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A viral lectin encoded in Cotesia plutellae bracovirus and its immunosuppressive effect on host hemocytes
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Comparative Biochemistry and Physiology, Part A 149 (2008) 351 – 361 www.elsevier.com/locate/cbpa
A viral lectin encoded in Cotesia plutellae bracovirus and its immunosuppressive effect on host hemocytes Sunyoung Lee, Madanagopal Nalini, Yonggyun Kim ⁎ Department of Bioresource Sciences, Andong National University, Andong 760-749, Republic of Korea Received 16 November 2007; received in revised form 9 January 2008; accepted 9 January 2008 Available online 14 January 2008
Abstract An endoparasitoid wasp, Cotesia plutellae, induces immunosuppression of the host diamondback moth, Plutella xylostella. To identify an immunosuppressive factor, the parasitized hemolymph of P. xylostella was separated into plasma and hemocyte fractions. When nonparasitized hemocytes were overlaid with parasitized plasma, they showed significant reduction in bacterial binding efficacy. Here, we considered a viral lectin previously known in other Cotesia species as a humoral immunosuppressive candidate in C. plutellae parasitization. Based on consensus regions of the viral lectins, the corresponding lectin gene was cloned from P. xylostella parasitized by C. plutellae. Its cDNA is 674 bp long and encodes 157 amino acid residues containing a signal peptide (15 residues) and one carbohydrate recognition domain. Open reading frame is divided by one intron (156 bp) in its genomic DNA. Amino acid sequence shares 80% homology with that of C. ruficrus bracovirus lectin and is classified into C-type lectin. Southern hybridization analysis indicated that the cloned lectin gene was located at C. plutellae bracovirus (CpBV) genome. Both real-time quantitative RT-PCR and immunoblotting assays indicated that CpBV-lectin showed early expression during the parasitization. A recombinant CpBV-lectin was expressed in a bacterial system and the purified protein significantly inhibited the association between bacteria and hemocytes of nonparasitized P. xylostella. In the parasitized P. xylostella, CpBV-lectin was detected on the surface of parasitoid eggs after 24 h parasitization by its specific immunostaining. The 24 h old eggs were not encapsulated in vitro by hemocytes of P. xylostella, compared to newly laid parasitoid eggs showing no CpBV-lectin detectable and easily encapsulated. These results support an existence of a polydnaviral lectin family among Cotesia-associated bracovirus and propose its immunosuppressive function. © 2008 Elsevier Inc. All rights reserved. Keywords: Cotesia plutellae; Plutella xylostella; CpBV; Encapsulation; Viral lectin; Polydnavirus; Immune
1. Introduction In response to invading pathogens, insects can express various defense responses including both cellular and humoral immune reactions (Ratcliffe et al., 1985). Endoparasitoid wasps develop within the general cavity of other insects and must overcome the immune responses of target insects. Some braconid or ichneumonid wasps possess symbiotic polydnavirus, which has been regarded as a potent agent to suppress immune response of parasitized hosts (Webb, 1998). Polydnavirus is an insect DNA virus exhibiting obligate mutualism with host insects (Whitfield, 1990). It is classified
⁎ Corresponding author. Tel.: +82 54 820 5638; fax: +82 54 823 1628. E-mail address: [email protected] (Y. Kim). 1095-6433/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2008.01.007
into bracovirus and ichnovirus according to its host wasp family and virion morphology. As a provirus, its segmented genome is located on host wasp chromosome (Belle et al., 2002). During replication, different viral DNA segments are excised from host chromosome and circularized, which finally become nucleocapsids (Krell et al., 1982). Viral replication occurs in ovarian calyx region probably in response to ecdysteroid signal during pupal period (Webb and Summers, 1992). Virus particles in the calyx lumen of the parasitoid are delivered into the hemocoel of parasitized insects along with laying eggs and express their encoded genes after getting entry into target tissues (Edson et al., 1981). Various viral products have been implicated to suppress insect immunity by inducing hemocyte apoptosis (Strand and Pech, 1995), altering hemocyte morphology and behavior (Tanaka, 1987; Strand and Pech, 1995), and destabilization of hemocyte cytoskeleton (Asgari et al., 1997).
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Cotesia plutellae (Braconidae: Hymenoptera) is an endoparasitoid of the diamondback moth, Plutella xylostella, in which a symbiotic polydnavirus (C. plutellae bracovirus: CpBV) plays a critical role in altering physiological status favorable for wasp survival and development (Bae and Kim, 2004; Kim et al., 2007). Parasitized P. xylostella exhibits significant immunosuppressive effect in both humoral and cellular reactions (Bae and Kim, 2004; Ibrahim and Kim, 2008) and fails to undergo metamorphosis (Lee and Kim, 2004). Replication of CpBV within the parasitoid begins at late pupal stage (Kim et al., 2004) so that the wasp can parasitize successfully even just after adult emergence. The replicated CpBV genome consists of at least 27 DNA segments, in which more than 470 kb nonoverlapping nucleotide sequences have been determined and have been known to encode several putative gene families (Choi et al., 2005; Kim, 2006; Kim et al., 2007). CpBV-PTPs are the largest family in CpBV and play significant role in immunosuppression probably by altering phosphorylation status of the infected hemocytes (Ibrahim et al., 2007; Ibrahim and Kim, 2008). CpBV15β is a 15 kDa protein-encoding CpBV gene, which inhibits hemocyte-spreading by modifying the cytoskeletal structure of P. xylostella hemocytes (Nalini and Kim, 2007). This study was focused on identifying immunosuppressive factor from C. plutellae bracovirus especially in terms of nonself recognition capacity by hemocytes of parasitized P. xylostella. To this end, we analyzed the source of the inhibiting factor(s) using a binding assay between hemocytes and bacteria. A suggestion that there would be a humoral interrupting factor in parasitized plasma led us to clone viral lectin gene, which has been proposed to be a plausible candidate (Glatz et al., 2003) for interrupting hemocyte recognition. Finally, we proved the immunosuppressive effect of the viral lectin in preventing hemocytic encapsulation against C. plutellae eggs. 2. Materials and methods 2.1. Rearing insects and parasitization P. xylostella larvae were reared at 25 ± 1 °C with 16:8 h (L:D) photoperiod on cabbage leaves. Late second instar larvae were parasitized by C. plutellae at 1:2 (wasp:host) density for 12 h and reared under the above rearing condition. Wasp cocoons were collected and held in plastic cage until their emergence. The emerged adult wasps were collected every day and allowed to mate for 24 h before using for parasitization. Adult wasps were fed 40% sucrose. 2.2. Hemocyte-bacterial binding assay Adhesion of bacteria on hemocytes was determined by using hemocyte monolayers overlaid with Safranin-O-stained Escherichia coli under in vitro conditions. Hemolymph from 20 larvae of P. xylostella was separated into hemocytes and plasma by centrifugation (1000 ×g, 3 min, 4 °C). The hemocytes were further washed by centrifugation and resuspended in 100 μL of Grace's insect medium (Sigma-Aldrich, MO, USA). Heat-killed E. coli (70 °C for 60 min) stained in 10% Safranin-
