The authors have declared that no competing interests exist.
Conceived and designed the experiments: HCW. Performed the experiments: MAS YTH ITC DYL YCH CYL YAC SuYL ShYL SWH THN. Analyzed the data: MAS YTH HTY KHK GDC CFL HCW. Contributed reagents/materials/analysis tools: KHK GDC CFL. Wrote the paper: MAS HCW.
In this study, we used a systems biology approach to investigate changes in the proteome and metabolome of shrimp hemocytes infected by the invertebrate virus WSSV (white spot syndrome virus) at the viral genome replication stage (12 hpi) and the late stage (24 hpi). At 12 hpi, but not at 24 hpi, there was significant up-regulation of the markers of several metabolic pathways associated with the vertebrate Warburg effect (or aerobic glycolysis), including glycolysis, the pentose phosphate pathway, nucleotide biosynthesis, glutaminolysis and amino acid biosynthesis. We show that the PI3K-Akt-mTOR pathway was of central importance in triggering this WSSV-induced Warburg effect. Although dsRNA silencing of the mTORC1 activator Rheb had only a relatively minor impact on WSSV replication,
The Warburg effect (or aerobic glycolysis) is a metabolic shift that was first found in cancer cells, but has also recently been discovered in vertebrate cells infected by viruses. The Warburg effect facilitates the production of more energy and building blocks to meet the enormous biosynthetic requirements of cancerous and virus-infected cells. To date, all of our knowledge of the Warburg effect comes from vertebrate cell systems and our previous paper was the first to suggest that the Warburg effect may also occur in invertebrates. Here, we use a state-of-the-art systems biology approach to show the global metabolomic and proteomic changes that are triggered in shrimp hemocytes by a shrimp virus, white spot syndrome virus (WSSV). We characterize several critical metabolic properties of the invertebrate Warburg effect and show that they are similar to the vertebrate Warburg effect. WSSV triggers aerobic glycolysis via the PI3K-Akt-mTOR pathway, and during the WSSV genome replication stages, we show that the Warburg effect is essential for the virus, because even when the TCA cycle is boosted in mTOR-inactivated shrimp, this fails to provide enough energy and materials for successful viral replication. Our study provides new insights into the rerouting of the host metabolome that is triggered by an invertebrate virus.
The Warburg effect, which was first described by Warburg in the 1930s, is a metabolic rerouting used by tumor cells and cancer cells to support their high energy requirements and high rates of macromolecular synthesis
WSSV is a large unique, complex, dsDNA virus, and in shrimp hemocytes, its complete
To understand the global changes triggered by WSSV infection, hemocytes were collected from PBS- and WSSV-injected shrimp at the genome replication stage (12 hpi) and the late stage (24 hpi) of the first WSSV replication cycle
To further understand the cellular responses after WSSV infection, we also used a global metabolomic platform to measure the metabolic changes in shrimp during WSSV infection. In this study, LC-ESI-MS data on over 100 metabolites were collected at 12 and 24 h after WSSV- or PBS-injection. However, since we were interested primarily in host processes that are involved in the Warburg effect, we focused particularly on a limited number of important host pathways, including glycolysis, the PPP, nucleotide metabolism and the TCA cycle. Our metabolomic and proteomic data are given in Supplementary
Changes in the levels of enzymes and proteins (ellipses) and metabolites (rectangles) relative to PBS-injected controls are color-coded to represent up- (red) or down- (green) regulation. Yellow represents no change. Colorless boxes and ellipses indicate that no data was detected. Protein data were collected from 3–5 pooled samples of 5 shrimp using quantitative label-free proteomics and expressed on a logarithmic scale. Metabolomic data were collected from 5–6 pooled samples of 10 shrimp using LC-ESI/MS. Numeric values for the proteomic and metabolomic data are given in
In mammalian cells, the two main pathways of carbon metabolism, glycolysis and the TCA cycle, oxidize hexose sugars to form ATP and NADPH, or else convert the same sugars to precursors of nucleotides, amino acids, and lipids. In shrimp hemocytes, WSSV infection at 12 hpi has previously been shown to increase glucose consumption and lactate production in ways that resemble the Warburg effect, but details of the intracellular changes in the carbon metabolism have not yet been investigated.
