAC). In addition to providing the major pathway for metabolite flux through the outer mitochondrial membrane, VDAC also binds to any hexokinase (HK) that translocates to the mitochondrial membrane. VDAC-bound HK is then able to use intramitochondrial ATP to phosphorylate glucose, which is the first step of glycolysis [42,43]. VDAC-bound HK also has an anti-apoptotic function [44,45], but even through apoptosis is inhibited in WSSV-infected cells [27], we did not find any evidence here that HK was up-regulated.
4.5. Cellular signaling
14-3-3b protein was also up-regulated after WSSV infection. Proteins in the 14-3-3 family are involved in cellular processes such as signal trans-duction, cell-cycle control, apoptosis, stress re-sponse and malignant transformation by binding to specific phosphorylated sites on diverse target proteins [46,47]. In our previous study (Wu et al., submitted), we found that WSSV infection led to elevated 14-3-3 protein levels in lymphoid organ cells and, to a lesser extent, in stomach cells as well. Fig. 3. Temporal 2-DE profiles and quantitative relative
abundance for the same 7 genes (12 spots) shown inFig. 2. (A) 2-DE spots of shrimp stomachs experimentally infected with WSSV for the indicated times. The WSSV non-structural protein ICP11 and beta-actin are also shown and used as reference proteins. (B) Plotted data represent the intensity of each protein spot after normalization relative to the beta-actin protein intensity (spot 34) in the same gel. Each bar represents the average relative abundance of 2-DE gels from two independent experiments. An asterisk indicates a marked (450%) increase or decrease relative to the immediately previous sampling time.
However, in the lymphoid cells, up-regulation of 14-3-3 protein was related to apoptosis, whereas in the stomach cells, apoptosis did not occur. All these results show that 14-3-3 proteins are up-regulated in stomach cells, but suggest that the up-regulation of 14-3-3b protein is not related to apoptosis. Instead, we tentatively conclude that it must be related to other cellular processes that are often modulated during virus infection, such as, for instance, cell-cycle control.
4.6. Possible multifaceted roles of glycolytic
enzymes: GAPDH, fructose biphosphate aldolase and enolase
As noted above, the protein profiling of WSSV-infected cells revealed an increase in GAPDH and fructose biphosphate aldolase (aldolase) proteins and their corresponding mRNAs. GAPDH is one of the key enzymes in the glycolysis pathway. After aldolase breaks fructose-1,6-diphosphate into gly-ceraldehyde-3-phosphate (G3P) and dihydroxyace-tone phosphate, GAPDH oxidizes G3P and reduces NAD+ to NADH. This not only facilitates energy production but also creates a reducing environment to protect macromolecules from being damaged by free radical/reactive oxygen species during virus infection. There was also an increase in another NAD+/NADH-dependent enzyme, isocitrate dehy-drogenase, which is additional evidence of the importance of NADH in the WSSV-infected cells. Lastly, an increase in the levels of NADH would be useful to WSSV because such a large virus would be expected to use the cell machinery to synthesize many organic molecules, and NADH can supply the reducing equivalents that are critical for the biogenesis of carbohydrates and fats.
An increasing number of diverse non-glycolytic activities of GAPDH have been reported, including macromolecular transport, microtubule bundling, nuclear tRNA transport and apoptosis [48,49]. GAPDH has also been variously implicated in virus infections: in the replication of Hepatitis Delta Virus, GAPDH interacts with viral RNA and enhances ribozyme catalysis [50], and it has also been reported that the GAPDH mRNA level of human adherent monocytes is markedly increased during vaccinia virus infection [49]. GAPDH interacts with multi-function 14-3-3 proteins in plants [51] and it is curious to note that the expression of a 14-3-3-like protein also increased after WSSV infection, although there is not yet
any evidence that this binds with GAPDH to regulate apoptosis or any other of its multi-functions.
Finally, it has recently been shown that the muscle-specific calmodulin-dependent protein ki-nase forms a complex with the glycolytic enzymes GAPDH, aldolase and enolase at the sacroplasmic reticulum membrane[52]. This may provide another mechanism for controlling Ca2+release and signal-ing. Since we have shown here that GAPDH, aldolase and enolase are the only three glycolytic enzymes to be up-regulated (at both the mRNA and protein levels) during WSSV infection, it will be interesting to determine whether such a complex exists in WSSV-infected cells.
In conclusion, the results presented here are basic data that will be a useful starting point for many subsequent studies. In particular, the pro-teome maps (Fig. 1), which were very consistent across multiple replications, should provide a reliable baseline reference for identifying potential biomarkers in future immuno-stimulant or vaccina-tion studies, as well as in assays of anti-viral drugs.
Acknowledgments
This investigation was supported financially by National Science Council grants (NSC94-2317-B-002-010 and NSC94-2311-B-002-021). Proteomic mass spectrometry analyses were performed by the Core Facilities for Proteomics Research located at the Institute of Biological Chemistry, Academia Sinica. The authors would like to thank Prof. S.H. Chiou for technical support of 2-DE work. We are also indebted to Paul Barlow for his helpful criticism.
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