O dye solution was washed and resuspended in parasitized or nonparasitised plasma at a concentration of ~ 103 cells/μL. Hemocyte monolayers were made by using 10 μL of hemocyte suspension and left for 1 h at room temperature under humid condition. After attachment of hemocytes, the medium was replaced with 10 μL of E. coli suspension in parasitized or nonparasitized plasma and incubated for 30 min. The monolayers were washed with fresh Grace's insect medium and observed under light microscope at 400× magnification. Hemocytes adhered with bacteria were counted by counting 100 hemocytes from ten random fields and was recorded as percent bacteria-bound hemocytes. Each treatment was determined based on three independent replications. 2.3. CpBV DNA purification CpBV DNA was isolated from the ovary of female wasps. For this, 200 abdomens of female wasps were homogenized in TEK (11 mM Na2EDTA·2H2O, 5 mM KCl, 100 mM Tris, pH 7.5) using a 3 mL syringe with a 21G needle. The suspension was passed through 0.45 μm syringe filter (MFS, CA, USA), 15 μL proteinase K (2 ng/μL) and 25 μL of 10% sarcosyl solutions was added, and incubated at 50 °C overnight. The lysate was treated with RNase A (0.3 μg/μL) at 37 °C for 2 h and then extracted with phenol/chloroform. The viral DNA was precipitated by adding 2 vol. of ethanol and centrifugation (14,000 ×g, 20 min, 4 °C). The obtained pellet was washed with 70% ethanol, air-dried, and dissolved in TE buffer (1 mM Tris, 0.1 mM EDTA, pH 8.0). 2.4. RNA extraction and cDNA construction Total RNA was extracted from parasitized P. xylostella larvae from day 1 to day 8 post-parasitization using Trizol reagent (MRC, OH, USA) and reverse-transcribed with RT-premix (Bioneer, Daejon, Korea) using an oligo-dT primer (5′-CCAGT GAGCA GAGTG ACGAG GACTC GAGCT CAAGC TTTTT TTTTT TTTTT T-3′) after RNase H treatment. For a control, nonparasitized larvae were collected at their 4th instar and used for constructing cDNA. 2.5. CpBV-lectin gene cloning Two consensus regions (‘FHSTPAT’ and ‘KCGSLLK’) were chosen from viral lectin genes of C. rubecula and C. ruficrus (Glatz et al., 2003; Teramoto and Tanaka, 2003). The corresponding degenerate primers of forward (5′-TTYCA YAGYA CNCCN GCNAC-3′) and reverse (5′-TTNAR NARRC TNCCR CAYTT3′) were designed and used for amplifying the region from cDNA of parasitized P. xylostella under following conditions: 94 °C for 1 min, 50 °C for 1 min and 72 °C for 2 min with 35 cycles. The amplified PCR products were ligated into pGEM-T Easy cloning vector (Promega, WI, USA). Both T7 and Sp6 sequencing primers were used to sequence the inserts by automatic DNA sequencer (Bionics, Seoul, Korea). The resulting sequences were compared with the known viral lectin genes using NCBI-BLAST search program. Based on the resulting partial sequence, gene-specific
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primers were designed for 5′-RACE and 3′-RACE: 5′-1 (5′GGTAA CTACA GCAAG ACTTC-3′), 5′-2 (5′-TCTTA GCTTC GTCGA AGGTT-3′), 3′-1 (5′-TGAAA TTACA AGCGT ACATT-3′), and 3′-2 (5′-CGAGC ACTCC TAGTG TTCAA-3′). 5′-RACE was performed using 5′-RACE kit (Invitrogen, CA, USA) according to manufacturer's method. Total RNA was reverse-transcribed with 5′-1 primer. After RNase H treatment, the first-strand cDNA was tailed with dCTP by terminal deoxynucleotidyl transferase and PCR-amplified with oligo-dG (5′-GGCCA CGCGT CGACT AGTAC GGGII GGGII GGGII G-3′) and 5′-1 primers, where PCR was performed under the described condition above except 55 °C annealing temperature. For 3′-RACE, total RNA was reverse-transcribed with RTpremix (Bioneer) using an oligo-dT primer. Nested RT-PCR was then performed by the sequential primer pairs of 3′-1/ oligo-dT and then 3′-2/oligo-dT. PCR was performed under the described condition above except 52 °C annealing temperature. The amplified PCR products were ligated and sequenced as described above. The sequence data were aligned and corrected by DNAstar program (Version 5.01, DNAstar Inc, Madison, USA). The functional domains including open reading frame (ORF), 5′-untranslated region (UTR), and 3′UTR were searched using Smart/Pfam domain search program (http://elm.eu.org/basicELM/). 2.6. Hybridization analyses of CpBV-lectin For Southern hybridization, DNA samples were separated on agarose gel and transferred to a nylon membrane (Nytran, NH, USA). A biotinylated DNA probe encompassing ORF was prepared by incorporating biotin-N4-dCTP (KPL, MD, USA) through random primer extension as described by the manufacturer. The blot was then hybridized with the labeled probe in hybridization solution (50% formamide, 6×SSC, 5×Denhardt's reagent, 0.5% SDS) overnight at 42 °C. The blot was washed twice in 2×SSC containing 0.5% SDS at 42 °C for 20 min each, then twice in 0.2×SSC containing 0.5% SDS at 55 °C for 20 min each, and finally twice in 1×SSC at 37 °C for 10 min each. CDP-star chemiluminescent substrate (KPL, MD, USA) was used for detection after binding alkaline phosphatase-labeled streptavidin. Then, the probed membrane was exposed to hyperfilm (Amersham Biosciences, UK) and developed by dipping in Lucky MQ (Poohung, Ahnsan, Korea). For immunoblotting assay, P. xylostella larvae were ground in 50 mM phosphate buffer (pH 7.0) and centrifuged (14,000 ×g, 5 min). The resulting supernatant was mixed with the same volume of denaturing buffer (4% SDS, 20% glycerol, 10% β-mercaptoethanol in 62.5 mM Tris–HCl, pH 6.8). Electrophoresis was performed on 15% SDS–PAGE under denaturing condition (Laemmli, 1970). The separated proteins were transferred onto a nitrocellulose membrane according to Towbin et al. (1979). Non-specific sites were blocked by 5% skim milk at room temperature for 1 h. After three washes with PBS, the membrane was incubated at room temperature with primary antibody raised against V5 (Invitrogen) or CpBV-lectin for 2 h. Subsequently, the membrane was washed with PBS thrice and incubated with secondary antibody (1/2000 dilution)
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conjugated with alkaline phosphatase (Sigma). Finally after three times washing with PBS, the membrane was stained with an alkaline phosphatase substrate solution containing nitro blue tetrazolium/5-mono-4-chloro-3-indolyl phosphate (Sigma) in 10 mM phosphate buffer, pH 9.5. 2.7. Quantitative RT-PCR of CpBV-lectin expression Real-time quantitative RT-PCR was performed on a Bioneer Exicycler™ Quantitative Thermal Block (Bioneer) using SYBR green chemistry and real-time fluorescence measurements using Accupower Greenstar™ PCR premix (Bioneer). Specific primers for CpBV-lectin were designed for real-time PCR: RTFP (5′-ATGAG TATTG GGAAG AGACT-3′), and RT-RP (5′GGTAA CTACA GCAAG ACTTC-3′). Template cDNAs were constructed as described above. The reaction mixture (20 μL) consisted of 1× Greenstar™ PCR Master mix, 10 mM MgCl2, each 0.5 μM of RT-FP and RT-RP primers, and 250 ng of cDNA. The reaction was performed and monitored under following conditions: one cycle (95 °C for 15 min) for activation of Hotstart Taq DNA polymerase, subsequently 40 cycles (94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min), and finally one cycle (72 °C for 10 min). Each RT-PCR amplification was independently replicated three times. In each replication as an internal control for equivalence of template, β-actin gene was amplified with forward (5′-TGGCA CCACA CCTTC TAC-3′) and reverse (5′-CATGA TCTGG GTCAT CTTCT-3′) primers. Fluorescence values were measured and amplification plots were generated in real-time by the Exicycler™ program. Quantitative analysis of CpBV-lectin expression followed a comparative CT (ΔCT) method (Livak and Schmittgen, 2001). 2.8. Expression and purification of CpBV-lectin in bacteria Two gene-specific primers (Lec-FP: 5′-ATGAG TATTG GGAAG AGACT-3′, Lec-RP: 5′-CACGC TTGTG CAGAA GAAGG-3′) were designed to amplify ORF of CpBV-lectin gene excluding putative signal sequence corresponding to the first 17 amino acids of the protein. PCR product was ligated into pBADTOPO expression vector (Invitrogen) according to the protocol provided by the manufacturer. The ligation product was transformed into Top10 strain of E. coli by heat shock. Colonies containing recombinant vectors were identified by digestion of Pmi I and Nco I. For a pilot expression, a positive clone was cultured at 37 °C with 200 rpm and over-expressed until OD600 = 0.5 after adding L-arabinose (final concentration of 0.002%). Fusion protein was identified by immunoblot using V5 antibody (Invitrogen). Bacterial CpBV-lectin was identified in the insoluble fraction of total bacterial proteins, with only a small amount being soluble. For large culture and expression, bacteria culture (30 L) was centrifuged (4000 ×g, 20 min, 4 °C). Cells were then resuspended in lysis buffer (8 M urea, 100 mM NaH2PO4, 10 mM Tris, pH 8.0) and gently rocked for 1 h. After centrifugation (14,000 ×g, 20 min, 4 °C), 2 mL of 5% Ni-NTA resin (Qiagen, CA, USA) was added to the resulting supernatant and mixed gently by shaking at 200 rpm for 1 h at room temperature. Lysate-resin mixture was carefully loaded into an empty column, and washed with a low pH