In WSSV-injected shrimp hemocytes at 12 hpi, there was a significant increase (p<0.05) in the glycolytic pathway metabolites glucose, dihydroxyacetone phosphate (DHAP), glyceraldehyde-3-P, 3-phosphoglycerate (3-PG), 2-phosphoglycerate (2-PG), phosphoenolpyruvate (PEP) and pyruvate (
The levels of lactate production were profiled and measured by LC-ESI-MS. Each bar represents the mean ± SD from four or five independent samples, with each sample pooled from 10 shrimp. The asterisks indicates a statistically significant difference in lactate production (p<0.05). (B) WSSV-induced phosphorylation of 4E-BP1 was suppressed by treatment with the mTOR inhibitors Rapamycin and Torin 1. Two hours before WSSV injection, shrimp were treated with volume-matched solvent (PEG), Rapamycin (RAP; 0.02 µg/g shrimp) or Torin 1 (TR 1, 25 µg/g shrimp). At 24 h post WSSV injection, twelve shrimp were selected from each group and divided into four sets (A–D, E–H, I–L, M–P). From each set, four pooled samples (3 shrimp in each pool) were prepared by collecting gill samples and extracting the total proteins. Each pooled sample was then subjected to Western blotting with antibodies to phosphorylated 4E-BP1-PT37/46 and actin. (C) Shrimp LvRheb is induced after WSSV infection. At the indicated time points after WSSV injection, total protein was extracted from the gills of individual shrimp and subjected to Western blotting. The expression of LvRheb was significantly induced at 24 hpi. ICP11, which is a major WSSV very late protein, was used as a proxy to indicate the WSSV infection state. Actin was used as an internal control. (D) Time series showing that LvRheb dsRNA treatment successfully silenced LvRheb expression in the hemocytes of WSSV-infected shrimp. Three days after LvRheb dsRNA injection, shrimp were injected with WSSV. Hemocytes were collected after the indicated number of days and subjected to cDNA synthesis and real-time PCR. Injections of enhanced green fluorescent protein-dsRNA (EGFP dsRNA) and phosphate-buffered saline (PBS) were used as dsRNA controls. Each bar represents the mean ± SD from four pooled samples with 3 shrimp in each sample. An asterisk indicates a significant statistical difference between groups (p<0.05). (E) Gene silencing of shrimp LvRheb has no significant effect on the expression of the WSSV genes IE1 and VP28 during the first replication cycle (∼24 hpi). The mRNA expression of IE1 gene and VP28 were used as proxies to indicate the WSSV infection state. Each bar represents the mean ± SD from four pooled samples (3 shrimp in each sample). An asterisk indicates a significant statistical difference between groups (p<0.05). (F) Gene silencing of shrimp LvRheb also has no significant effect on the number of WSSV genome copies during the first replication cycle (∼24 hpi). Experimental conditions were as described above. The IQ Real WSSV Quantitative System was used to measure the number of copies of the WSSV genomic DNA. Each bar represents the mean ± SD from four pooled samples (3 shrimp in each sample). An asterisk indicates a significant statistical difference between groups (p<0.05).
At 12 hpi, we noticed that, even though glycolysis was enhanced, three glycolytic metabolites, glucose-6-P, fructose-6-P and fructose-1,6-BP, remained unchanged relative to the PBS-injected controls (
At 12 hpi, we also observed an apparent boost in nucleotide metabolism, as suggested by increases in the levels of the purine biosynthetic intermediates in ATP synthesis (ADP, ATP, dATP) and dGTP synthesis (GDP, GTP and dGTP) (
All of the above changes would generate building blocks for macromolecular synthesis that could be used by the virus for genome replication. We further note that at 24 hpi, almost all of these increases in the PPP and nucleotide biosynthesis had dissipated.