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buffer (8 M urea, 100 mM NaH2PO4, 10 mM Tris, pH 6.3). Bound proteins were eluted with acid elution buffer (8 M urea, 100 mM NaH2PO4, 10 mM Tris, pH 4.5) and confirmed by immunoblotting with V5 antibody as described above. The purified sample was sent to Peptron, Inc. (Daejon, Korea) for raising a polyclonal antibody using mouse. 2.9. In vitro encapsulation assay C. plutellae eggs were collected from parasitized P. xylostella at 0 and 24 h post-parasitization and washed with TBS (50 mM Tris–HCl, 100 mM dextrose, 5 mM KCl, 2.5 mM MgCl2, 50 mM NaCl, pH 7.5) to clear off any plasma. Hemocyte suspension was prepared by collecting hemolymph from 50 larvae in 200 μL of anticoagulant buffer (TBS containing 5 mM cysteine hydrochloride, 300 mOsm). Encapsulation assays were performed in flat-bottom microtiter plates (Falcon, Becton Dickinson Labware,
NJ, USA) by adding 300 μL of the hemocyte suspension to 12 washed eggs. The plates were held at 25 °C for 4 h with gentle rocking at every 15 min in order to facilitate the association of eggs and hemocytes. After incubation, these suspensions were transferred on glass slide and left undisturbed for 15 min for the encapsulated eggs to settle. The eggs were scored positive for encapsulation only if at least 15 hemocytes were adhered on the egg surface. Each treatment was replicated three times. In order to check the presence of CpBV-lectin on the egg surface, immunostaining was performed. The washed eggs were incubated with antibody of CpBV-lectin for 1 h and then subsequent steps according to immunoblotting method described above. 2.10. Statistical analysis Treatment means and variances were analyzed in one-way ANOVA by PROC GLM of SAS program (SAS Institute,
Fig. 1. Immunosuppressive agent(s) in the plasma of Plutella xylostella parasitized by Cotesia plutellae. (A) Hemocyte-bacterial binding assay. Hemocytes were fixed on the cover glass, where parasitized (‘P’) or nonparasitized (‘NP’) plasma was overlaid and incubated with Safranin-O-stained Escherichia coli for 30 min. Inset figure illustrate hemocyte attached by stained bacteria (arrows). Scale bar represents 10 μm. (B) Inhibitory effect of ‘P’ plasma on mediating role of ‘NP’ plasma in hemocyte and bacterial binding. (C) Heat-labile property of plasma factor(s) mediating hemocyte-bacterial binding, where asterisk indicates heat-treatment of plasma at 95 °C for 5 min. Each treatment consisted of three measurements. Different letters above standard deviation bars indicate significant difference between means at Type I error = 0.05 (LSD test).
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1989). All means were compared by least squared difference (LSD) test at Type I error = 0.05. 3. Results 3.1. Reduction in bacterial binding ability of P. xylostella hemocytes parasitized by C. plutellae Interaction between hemocytes and bacteria was measured as frequency of bacteria-bound hemocytes (Fig. 1). For this assay, immobilized hemocytes of P. xylostella were used by preparing monolayer, most of which consisted of granular cells and plasmatocytes based on their cell morphology. This assay showed that Safranin-O-stained bacteria resuspended in nonparasitized plasma could be easily attached to nonparasitized hemocytes (inset in Fig. 1A). However, parasitized hemocytes incubated with parasitized plasma significantly lost this binding ability. To clarify the source of inhibitory factor(s), parasitized or nonparasitized hemolymph was separated into hemocyte and plasma. Bacteria did not bind well to parasitized hemocytes
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even in the presence of nonparasitized plasma (Fig. 1A). Also, parasitized plasma significantly inhibited the ability of nonparasitized hemocytes to bind bacteria. Inhibitory factor(s) of parasitized plasma was further analyzed for dosimetric effect (Fig. 1B). When nonparasitized plasma was mixed with parasitized plasma in different ratios, the efficacy of nonparasitized hemocytes to bind bacteria decreased with increase in proportion of parasitized plasma. This suggests that parasitized plasma possesses inhibitory factor (s) to prevent hemocytes to bind bacteria. The plasma factor(s) was heat-labile because nonparasitized hemocytes overlaid with heat-treated nonparasitized plasma lost the bacterial binding ability just like the inhibition induced by parasitized plasma (Fig. 1C). 3.2. cDNA cloning and sequence of CpBV-lectin Heat-labile bacterial binding factor(s) in nonparasitized plasma and inhibitory effect of parasitized plasma on the hemocytes to bind bacteria suggest a hypothesis that there is an
Fig. 2. Gene structure of Cotesia plutellae bracovirus (CpBV) lectin (NCBI accession No. AY461733). (A) DNA sequence and predicted amino acid sequence of CpBV-lectin. Initiation and termination codons are shown in black box. The degenerate primers (forward and reverse) were designed from the consensus regions of Cotesia-associated spp. lectin genes. Gene-specific primers (5′-1, 5′-2, 3′-1 and 3′-2) used for 5′ and 3′ RACEs are indicated by underlines. RT-FP and RT-RP primers used for the real-time quantitative RT-PCR. The signal peptide is indicated by bold letters. Intron sequence is written in small and italic letter. (B) Intron/exon structure and restriction map, where one intron was positioned by dotted lines between cDNA and genomic DNA (‘gDNA’). Open reading frame (ORF, black bar) and untranslated region (UTR, white bar) are also denoted.
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inhibitory factor to interrupt nonself recognition of hemocytes in parasitized plasma. As an inhibitory candidate in nonself recognition, viral lectin genes have been reported in other Cotesiaassociated polydnaviruses (Glatz et al., 2003). Here, we cloned the viral lectin gene from C. plutellae-parasitized P. xylostella. CpBV-lectin mRNA was amplified by RT-PCR using degenerate primers (forward and reverse in Fig. 2A), which were designed from the consensus residues of other lectin genes from Cotesia spp. An approximately 260 bp cDNA amplicon was obtained and showed high homology with that of other Cotesia lectin genes. Four nested gene-specific primers were designed and produced 5′-RACE product of approximately 200 bp, and 3′RACE product of approximately 230 bp. The complete cDNA of the CpBV-lectin gene was 674 bp long. The cDNA has an ORF of 474 nucleotides encoding the entire CpBV-lectin of 157 amino acids (Fig. 3A). Fifteen amino acid residues at the Nterminus were predicted as a signal peptide by SignalP program (http://www.cbs.dtu.dk/services/SignalP/). The molecular weight of the CpBV-lectin from the predicted amino acid sequence, after removal of the putative signal peptide, was 17,270 Da. A potential N-linked glycosylation site is located at Asn-113, judged by NXS or NXT sequence, where X is any amino acid except proline. PCR using genomic DNA of C. plutellae bracovirus produced longer product than the expected size of cDNA, indicating an intron in ORF (156 bp) (Fig. 2B). The junctions between exons and intron showed a consensus eukaryotic splicing signal (‘GT-AG rule’). CpBV-lectin showed high sequence homologies with other bracovirus lectins based on predicted amino acid sequence; for example, 80% homology with C. ruficrus bracovirus lectin (‘Crf111’ in Fig. 3). CpBV-lectin also shared homology with insect and mammalian lectins (Fig. 3B). Whole sequence except the predicted signal peptide represents a single C-type lectin (CTL) with a single carbohydrate recognition domain (CRD, residues 17–139 after signal peptide cleavage) like other polydnaviral lectins, whereas lepidopteran lectins contains two CRDs (Fig. 3B).
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Fig. 4. Viral lectin gene in Cotesia plutellae bracovirus (CpBV) genome. (A) Southern hybridization (right box) of the viral lectin against CpBV genome (left box). The DNA segments were separated on 5% agarose gel for 24 h and visualized under UV after ethidium bromide staining. Numbering segments follows alphabetic order from anode to cathode. (B) RT-PCR of the CpBV-lectin was performed with RNA extracts from nonparasitized (‘NP’) or parasitized (‘P’) Plutella xylostella. Primers used ‘Lec-FP’ and ‘Lec-RP’ as described in Materials and methods (Section 2.8). ‘M’ indicates molecular weight marker. β-Actin gene was used as an internal control of RT-PCR.
3.3. Encoding viral lectin gene in CpBV and its expression in parasitized P. xylostella By a Southern hybridization analysis, CpBV-lectin gene was confirmed to be present in the CpBV genome (Fig. 4A). Two segments (‘B’ and ‘I’) of CpBV genome (Kim, 2006) were hybridized by CpBV-lectin probe. RT-PCR analysis showed that CpBV-lectin gene was expressed only in the parasitized P. xylostella (Fig. 4B). A temporal expression pattern of CpBV-lectin in parasitized P. xylostella was analyzed by real-time quantitative RT-PCR using gene-specific primers (‘RT-FP’ and ‘RT-RP’ in
Fig. 3). The estimated fluorescence levels of CpBV-lectin expression were normalized by concomitant fluorescence levels produced by β-actin expression as an endogenous reference. Based on the normalized expression level at 7 days post-parasitization as a calibrator because of the lowest detectable expression level, relative CpBV-lectin expression values of all other samples were calculated and compared (Table 1). The expression of the CpBV-lectin gene was detected at the first day after parasitization and its level increased up to 4 days, and then decreased.
Fig. 3. Structural analysis of putative amino acid sequence of Cotesia plutellae bracovirus (CpBV) lectin. (A) Sequence comparison of CpBV-lectin with those of Manduca sexta (MsIML-2), Hyphantria cunea (Hdd15), Periplaneta americana (LPSBP), Mus musculus (MmMBP-A), Homo sapiens (HsMBP-2) and other Cotesia spp. (CrV3, Crf111, and Cky811). Conserved regions are black-boxed. Gaps were introduced to optimize alignment by a Clustal W program with a parameter set of gap penalty (10.00), gap length penalty (0.20), delay divergent seqs (30%), and DNA transition weight (0.50). (B) Their phylogenetic tree of the selected lectins and comparison of their carbohydrate recognition domain (‘CRD’) regions. Numbers on the phylogenetic tree indicate the percentage of similarity index on each branch analyzed by a Clustal W program. CrV3 (NCBI accession No. AY234855); Crf111 (BAC55179); Cky811 (BAC55180); MsIML-2 (AAF91316); Hdd15 (AAD09286); LPSBP (P26305); MmMBP-A (LNMSMA); HSMBP-2 (AAC31937).
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Table 1 Relative quantification of Cotesia plutellae bracovirus (CpBV) lectin gene expression in parasitized host (Plutella xylostella) by real-time quantitative RT-PCR a using the comparative CT method Days after parasitization
Viral lectin (average CT)
Actin (average CT)
ΔCT (CT(Viral
1 2 3 4 5 6 7 8
25.6 ± 0.9 23.0 ± 2.1 22.7 ± 1.5 20.5 ± 2.2 23.5 ± 1.2 24.4 ± 1.6 25.3 ± 2.0 24.5 ± 1.6
26.0 ± 0.6 25.8 ± 0.7 26.6 ± 1.0 26.1 ± 0.9 27.1 ± 0.8 25.8 ± 1.1 25.7 ± 1.9 24.7 ± 1.9
−1.5 ± 0.5 −2.9 ± 1.6 −3.9 ± 0.7 −5.5 ± 1.5 −3.6 ± 0.7 −1.4 ± 0.8 −0.3 ± 0.3 −0.4 ± 0.1
lectin) − CT(Actin))
ΔΔCT b (ΔCT − ΔCT(P7))
2− ΔΔCT
− 1.1 ± 0.5 − 2.5 ± 1.6 − 3.6 ± 0.7 − 5.2 ± 1.5 − 3.2 ± 0.7 − 1.0 ± 0.8 0.0 ± 0.3 − 0.1 ± 0.1
2.3 ± 0.7 9.0 ± 10.2 12.9 ± 6.8 52.8 ± 55.6 10.1 ± 4.9 2.3 ± 1.4 1.0 ± 0.2 1.1 ± 0.0
Amplified products are shown below table, where the products with post-parasitization are compared in quantity with reference actin products. a The primers used for amplifying viral lectin and actin are described in Materials and methods. b Deviation from ΔCT at 7 days after parasitization (P7).
3.4. Expression levels of CpBV-lectin protein in parasitized P. xylostella To raise polyclonal antibody of CpBV-lectin, its ORF excluding a signal peptide was cloned into pBAD-TOPO bacterial expression system. The recombinant CpBV-lectin was expected as 20,240 Da in molecular weight because it contained 140 residues of the viral lectin and 28 residues of enterokinase recognition, V5 epitope, and polyhistidine sites. As expected, the expressed and purified recombinant viral lectin was detected at just above 20 kDa and confirmed by immunoblotting using V5 antibody (Fig. 5A). This purified protein was used to raise antibody, which was then used to analyze CpBV-lectin protein levels in the parasitized P. xylostella (Fig. 5B). The CpBV-lectin protein was detected as early as 12 h after parasitization and maintained up to 2 days post-parasitization. Unlike mRNA levels measured by quantitative RT-PCR, the CpBV-lectin protein did not appear after 48 h post-parasitization.
old eggs (data not shown). Quantitative analysis of encapsulation response against early (0 h) and late (24 h) parasitized eggs showed a complete inhibition of the recognition efficacy of hemocytes against late parasitized eggs. 4. Discussion Endoparasitoids can manipulate host immune capacity to defend themselves and accommodate their development (Schmidt et al., 2001). Especially in some endoparasitoids with symbiotic polydnavirus like C. plutellae, host immunosuppression can be induced by the help of viral products (Li and Webb, 1994; Summers and Dib-Hajj, 1995; Asgari et al., 1997). Generally, hemocytes mediate and express various immune responses after their nonself recognition (Gillespie et al., 1997). Thus, if the
3.5. Inhibitory effect of CpBV-lectin on hemocyte responses As shown above, plasma from parasitized P. xylostella inhibited hemocyte and bacterial association. To demonstrate this inhibition caused by CpBV-lectin, bacteria were preincubated with purified recombinant CpBV-lectin and incubated with nonparasitized hemocytes (Fig. 6A). As expected, untreated bacteria were easily bound on hemocytes, but bacteria treated with CpBV-lectin did not well attach to the hemocytes. To examine if CpBV-lectin protects C. plutellae eggs from hemocytic encapsulation of P. xylostella, encapsulation analyses were performed (Fig. 6B). A preliminary in vitro encapsulation assay showed that hemocytes of P. xylostella exhibited in vitro encapsulation against agarose beads (P-60, Biorad, Hercules, CA, USA) (data not shown). When parasitoid eggs were incubated with hemocyte suspension, newly laid eggs (‘0 h’) were encapsulated by the hemocytes, compared to inhibition of encapsulation response against 24 h old eggs. Immunostaining using CpBV-lectin antibody showed strong positive reaction on surface of 24 h old eggs, but not on newly laid eggs. For a control, pre-immune serum was used for the primary antibody and did not detect any CpBV-lectin on 24 h
Fig. 5. A recombinant viral lectin of Cotesia plutellae bracovirus (CpBV). (A) The CpBV-lectin was produced using a bacterial expression system and purified by NiNTA column against His-tag. The protein was separated on 15% SDS–PAGE and visualized by immunoblotting against V5 antibody. (B) Immunoblot of CpBVlectin from whole body extracts of nonparasitized (‘NP’) and parasitized Plutella xylostella. Each protein extract (10 μg) was run on 15% SDS–PAGE and transferred to a membrane, which were followed by incubation with anti-mouse IgG (1000× dilution) against CpBV-lectin.