The end product of glycolysis, pyruvate, can be further converted into two metabolites, alanine and the important metabolite acetyl-CoA, which occupies a central position between glycolysis, the mitochondrial TCA cycle, beta-oxidation, amino acid biosynthesis and lipid biosynthesis. At 12 hpi, although pyruvate levels were significantly increased, surprisingly there was a decrease in the levels of acetyl-CoA (
At 12 hpi, WSSV infection induced an increase in several of the detected amino acids, including alanine, histidine, tryptophan, glutamate, proline and aspartate (
Recent studies have shown that numerous cancer/tumor cells depend on the mTOR signaling pathway to trigger the Warburg effect for efficient cellular proliferation
Here we found that the level of phosphorylated 4E-BP1 in pooled samples of shrimp gills was elevated after WSSV injection compared to the PBS controls (
Our proteomic data also suggests that the mTORC1 pathway is activated, as shown by the increased expression of several proteins in the mTOR pathway at 12 hpi, including the mTOR activator Rheb (
To further determine if the increase in Rheb expression was important for WSSV replication, shrimp were treated with Rheb dsRNA to silence Rheb expression before being injected with WSSV (
To investigate whether activation of the mTOR pathway might regulate the WSSV-induced Warburg-like effect, we pre-treated shrimp with Rapamycin and Torin 1 to respectively suppress mTORC1- and mTORC1/C2- activation before injection with WSSV. At the WSSV genome replication stage (12 hpi), although lactate levels were elevated in the hemolymph of the non-suppressed WSSV-infected shrimp controls, there was no significant lactate accumulation in WSSV-infected shrimp when mTOR activation was suppressed either by Rapamycin or by Torin 1 (
(A) At 12 h post WSSV injection, the accumulation of plasma lactate that was seen in the PEG-injected control group was suppressed by both Rapamycin (RAP) and Torin 1 (TR1). Each bar represents the mean ± SD from four pooled samples of hemolymph (3 shrimp in each sample). An asterisk indicates a significant statistical difference between groups (p<0.05). The effect of Rapamycin and Torin1 on (B) the expression of the WSSV genes IE1, DNA pol, VP28 and ICP11, and (C) WSSV genome copy number. Data were based on pooled samples of hemocytes (gene expression) or pleopods (genome copy number), with all samples being taken from the same sets of shrimp as those used in
Having shown that mTOR activation plays an essential role in triggering the Warburg-like effect during WSSV infection, we next explored what happens to WSSV viral gene expression and viral DNA genome replication when the Warburg-like effect is suppressed by inhibition of the mTOR pathway. As shown in
Since mTOR activation can be stimulated by the upstream PI3K-Akt pathway
To determine if the PI3K-Akt-mTOR pathway is critical for the promotion of WSSV gene expression and genome replication, we investigated the effect of the three inhibitors, LY294002, MK2206, and Torin 1. As shown in
Shrimps were pretreated with LY294002 and samples were collected at 24(A–D) show the expression of WSSV (A) IE1, (B) VP28, and (C) ICP11 in shrimp hemocytes and (D) the viral copy number in pleopods from the same groups of LY294002-pretreated shrimp (n = 6–10 in each group). Graph (E) shows the mean WSSV copy number in five pooled samples of pleopods (3 shrimp in each pooled sample) collected from another batch of experimentally infected shrimp after pretreatment with the indicated inhibitor. Bars labeled with different letters indicate significantly different values (p<0.05).