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endoparasitoids interrupt the ability of hemocyte to recognize pathogen, they can effectively induce their host to be in an immunosuppressive state. For the analysis of nonself recognition, we used a binding assay between hemocytes and bacteria. In this assay, most hemocytes in nonparasitized P. xylostella exhibited strong affinity to the bacteria. Most hemocytes fixed on the slide glass during incubation were granular cells and plasmatocytes in cell morphology. These two cells possess phagocytotic activity in
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P. xylostella (Ibrahim and Kim, 2008), suggesting their binding capacities to bacteria. Moreover, granular cells have been known to strongly adhere to culture plate, which is usually used for separating granular cells from other hemocytes by a short incubation in culture plate (Pech and Strand, 1996). Even though our assay was not direct measurement of nonself recognition of the hemocytes, C. plutellae-parasitized P. xylostella hemocytes clearly lost their binding efficacy to bacteria. The inhibitory factors by parasitization were present in both hemocytes and plasma because the reciprocal compensation with nonparasitized hemocytes or plasma did not completely recover the suppressed efficacy of the hemocytes to bind bacteria. Several hemocyte-inhibitory factors have been isolated in other endoparasitoid systems. VHv1.1, derived from Campoletis sonorensis ichnovirus, targets granular cells and reduces host egg encapsulation by 50% (Li and Webb, 1994). In Pseudoplusia includens parasitized by Microplitis demolitor, MdBV directly induces cell death of granular cells (Strand and Pech, 1995). CrV1 is a viral product of C. rubecula and has been regarded as a hemocyte-inhibitory factor by mediating cytoskeleton breakdown (Asgari et al., 1996). In this study, we had interest in the inhibitory factor(s) present in the parasitized plasma. Without parasitization, plasma seemed to mediate pathogen recognition because bacteria did not bind to hemocytes in heat-treated plasma. This suggests a presence of opsonin in the plasma that mediates the association of coupling pathogen and hemocytes. Insects can recognize several pathogens according to their general molecular characteristics such as peptidoglycan against Gram-positive bacteria, lipopolysaccharides against Gram-negative bacteria, and β-1,3-glucan against fungi (Janeway, 1989; Hoffmann et al., 1999). To perform this recognition function, pattern recognition proteins (PRPs) have been proposed and identified in insect plasma (Yu et al., 2002). Among these PRPs, C-type lectins have been proved to function as an opsonin to induce phenoloxidase activation, hemocyte nodule formation, and encapsulation (Koizumi et al., 1999; Yu and Kanost, 1999, 2000, 2004). Interestingly, some polydnaviruses possess C-type lectin in their genome, which is expressed during parasitization (Glatz et al., Fig. 6. Immunosuppressive effects of Cotesia plutellae bracovirus (CpBV) lectin. (A) Inhibitory activity of recombinant CpBV-lectin on the bacterial binding capacity of nonparasitized hemocytes of Plutella xylostella. Escherichia coli were stained with Safranin-O and incubated with CpBV-lectin (10 ng protein plus 105 bacterial cells in 1 mL Grace's medium). The unbound lectin was washed off by three times washings with fresh Grace's medium. Ten μl of the bacterial preparation was overlaid on the hemocytes (≈ 100 cells) and incubated for 30 min at room temperature. After three times washings, the bound hemocytes were counted. (B) Encapsulation assay of C. plutellae eggs (‘E’) by hemocytes (‘H’) of P. xylostella. Newly laid (‘0 h’) and 24 h old eggs were collected from the parasitized larvae and washed with TBS to clear off any plasma. For encapsulation assay, the washed eggs were incubated with hemocyte suspension for 4 h and washed with TBS. Arrow indicates aggregated hemocytes encapsulating the parasitoid egg. For immunostaining assay, the washed eggs were incubated with antibody of CpBV-lectin for 1 h and followed by incubation in secondary antibody conjugated with alkaline phosphatase. For a control, pre-immune serum was used for the primary antibody. Scale bar represents 100 μm. In both graph data, each treatment consisted of three measurements and different letters above standard deviation bars indicate significant difference between means at Type I error = 0.05 (LSD test).
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2003). But, its clear inhibitory implication in immune recognition has not been proved. Here, we hypothesized that an inhibitory factor in the plasma of C. plutellae-parasitized P. xylostella is CpBV-lectin, which interrupts normal opsonin role of host lectin presumably by its structural similarity. This was supported indirectly by heat experiment of nonparasitized plasma and by the fact that the capacity of the nonparasitized hemocytes to bind bacteria decreased as the proportion of parasitized plasma increased in the overlaying plasma volume. With these speculation and indirect evidence, we began to clone viral lectin from the parasitized P. xylostella. With both directional RACEs, a complete cDNA of lectin gene was identified after primary RT-PCR using consensus regions of several viral lectin genes. A Southern hybridization indicated that this CpBV-lectin gene was located in the CpBV genome and RTPCR analysis showed that its expression was only detected in the parasitized P. xylostella. Southern hybridization of CpBV-lectin in two independent viral segments suggests a presence of another related gene or by segment nesting process reported in ichnoviruses (Xu and Stoltz, 1993; Cui and Webb, 1997). Considering that polydnaviruses do not replicate in the target host tissues, segment nesting can be exploited to increase expression level of a functionally significant gene. However, a current genome study did not show any clear evidence in segment nesting in CpBV (Kim et al., 2007). This suggests a presence of another homologous gene, which needs to be verified. Predicted amino acid sequence of CpBV-lectin contains a signal peptide, suggesting a secretory protein and exhibits high homologies with ORF size and sequence with those of Cotesia spp.: 162 residues in C. kariyai, 157 residues in C. ruficrus, and 159 residues in C. rubecula (Teramoto and Tanaka, 2003; Glatz et al., 2003). This supports an existence of a novel polydnaviral lectin family in Cotesia-associated bracoviruses (Glatz et al., 2003). Expression of CpBV-lectin was analyzed by a real-time quantitative RT-PCR and showed that it increased during early parasitization, reached a maximal level at 4 days, and then declined. Protein levels of CpBV-lectin showed much transient expression pattern for only 2 days. This transient expression pattern was also found in other analyzed related viral lectins. For examples, maximal transcription of Cky811 gene was found in 6 h after parasitization and declined (Teramoto and Tanaka, 2003), and CrV3 in the parasitized hemolymph showed a maximal level at 6 h after parasitization and disappeared at 24 h (Glatz et al., 2003). The discrepancy between CpBV-lectin mRNA and protein levels was evident through several re-measurements. We speculate that relatively transient protein level may be due to proteolytic degradation after 48 h by teratocytes of C. plutellae because teratocytes, derived from embryonic serosal membrane, are well known to secrete proteolytic enzymes (Strand et al., 1988) and are in fact detected in the hemolymph of P. xylostella with egg hatch of C. plutellae after 36 h post-parasitization (Basio and Kim, 2005). But we do not know physiological significance in transcriptional activation of CpBV-lectin after 48 h from this study. Bacterial binding assay and CpBV-lectin expression pattern suggest that CpBV-lectin plays a role in inducing host immunosuppression by inhibiting nonself recognition of
P. xylostella hemocytes. First of all, we showed that purified CpBV-lectin significantly interfered with association between hemocytes and bacteria. This indicates that the suppression of hemocyte and bacterial association observed in the parasitized plasma treatment may be contributed by CpBV-lectin. To prove this speculation, we performed in vitro encapsulation assay with different ages of parasitoid eggs from the parasitized P. xylostella. Though we did not observe full encapsulation shown in in vivo conditions (Ibrahim and Kim, 2008), in vitro encapsulation assay used in this study showed hemocyte clumpings around beads or early (0 h) parasitoid eggs. Compared to newly laid eggs, 24 h old eggs were not encapsulated. As expected, the encapsulated eggs contained CpBV-lectin on their surface detectable by immunostaining, but the newly laid eggs did not. Based on these results on CpBV-lectin, we propose a working hypothesis how the viral lectin interrupts immune response of parasitized P. xylostella in terms of pathogen recognition. Generally, other viral lectins are surface proteins that are involved in virus entry to target tissues with specific sugar determinants (Sharon and Lis, 2004). However, polydnaviral lectins are expressed and secreted, which result in soluble proteins in the plasma as shown in CrV3 (Glatz et al., 2003). Signal peptide in putative ORF and detection of CpBVlectin proteins on the parasitoid egg surface suggest that CpBVlectin is produced and secreted into plasma. All lepidopteran CTLs identified have two CRDs, which are contrasting with other animal CTLs that contain only a single CRD (Yu et al., 2002). The single CRD of CpBV-lectin shows homology with C-terminal CRD of Hyphantria cunea, which is another host of C. plutellae (Shin et al., 2000; Bae and Kim, 2004), representing a truncated structure of host counterpart. This suggests that CpBV-lectin and other polydnaviral lectins, have structural and sequence similarities to host lectins, enabling to compete with host lectins for binding sites that are pathogen-specific moieties for recognition or involved in induction of immune reactions. This competitive inhibition may lead to immunosuppression of the parasitized P. xylostella in terms of nonself recognition. Acknowledgments This study was supported by the Korea Research Foundation Grant funded by the Korean Government (R0520020000 48802004) and Biogreen 21 program of Rural Development Administration. NM was financially supported by the 2nd stage of BK21. We also appreciate Youngim Song for supplying research materials and valuable encouragement. References Asgari, S., Hellers, M., Schmidt, O., 1996. Host hemocyte inactivation by an insect parasitoid: transient expression of a polydnavirus gene. J. Gen. Virol. 77, 2653–2662. Asgari, S., Schmidt, O., Theopold, U., 1997. A polydnavirus-encoded protein of an endoparasitoid wasp is an immune suppressor. J. Gen. Virol. 78, 3061–3070. Bae, S., Kim, Y., 2004. Host physiological changes due to parasitism of a braconid wasp, Cotesia plutellae, on diamondback moth, Plutella xylostella. Comp. Biochem. Physiol. A 138, 39–44. Basio, N.A., Kim, Y., 2005. A short review of teratocytes and their characters in Cotesia plutellae (Braconidae: Hymenoptera). J. Asia-Pac. Entomol. 8, 211–217.
S. Lee et al. / Comparative Biochemistry and Physiology, Part A 149 (2008) 351–361 Belle, E., Beckage, N.E., Rousselet, J., Poirie, M., Lemeunier, F., Drezen, J., 2002. Visualization of polydnavirus sequences in a parasitoid wasp chromosome. J. Virol. 76, 5793–5796. Choi, J.Y., Rho, J.Y., Kang, J.N., Shim, H.J., Woo, S.D., Jin, B.R., Li, M.S., Je, Y.H., 2005. Genomic segments cloning analysis of Cotesia plutellae polydnavirus using plasmid capture system. Biochem. Biophys. Res. Commun. 332, 487–493. Cui, L., Webb, B.A., 1997. Homologous sequences in the Campoletis sonorensis polydnavirus genome are implicated in replication and nesting of the W segment family. J. Virol. 71, 8504–8513. Edson, K.M., Vinson, S.B., Stoltz, D.B., Summers, M.D., 1981. Virus in a parasitoid wasp: suppression of the cellular immune response in the parasitoid's host. Science 211, 582–583. Gillespie, J.P., Kanost, M.R., Trenczek, T., 1997. Biological mediators of insect immunity. Annu. Rev. Entomol. 42, 611–643. Glatz, R., Schmidt, O., Asgari, S., 2003. Characterization of a novel protein with homology to C-type lectins expressed by the Cotesia rubecula bracovirus in larvae of the lepidopteran host, Pieris rapae. J. Biol. Chem. 278, 19743–19750. Hoffmann, J.A., Kafatos, F.C., Janeway, C.A., Ezekowitz, R.A.B., 1999. Phylogenetic perspectives in innate immunity. Science 284, 1313–1318. Ibrahim, A.M.A., Kim, Y., 2008. Transient expression of protein tyrosine phosphatases encoded in Cotesia plutellae bracovirus inhibits insect cellular immune responses. Naturwissenschaften 95, 25–32. Ibrahim, A.M.A., Choi, J.Y., Je, Y.H., Kim, Y., 2007. Protein tyrosine phosphatases encoded in Cotesia plutellae bracovirus: sequence analysis, expression profile, and a possible biological role in host immunosuppression. Dev. Comp. Immunol. 31, 978–990. Janeway, C.A., 1989. Approaching the asymptote? Evolution and revolution in immunology. Cold Spring Harbor Symp. Quant. Biol. 54, 1–13. Kim, Y., 2006. Polydnavirus and its novel application to insect pest control. Korean J. Appl. Entomol. 45, 241–259. Kim, Y., Bae, S., Lee, S., 2004. Polydnavirus replication and ovipositional habit of Cotesia plutellae. Korean J. Appl. Entomol. 43, 225–231. Kim, Y., Choi, J.Y., Je, Y.H., 2007. Cotesia plutellae. Bracovirus genome and its function in altering insect physiology. J. Asia-Pac. Entomol. 10, 181–191. Koizumi, N., Imamura, M., Kadotani, T., Yaoi, K., Iwahana, H., Sato, R., 1999. The lipopolysaccharide-binding protein participating in hemocyte nodule formation in the silkworm Bombyx mori is a novel member of the C-type lectin superfamily with two different tandem carbohydrate-recognition domains. FEBS Lett. 443, 139–143. Krell, P.J., Summers, M.D., Vinson, S.B., 1982. Virus with a multipartite superhelical DNA genome from the ichneumonid parasitoid Campoletis sonorensis. J. Gen. Virol. 43, 859–870. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Lee, S., Kim, Y., 2004. Activity of juvenile hormone esterase of diamondback moth, Plutella xylostella, is not inhibited by parasitism of Cotesia plutellae. J. Asia-Pac. Entomol. 7, 283–287. Li, X., Webb, B.A., 1994. Apparent functional role for a cysteine-rich polydnavirus protein in suppression of insect cellular immunity. J. Virol. 68, 7482–7489. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402–408.
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Comparative Biochemistry and Physiology, Part A 149 (2008) 351 – 361 www.elsevier.com/locate/cbpa
A viral lectin encoded in Cotesia plutellae bracovirus and its immunosuppressive effect on host hemocytes Sunyoung Lee, Madanagopal Nalini, Yonggyun Kim ⁎ Department of Bioresource Sciences, Andong National University, Andong 760-749, Republic of Korea Received 16 November 2007; received in revised form 9 January 2008; accepted 9 January 2008 Available online 14 January 2008
Abstract An endoparasitoid wasp, Cotesia plutellae, induces immunosuppression of the host diamondback moth, Plutella xylostella. To identify an immunosuppressive factor, the parasitized hemolymph of P. xylostella was separated into plasma and hemocyte fractions. When nonparasitized hemocytes were overlaid with parasitized plasma, they showed significant reduction in bacterial binding efficacy. Here, we considered a viral lectin previously known in other Cotesia species as a humoral immunosuppressive candidate in C. plutellae parasitization. Based on consensus regions of the viral lectins, the corresponding lectin gene was cloned from P. xylostella parasitized by C. plutellae. Its cDNA is 674 bp long and encodes 157 amino acid residues containing a signal peptide (15 residues) and one carbohydrate recognition domain. Open reading frame is divided by one intron (156 bp) in its genomic DNA. Amino acid sequence shares 80% homology with that of C. ruficrus bracovirus lectin and is classified into C-type lectin. Southern hybridization analysis indicated that the cloned lectin gene was located at C. plutellae bracovirus (CpBV) genome. Both real-time quantitative RT-PCR and immunoblotting assays indicated that CpBV-lectin showed early expression during the parasitization. A recombinant CpBV-lectin was expressed in a bacterial system and the purified protein significantly inhibited the association between bacteria and hemocytes of nonparasitized P. xylostella. In the parasitized P. xylostella, CpBV-lectin was detected on the surface of parasitoid eggs after 24 h parasitization by its specific immunostaining. The 24 h old eggs were not encapsulated in vitro by hemocytes of P. xylostella, compared to newly laid parasitoid eggs showing no CpBV-lectin detectable and easily encapsulated. These results support an existence of a polydnaviral lectin family among Cotesia-associated bracovirus and propose its immunosuppressive function. © 2008 Elsevier Inc. All rights reserved. Keywords: Cotesia plutellae; Plutella xylostella; CpBV; Encapsulation; Viral lectin; Polydnavirus; Immune
1. Introduction In response to invading pathogens, insects can express various defense responses including both cellular and humoral immune reactions (Ratcliffe et al., 1985). Endoparasitoid wasps develop within the general cavity of other insects and must overcome the immune responses of target insects. Some braconid or ichneumonid wasps possess symbiotic polydnavirus, which has been regarded as a potent agent to suppress immune response of parasitized hosts (Webb, 1998). Polydnavirus is an insect DNA virus exhibiting obligate mutualism with host insects (Whitfield, 1990). It is classified
⁎ Corresponding author. Tel.: +82 54 820 5638; fax: +82 54 823 1628. E-mail address: [email protected] (Y. Kim). 1095-6433/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2008.01.007
into bracovirus and ichnovirus according to its host wasp family and virion morphology. As a provirus, its segmented genome is located on host wasp chromosome (Belle et al., 2002). During replication, different viral DNA segments are excised from host chromosome and circularized, which finally become nucleocapsids (Krell et al., 1982). Viral replication occurs in ovarian calyx region probably in response to ecdysteroid signal during pupal period (Webb and Summers, 1992). Virus particles in the calyx lumen of the parasitoid are delivered into the hemocoel of parasitized insects along with laying eggs and express their encoded genes after getting entry into target tissues (Edson et al., 1981). Various viral products have been implicated to suppress insect immunity by inducing hemocyte apoptosis (Strand and Pech, 1995), altering hemocyte morphology and behavior (Tanaka, 1987; Strand and Pech, 1995), and destabilization of hemocyte cytoskeleton (Asgari et al., 1997).