To investigate the role of PI3K only, we used the selective pan-class I PI3K inhibitor BKM120. Although we were only able to conduct a pilot study, our results show that pretreatment with 0.625 µg BKM120 per g shrimp significantly reduced WSSV copy number (
To investigate whether the decrease in WSSV gene expression and viral genome copies in Torin 1-pretreated shrimp (
Two hours after treatment with Torin 1, shrimp were injected with PBS or a WSSV inoculum. At 12(10 shrimp per pool) were collected from each group. Changes in the metabolomic levels of the WSSV-infected samples relative to the PBS controls are color-coded as described in
Although only a limited number of vertebrate viruses (HCMV, HCV, Dengue virus, influenza virus, HSV and KSHV) have been studied using a systems biology approach
The hallmark of the Warburg effect is aerobic glycolysis, i.e. a high rate of glycolysis accompanied by lactic acid production, despite readily available oxygen
We previously reported that the activity of G6PDH was increased at the WSSV genome replication stage
In cancer cells, the PPP is usually up-regulated to balance cellular redox conditions and to ensure an adequate supply of ribose-5-phosphate (R5P) for nucleotide synthesis, a rate-limiting step in cancer cell proliferation
WSSV also induced an increase in the levels of the free amino acids, most of which are either derived from TCA cycle intermediates or converted from pyruvate and glutamine. Mazurek
Acetyl-CoA, which can be converted from pyruvate, occupies a nodal position at the bifurcation of the anabolic and catabolic pathways
With such a marked decrease in the amount of acetyl-CoA entering the TCA cycle, it was surprising to see an accumulation of the TCA cycle intermediates succinate, fumarate, malate and oxaloacetate at 12 and 24 hpi. Two possible mechanisms might account for this. First, in proliferating glioblastoma cells, glutaminolysis, which ultimately results in the conversion of glutamine to lactate, provides an anaplerotic source for the TCA cycle and allows it to produce enough NADPH to support fatty acid synthesis
The phosphorylation of 4E-BP1 (
When the Warburg effect was shut down by using PI3K-Akt-mTOR inhibitors, viral gene expression and viral copy number were significantly reduced (
Our results show that when the PI3K-Akt-mTOR pathway is entirely blocked, i.e. by either an upstream inhibitor of PI3K or Akt (
Interestingly, silencing Rheb, which acts as a positive regulator of mTORC1 signaling, does not significantly affect virus propagation during the first replication cycle (
In this study, in addition to providing a global metabolic view of the invertebrate Warburg effect during WSSV infection, we have developed a model of how WSSV may trigger these metabolic changes. A schematic is presented in
For the proteins investigated in this study, red indicates a positive involvement and gray indicates a partial involvement. Blue indicates important upregulated proteins and intermediates from previous studies: Glucose transporter (GLU1) is from Huang
The virus used in this study was the WSSV Taiwan isolate. To prepare the WSSV stock, hemolymph was extracted from WSSV-infected moribund shrimp and subjected to centrifugation (10,000×g). The supernatant was then diluted with phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4), and stored at −80°C. The experimental inoculum was prepared from this stock by dilution (10−4) with PBS.
All of the shrimp used in this study were obtained from the Aquatic Animal Center at National Taiwan Ocean University. These animals were specifically raised for research purposes and Taiwan does not require any additional permit or permission. Because the experimental animals were invertebrates, no specific permits were required for this study, and there is no official recommendation for the use of shrimp for scientific purposes in Taiwan. Nevertheless all of our experimental procedures, including animal sacrifice, were designed to be as humane as possible, and all animals were treated so as to minimize suffering at all times.
Primary antibodies used in this study include phospho-4E-BP1 (Thr37/46) (Cell Signaling; Catalog No. 2855), Rheb (Cell Signaling; Catalog No. 4935), and Actin (Millipore). The antibody that recognizes the major WSSV late protein ICP11 was prepared in the lab as described previously
Rapamycin (sirolimus) stock was prepared by dissolving Rapamycin powder (Sigma-Aldrich Co.) in 99% ethyl alcohol. Before use, this stock was diluted with PEG solvent (0.25% polyethylene glycol, 0.25% Tween 20 and 0.15 M NaCl). Torin 1 (Tocris Bioscience) was dissolved in dimethyl sulfoxide (DMSO) to provide a stock solution. Before use, this stock was diluted with PEG solvent.