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Cotesia plutellae (Braconidae: Hymenoptera) is an endoparasitoid of the diamondback moth, Plutella xylostella, in which a symbiotic polydnavirus (C. plutellae bracovirus: CpBV) plays a critical role in altering physiological status favorable for wasp survival and development (Bae and Kim, 2004; Kim et al., 2007). Parasitized P. xylostella exhibits significant immunosuppressive effect in both humoral and cellular reactions (Bae and Kim, 2004; Ibrahim and Kim, 2008) and fails to undergo metamorphosis (Lee and Kim, 2004). Replication of CpBV within the parasitoid begins at late pupal stage (Kim et al., 2004) so that the wasp can parasitize successfully even just after adult emergence. The replicated CpBV genome consists of at least 27 DNA segments, in which more than 470 kb nonoverlapping nucleotide sequences have been determined and have been known to encode several putative gene families (Choi et al., 2005; Kim, 2006; Kim et al., 2007). CpBV-PTPs are the largest family in CpBV and play significant role in immunosuppression probably by altering phosphorylation status of the infected hemocytes (Ibrahim et al., 2007; Ibrahim and Kim, 2008). CpBV15β is a 15 kDa protein-encoding CpBV gene, which inhibits hemocyte-spreading by modifying the cytoskeletal structure of P. xylostella hemocytes (Nalini and Kim, 2007). This study was focused on identifying immunosuppressive factor from C. plutellae bracovirus especially in terms of nonself recognition capacity by hemocytes of parasitized P. xylostella. To this end, we analyzed the source of the inhibiting factor(s) using a binding assay between hemocytes and bacteria. A suggestion that there would be a humoral interrupting factor in parasitized plasma led us to clone viral lectin gene, which has been proposed to be a plausible candidate (Glatz et al., 2003) for interrupting hemocyte recognition. Finally, we proved the immunosuppressive effect of the viral lectin in preventing hemocytic encapsulation against C. plutellae eggs. 2. Materials and methods 2.1. Rearing insects and parasitization P. xylostella larvae were reared at 25 ± 1 °C with 16:8 h (L:D) photoperiod on cabbage leaves. Late second instar larvae were parasitized by C. plutellae at 1:2 (wasp:host) density for 12 h and reared under the above rearing condition. Wasp cocoons were collected and held in plastic cage until their emergence. The emerged adult wasps were collected every day and allowed to mate for 24 h before using for parasitization. Adult wasps were fed 40% sucrose. 2.2. Hemocyte-bacterial binding assay Adhesion of bacteria on hemocytes was determined by using hemocyte monolayers overlaid with Safranin-O-stained Escherichia coli under in vitro conditions. Hemolymph from 20 larvae of P. xylostella was separated into hemocytes and plasma by centrifugation (1000 ×g, 3 min, 4 °C). The hemocytes were further washed by centrifugation and resuspended in 100 μL of Grace's insect medium (Sigma-Aldrich, MO, USA). Heat-killed E. coli (70 °C for 60 min) stained in 10% Safranin-
O dye solution was washed and resuspended in parasitized or nonparasitised plasma at a concentration of ~ 103 cells/μL. Hemocyte monolayers were made by using 10 μL of hemocyte suspension and left for 1 h at room temperature under humid condition. After attachment of hemocytes, the medium was replaced with 10 μL of E. coli suspension in parasitized or nonparasitized plasma and incubated for 30 min. The monolayers were washed with fresh Grace's insect medium and observed under light microscope at 400× magnification. Hemocytes adhered with bacteria were counted by counting 100 hemocytes from ten random fields and was recorded as percent bacteria-bound hemocytes. Each treatment was determined based on three independent replications. 2.3. CpBV DNA purification CpBV DNA was isolated from the ovary of female wasps. For this, 200 abdomens of female wasps were homogenized in TEK (11 mM Na2EDTA·2H2O, 5 mM KCl, 100 mM Tris, pH 7.5) using a 3 mL syringe with a 21G needle. The suspension was passed through 0.45 μm syringe filter (MFS, CA, USA), 15 μL proteinase K (2 ng/μL) and 25 μL of 10% sarcosyl solutions was added, and incubated at 50 °C overnight. The lysate was treated with RNase A (0.3 μg/μL) at 37 °C for 2 h and then extracted with phenol/chloroform. The viral DNA was precipitated by adding 2 vol. of ethanol and centrifugation (14,000 ×g, 20 min, 4 °C). The obtained pellet was washed with 70% ethanol, air-dried, and dissolved in TE buffer (1 mM Tris, 0.1 mM EDTA, pH 8.0). 2.4. RNA extraction and cDNA construction Total RNA was extracted from parasitized P. xylostella larvae from day 1 to day 8 post-parasitization using Trizol reagent (MRC, OH, USA) and reverse-transcribed with RT-premix (Bioneer, Daejon, Korea) using an oligo-dT primer (5′-CCAGT GAGCA GAGTG ACGAG GACTC GAGCT CAAGC TTTTT TTTTT TTTTT T-3′) after RNase H treatment. For a control, nonparasitized larvae were collected at their 4th instar and used for constructing cDNA. 2.5. CpBV-lectin gene cloning Two consensus regions (‘FHSTPAT’ and ‘KCGSLLK’) were chosen from viral lectin genes of C. rubecula and C. ruficrus (Glatz et al., 2003; Teramoto and Tanaka, 2003). The corresponding degenerate primers of forward (5′-TTYCA YAGYA CNCCN GCNAC-3′) and reverse (5′-TTNAR NARRC TNCCR CAYTT3′) were designed and used for amplifying the region from cDNA of parasitized P. xylostella under following conditions: 94 °C for 1 min, 50 °C for 1 min and 72 °C for 2 min with 35 cycles. The amplified PCR products were ligated into pGEM-T Easy cloning vector (Promega, WI, USA). Both T7 and Sp6 sequencing primers were used to sequence the inserts by automatic DNA sequencer (Bionics, Seoul, Korea). The resulting sequences were compared with the known viral lectin genes using NCBI-BLAST search program. Based on the resulting partial sequence, gene-specific
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primers were designed for 5′-RACE and 3′-RACE: 5′-1 (5′GGTAA CTACA GCAAG ACTTC-3′), 5′-2 (5′-TCTTA GCTTC GTCGA AGGTT-3′), 3′-1 (5′-TGAAA TTACA AGCGT ACATT-3′), and 3′-2 (5′-CGAGC ACTCC TAGTG TTCAA-3′). 5′-RACE was performed using 5′-RACE kit (Invitrogen, CA, USA) according to manufacturer's method. Total RNA was reverse-transcribed with 5′-1 primer. After RNase H treatment, the first-strand cDNA was tailed with dCTP by terminal deoxynucleotidyl transferase and PCR-amplified with oligo-dG (5′-GGCCA CGCGT CGACT AGTAC GGGII GGGII GGGII G-3′) and 5′-1 primers, where PCR was performed under the described condition above except 55 °C annealing temperature. For 3′-RACE, total RNA was reverse-transcribed with RTpremix (Bioneer) using an oligo-dT primer. Nested RT-PCR was then performed by the sequential primer pairs of 3′-1/ oligo-dT and then 3′-2/oligo-dT. PCR was performed under the described condition above except 52 °C annealing temperature. The amplified PCR products were ligated and sequenced as described above. The sequence data were aligned and corrected by DNAstar program (Version 5.01, DNAstar Inc, Madison, USA). The functional domains including open reading frame (ORF), 5′-untranslated region (UTR), and 3′UTR were searched using Smart/Pfam domain search program (http://elm.eu.org/basicELM/). 