To evaluate the involvement of the mTOR complexes during WSSV infection, shrimp were pretreated with Rapamycin (0.02 µg/g shrimp) or Torin 1 (20 µg/g shrimp) by intramuscular injection 2 h before being challenged. Control shrimps were injected with PEG only. At 12 and 24 h after the pretreated shrimps were injected with WSSV or PBS, four pooled samples of gills, hemolymph, hemocytes, and pleopods were collected from each group with each pooled sample being taken from 3 shrimp. Western blotting was used to measure the protein levels of phospho-4E-BP1 in the gills. Lactate levels in the hemolymph were measured as described below. Real-time PCR was used to measure virus gene mRNA expression and virus copy number. See below for details of these procedures.
To evaluate the involvement of the PI3K-Akt-mTOR pathway during WSSV infection, shrimp were pretreated with inhibitors LY294002 (0.625–1.25 µg LY294002/g shrimp), MK2206 (0.625–1.25 µg MK2206/g shrimp) and BKM120 (buparlisib; 0.15625–25 µg/g shrimp) by intramuscular injection 2 h before being challenged. Stock solutions were prepared by dissolving LY294002 (Biovision), MK2206 (Biovision) and BKM120 (Selleckchem) in 10% DMSO, and these solutions were further diluted with PBS before use. Control shrimps were injected with 0.01% DMSO in PBS. At 24 h after the pretreated shrimps were injected with WSSV, hemocyte and pleopods samples were collected from 6–10 individual shrimp in each group. The hemocyte samples were subjected to real-time PCR to measure the mRNA levels of WSSV IE1, VP28 and ICP11 as described below. Real-time PCR was also used to measure the viral copy number in the pleopods samples.
At 12 and 24 h after shrimp were injected with PBS or WSSV, 4–5 pooled hemocyte samples (5 shrimp in each sample) were collected from each group using an anticoagulant (450 mM NaCl, 10 mM KCl, 10 mM EDTA, 10 mM Tris-HCl, pH 7.5). After centrifugation at 10,000×g for 1 min followed by washing twice with 1× PBS, hemocytes were resuspended with 0.25× PBS to extract total protein for LC-MS/MS based label-free quantitative proteomic analysis. The total protein content of the lysates was quantified using a Bradford protein assay kit (Bio-Rad) with bovine serum albumin (BSA) added as an internal quantitative standard for each analysis. The lyophilized hemocyte protein lysate (60 µg) was resolubilized in an 8 M urea/25 mM ammonium bicarbonate buffer, incubated for 1 h at 37°C with 2 mM dithioerythreitol, and alkylated for 1 h using 2.5 mM iodoacetamide at room temperature. Each sample was then diluted with 25 mM ammonium bicarbonate to a final urea concentration of 1 M and Trypsin (Promega) was added (1∶50 w/w). After overnight incubation, protease activity was quenched by acidification of the reaction mixture with formic acid solution (pH 1–2). Aliquots of the peptide mixtures were desalted and concentrated on a C18-StageTip (Proxeon Biosystems), and then eluted with 50% acetonitrile in 0.1% formic acid.
The resulting peptide mixtures were analyzed by online nanoflow liquid chromatography tandem mass spectrometry (LC-MS/MS) on a nanoAcquity system (Waters, Milford, MA) connected to an LTQ Orbitrap Velos hybrid mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a PicoView nanospray interface (New Objective, Woburn, MA). After loading onto a 75-µm×250-mm nanoACQUITY UPLC BEH130 column packed with C18 resin (Waters, Milford USA), the peptide mixtures were separated at a flow rate of 300 nl/min using a linear gradient from 5% to 40% solvent B (acetonitrile with 0.1% formic acid) for 90 min. The LTQ Orbitrap Velos instrument was operated in standard data-dependent acquisition mode, automatically switching between full-scan MS and CID (collision induced dissociation)-MS/MS acquisition.
For the CID-MS/MS top20 method, full scan MS spectra (from m/z 350–1600) were acquired in the Orbitrap analyzer at a resolution of 60,000 (at 400 m/z) and an AGC (automatic gain control) target value of 106. The 20 most intense peptide ions with charge states ≥2 were sequentially isolated to a target value of 5,000 and fragmented in the ion trap at 35% normalized collision energy, with an activation q of 0.25, 10 ms activation time, and minimum ion selection intensity of 500 counts.
Progenesis LC-MS (Nonlinear Dynamics, version 3.0) was used for label-free quantification analysis. All raw spectral files were first aligned and only high quality peptide features (charge state>1 and isotopic pattern> = 3) were used for the extraction of ion intensity data from the MS1 spectra. To adjust for system errors such as sample loading and intensity shift across LC-MS/MS runs, the peptide ion intensity was normalized using the Progenesis LC-MS robust mean, which was derived from the peptide log2 ratio distributions between a reference and the targeted LC-MS/MS run. The peak list generated from the qualified peptide features was used to search against a combined database that consisted of an in-house white shrimp database, the shrimp white spot syndrome virus database from NCBI, and the common Repository of Adventitious Proteins database downloaded from the Global Proteome Machine in the MASCOT 2.3 server (Matrix Science). To keep the false discovery rate below than 5% (as estimated from the target-decoy database), only peptides with a Mascot ion score greater than 17 were included for subsequent protein analysis. Proteins were automatically assigned to functional group, and only these proteins that met both of the following criteria were reported: 1) the protein had the most peptide hits within its group, and 2) the protein included at least one unique, quantifiable peptide. Protein quantitation was based on the sum of the total ion intensity of the unique peptides. Lastly, to map the results into the biological network, MetaCore network software (GeneGO) was used for pathway analysis of the expressed proteins.
In this experiment, 2 h before shrimp were challenged with WSSV or PBS, they were pretreated with PEG or Torin 1 (20 µg/g shrimp) by intramuscular injection to produce a total of four experimental groups: the PEG-PBS group, the PEG-WSSV group, the Torin 1-PBS group and the Torin 1-WSSV group. At 12 and 24 hpi, 5–6 pooled hemocyte samples (10 shrimp in each sample) were collected from each group using anticoagulant as described above. After centrifugation at 800×g for 1 min followed by washing twice with 1× PBS, the hemocytes were resuspended with 0.33× PBS and kept on ice for 10 min. The samples were then centrifuged at 10,000×g for 10 min, and 100% MeOH was added to the supernatant at a ratio 1∶ 2. After being centrifuged again at 10,000×g for 10 min, the supernatants were lyophilized, dissolved in 35 µl ddH2O and subjected to LC-ESI/MS metabolomic analysis as follows:
To enhance the detection of the carboxylic acid and organic phosphate signals, 5 µl aniline/HCl reaction buffer (0.3 M aniline [Sigma-Aldrich, USA] in 60 mM HCl) and 5 µl of 20 mg/ml N - (3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC; Sigma-Aldrich, USA) were added to each sample of the hemocyte residue. Each mixture was vortexed and incubated at 25°C for 2 h, after which the reaction was stopped by adding 5 µl of 10% ammonium hydroxide. The aniline derivatized samples were then analyzed using an LC-ESI-MS system consisting of an ultra-performance liquid chromatography (UPLC) system (Ultimate3000 RSLC, Dionex) and a quadrupole time-of-flight (TOF) mass spectrometer with an the electrospray ionization (ESI) source (maXis UHR-QToF system, Bruker Daltonics). The shrimp metabolites were separated by reversed-phase liquid chromatography (RPLC) on a BEH C18 column (2.1×100 mm, Walters). The LC parameters were as follows: autosampler temperature, 4°C; injection volume, 10 µl; and flow rate, 0.4 ml/min. After pre-starting with 1% mobile phase B (0.1% formic acid in ACN) for 4 min, the elution started from 99% mobile phase A (0.1% formic acid in ddH2O) and 1% mobile phase B (0.1% formic acid in ACN). After holding at 1% for 0.5 min and raising to 60% over 5 min, mobile phase B was further raised to 90% in another 0.5 min, held at 90% for 1.5 min, and then lowered back to 1% in 0.5 min. The column was then equilibrated by pumping 99% B for 4 min. The acquisition parameters for LC-ESI-MS chromatograms were as follows: dry gas temperature, 190°C; dry gas flow rate, 8 L/min; nebulizer gas, 1.4 bar and capillary voltage, 3,500 V. Mass spectra were recorded from m/z 100–1000 in the negative ion mode. Data were acquired by HyStar and micrOTOF control software (Bruker Daltonics) and processed by DataAnalysis and TargetAnalysis software (Bruker Daltonics). Each metabolite was identified by matching with its theoretical m/z value and with the isotope pattern derived from its chemical formula. The identified metabolites were quantified by summing the corresponding area of the extracted ion chromatogram, and metabolite signal levels were presented as the mean of the 5–6 pooled hemocyte samples from each experimental group at each time point.
To investigate the WSSV-induced metabolic changes in shrimp hemocytes, the fold changes in the PEG-WSSV group were calculated relative to the PBS injection group (PEG-PBS group). To investigate the WSSV-induced metabolic changes in the mTOR-inactivated shrimp, the fold changes in the Torin 1-WSSV group were calculated relative to the PBS injection group (Torin 1-PBS group). Lastly, the effect of Torin 1 pretreatment was shown by calculating the fold changes of the Torin 1-PBS group relative to the PEG pretreatment group (PEG-PBS group). Student's
Preparation of the dsRNA was done following Wang
For the gene silencing experiments, the experimental group was injected with LvRheb dsRNA (1 µg/g shrimp), while the control groups were injected with EGFP dsRNA or PBS only. To determine the efficiency of the gene silencing for pooled hemocytes samples (3 shrimp in each pool sample) were collected from each group at the indicated time points. Total RNA was extracted from these samples, and cDNA was synthesized using Superscriptase II Reverse Transcriptase (Invitrogen) with Anchor-dTv primer (
For this knockdown experiment, shrimp were randomly divided into 3 groups and injected with LvRheb dsRNA, EFGP dsRNA, or PBS. At 3 days post dsRNA injection, shrimp were then challenged with WSSV. Four pooled hemocyte samples were collected from each group at various time points (12, 24, 36, and 48 hpi), with each pooled sample taken from 3 shrimp. Total cDNA was then prepared from each sample as described above. To quantify the relative expression of the WSSV
Four pleopod samples (3 shrimp in each sample) were also collected from of the above experimental groups at the same time points. The samples were subjected to genomic DNA extraction using a DTAB/CTAB DNA extraction kit (GeneReach Biotechnology Corp.). WSSV genomic DNA copies were quantified using IQ Real WSSV quantitative system (GeneReach Biotechnology Corp.), which is a commercial real-time PCR based on the TaqMan assay.
At 12 and 24 h post WSSV injection, 4–5 hemolymph samples (3 shrimp in each sample) were collected from groups of shrimp pretreated with LY294002, MK2206, Rapamycin, Torin 1 or PEG/PBS (control) without using anticoagulant. After being kept at 4°C for 12–16 hours, the samples were centrifuged at 13000×g for 15 min at 4°C, and the supernatants were transferred to new tubes. The concentration of glucose and lactate in the supernatants was then determined using enzymatic colorimetric test kits (Fortress Diagnostics Limited).
After total hemocyte cDNA was prepared from all samples as described above, real-time PCR was performed with the specific primer sets IE1-qF/IE1-qR, DNApol-qF/DNApol-qR, VP28-qF/VP28-qR, ICP11-qF/ICP11-qR and EF1-α-qF/EF1-α-qR (
Genomic DNA was extracted from pleopod samples, and the number of WSSV genomic DNA copies was quantified by the IQ Real WSSV quantitative system (GeneReach Biotechnology Corp.) as described above. Data values were calculated, presented and statistically analyzed as described above.
Shrimp gill tissues were lysed in 0.33× PBS with protein inhibitor and phosphatase inhibitor (Roche). Protein concentrations in each lysate were measured by Bio-Rad Protein Assay. Approximately 25 µg of protein lysate per sample were separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto polyvinylidene fluoride (PDVF) membranes, blocked with 1–3% skim milk in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 hour at room temperature, and then incubated overnight in primary antibody in TBST at 4°C. Following three extensive washes with TBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz) for 1 hour at room temperature. After three more washes with TBST, the signals were developed by ECL detection agents (Amersham) and detected using chemiluminescence (Image Quant LAS 4000 mini).
Hierarchical K-means Clustering of ∼800 hemocyte protein expression profiles obtained from a large-scale, high-throughput, label-free, quantitative LC-MS/MS analysis. (A), (B) At 12 hours and 24 hours post injection, the protein profiles of the PBS and WSSV groups formed two distinct clusters based on their log2 protein abundance, suggesting that the host cell protein pattern was markedly changed after WSSV infection at both time points. (C) Although there was no significant difference between the PBS groups at 12 and 24 hpi, the protein profiles of (D) the WSSV groups at 12 and 24 hpi formed two distinct clades, indicating that the host responses were different after WSSV infection at 12 and 24 hpi. Two of the samples, 12-WSSV#1 and 24-WSSV#2, were not assigned to the corresponding cluster, and we therefore excluded these two mis-assigned samples from our subsequent analysis.
(TIF)
Proteomic data suggests that the mTOR pathway is activated at the replication stage (12 hpi) of WSSV infection. (A) Changes in the levels of enzymes and proteins (ellipses) relative to PBS-injected controls are color-coded to represent up- (red) or down- (green) regulation. Yellow represents no change. Colorless ellipses indicate that no data was detected. (B) WSSV-induced phosphorylation of 4E-BP1 was still detected even after Rheb was knocked down by Rheb dsRNA. Each lane shows the results for a pooled sample (n = 3) of total protein extracted from gills and probes with antibodies against 4E-BP1-PT37/46, ICP11 and actin. (C) WSSV-induced phosphorylation of 4E-BP1 was suppressed by pretreatment with the inhibitor LY294002. Each lane shows the result for a pooled sample (n = 3) of total protein subjected to Western blotting with antibodies against 4E-BP1-PT37/46 and actin. (D) WSSV replication was significantly reduced by specifically suppressing using pretreatment with 0.625 µg/g shrimp of the selective pan-class I PI3K inhibitor BKM120
(TIF)
In Torin 1-pretreated shrimp, the Warburg effect was not seen either at 24 hpi in WSSV-infected shrimp or at 12∼24 hpi in PBS-injected shrimp. (A) Two hours after treatment with Torin 1, shrimp were injected with PBS or a WSSV inoculum. At 24 hpi, 6 pooled hemocytes samples (10 shrimp per pool) were collected from each group. Changes in the metabolomic levels of the WSSV-infected samples relative to the PBS controls are color-coded as described in
(TIF)
Global changes in the shrimp hemocyte proteome after WSSV infection.
(DOCX)
Global changes in the shrimp hemocyte metabolome after WSSV infection.
(DOCX)
PCR primers used in this study.
(DOCX)
Metabolomic analysis was performed by the Technology Commons (TechComm) in the College of Life Science and Center for Systems Biology, National Taiwan University. The proteomic MS data were acquired at the former NRPGM Core Facilities for Proteomics and Glycomics, at the Core Facilities for Protein Structural Analysis at Academia Sinica supported by the Taiwan National Core Facility Program for Biotechnology, and at the Academia Sinica Common Mass Spectrometry Facilities. Computational analysis and data mining were performed using the system provided by the Bioinformatics Core, National Cheng Kung University. We are indebted to Paul Barlow, National Taiwan University, for his helpful criticism. We also warmly thank Prof. John Kastelic, University of Calgary, for editing this manuscript.