2.6. Hybridization analyses of CpBV-lectin For Southern hybridization, DNA samples were separated on agarose gel and transferred to a nylon membrane (Nytran, NH, USA). A biotinylated DNA probe encompassing ORF was prepared by incorporating biotin-N4-dCTP (KPL, MD, USA) through random primer extension as described by the manufacturer. The blot was then hybridized with the labeled probe in hybridization solution (50% formamide, 6×SSC, 5×Denhardt's reagent, 0.5% SDS) overnight at 42 °C. The blot was washed twice in 2×SSC containing 0.5% SDS at 42 °C for 20 min each, then twice in 0.2×SSC containing 0.5% SDS at 55 °C for 20 min each, and finally twice in 1×SSC at 37 °C for 10 min each. CDP-star chemiluminescent substrate (KPL, MD, USA) was used for detection after binding alkaline phosphatase-labeled streptavidin. Then, the probed membrane was exposed to hyperfilm (Amersham Biosciences, UK) and developed by dipping in Lucky MQ (Poohung, Ahnsan, Korea). For immunoblotting assay, P. xylostella larvae were ground in 50 mM phosphate buffer (pH 7.0) and centrifuged (14,000 ×g, 5 min). The resulting supernatant was mixed with the same volume of denaturing buffer (4% SDS, 20% glycerol, 10% β-mercaptoethanol in 62.5 mM Tris–HCl, pH 6.8). Electrophoresis was performed on 15% SDS–PAGE under denaturing condition (Laemmli, 1970). The separated proteins were transferred onto a nitrocellulose membrane according to Towbin et al. (1979). Non-specific sites were blocked by 5% skim milk at room temperature for 1 h. After three washes with PBS, the membrane was incubated at room temperature with primary antibody raised against V5 (Invitrogen) or CpBV-lectin for 2 h. Subsequently, the membrane was washed with PBS thrice and incubated with secondary antibody (1/2000 dilution)
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conjugated with alkaline phosphatase (Sigma). Finally after three times washing with PBS, the membrane was stained with an alkaline phosphatase substrate solution containing nitro blue tetrazolium/5-mono-4-chloro-3-indolyl phosphate (Sigma) in 10 mM phosphate buffer, pH 9.5. 2.7. Quantitative RT-PCR of CpBV-lectin expression Real-time quantitative RT-PCR was performed on a Bioneer Exicycler™ Quantitative Thermal Block (Bioneer) using SYBR green chemistry and real-time fluorescence measurements using Accupower Greenstar™ PCR premix (Bioneer). Specific primers for CpBV-lectin were designed for real-time PCR: RTFP (5′-ATGAG TATTG GGAAG AGACT-3′), and RT-RP (5′GGTAA CTACA GCAAG ACTTC-3′). Template cDNAs were constructed as described above. The reaction mixture (20 μL) consisted of 1× Greenstar™ PCR Master mix, 10 mM MgCl2, each 0.5 μM of RT-FP and RT-RP primers, and 250 ng of cDNA. The reaction was performed and monitored under following conditions: one cycle (95 °C for 15 min) for activation of Hotstart Taq DNA polymerase, subsequently 40 cycles (94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min), and finally one cycle (72 °C for 10 min). Each RT-PCR amplification was independently replicated three times. In each replication as an internal control for equivalence of template, β-actin gene was amplified with forward (5′-TGGCA CCACA CCTTC TAC-3′) and reverse (5′-CATGA TCTGG GTCAT CTTCT-3′) primers. Fluorescence values were measured and amplification plots were generated in real-time by the Exicycler™ program. Quantitative analysis of CpBV-lectin expression followed a comparative CT (ΔCT) method (Livak and Schmittgen, 2001). 2.8. Expression and purification of CpBV-lectin in bacteria Two gene-specific primers (Lec-FP: 5′-ATGAG TATTG GGAAG AGACT-3′, Lec-RP: 5′-CACGC TTGTG CAGAA GAAGG-3′) were designed to amplify ORF of CpBV-lectin gene excluding putative signal sequence corresponding to the first 17 amino acids of the protein. PCR product was ligated into pBADTOPO expression vector (Invitrogen) according to the protocol provided by the manufacturer. The ligation product was transformed into Top10 strain of E. coli by heat shock. Colonies containing recombinant vectors were identified by digestion of Pmi I and Nco I. For a pilot expression, a positive clone was cultured at 37 °C with 200 rpm and over-expressed until OD600 = 0.5 after adding L-arabinose (final concentration of 0.002%). Fusion protein was identified by immunoblot using V5 antibody (Invitrogen). Bacterial CpBV-lectin was identified in the insoluble fraction of total bacterial proteins, with only a small amount being soluble. For large culture and expression, bacteria culture (30 L) was centrifuged (4000 ×g, 20 min, 4 °C). Cells were then resuspended in lysis buffer (8 M urea, 100 mM NaH2PO4, 10 mM Tris, pH 8.0) and gently rocked for 1 h. After centrifugation (14,000 ×g, 20 min, 4 °C), 2 mL of 5% Ni-NTA resin (Qiagen, CA, USA) was added to the resulting supernatant and mixed gently by shaking at 200 rpm for 1 h at room temperature. Lysate-resin mixture was carefully loaded into an empty column, and washed with a low pH
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buffer (8 M urea, 100 mM NaH2PO4, 10 mM Tris, pH 6.3). Bound proteins were eluted with acid elution buffer (8 M urea, 100 mM NaH2PO4, 10 mM Tris, pH 4.5) and confirmed by immunoblotting with V5 antibody as described above. The purified sample was sent to Peptron, Inc. (Daejon, Korea) for raising a polyclonal antibody using mouse. 2.9. In vitro encapsulation assay C. plutellae eggs were collected from parasitized P. xylostella at 0 and 24 h post-parasitization and washed with TBS (50 mM Tris–HCl, 100 mM dextrose, 5 mM KCl, 2.5 mM MgCl2, 50 mM NaCl, pH 7.5) to clear off any plasma. Hemocyte suspension was prepared by collecting hemolymph from 50 larvae in 200 μL of anticoagulant buffer (TBS containing 5 mM cysteine hydrochloride, 300 mOsm). Encapsulation assays were performed in flat-bottom microtiter plates (Falcon, Becton Dickinson Labware,
NJ, USA) by adding 300 μL of the hemocyte suspension to 12 washed eggs. The plates were held at 25 °C for 4 h with gentle rocking at every 15 min in order to facilitate the association of eggs and hemocytes. After incubation, these suspensions were transferred on glass slide and left undisturbed for 15 min for the encapsulated eggs to settle. The eggs were scored positive for encapsulation only if at least 15 hemocytes were adhered on the egg surface. Each treatment was replicated three times. In order to check the presence of CpBV-lectin on the egg surface, immunostaining was performed. The washed eggs were incubated with antibody of CpBV-lectin for 1 h and then subsequent steps according to immunoblotting method described above. 2.10. Statistical analysis Treatment means and variances were analyzed in one-way ANOVA by PROC GLM of SAS program (SAS Institute,
Fig. 1. Immunosuppressive agent(s) in the plasma of Plutella xylostella parasitized by Cotesia plutellae. (A) Hemocyte-bacterial binding assay. Hemocytes were fixed on the cover glass, where parasitized (‘P’) or nonparasitized (‘NP’) plasma was overlaid and incubated with Safranin-O-stained Escherichia coli for 30 min. Inset figure illustrate hemocyte attached by stained bacteria (arrows). Scale bar represents 10 μm. (B) Inhibitory effect of ‘P’ plasma on mediating role of ‘NP’ plasma in hemocyte and bacterial binding. (C) Heat-labile property of plasma factor(s) mediating hemocyte-bacterial binding, where asterisk indicates heat-treatment of plasma at 95 °C for 5 min. Each treatment consisted of three measurements. Different letters above standard deviation bars indicate significant difference between means at Type I error = 0.05 (LSD test).
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1989). All means were compared by least squared difference (LSD) test at Type I error = 0.05. 3. Results 3.1. Reduction in bacterial binding ability of P. xylostella hemocytes parasitized by C. plutellae Interaction between hemocytes and bacteria was measured as frequency of bacteria-bound hemocytes (Fig. 1). For this assay, immobilized hemocytes of P. xylostella were used by preparing monolayer, most of which consisted of granular cells and plasmatocytes based on their cell morphology. This assay showed that Safranin-O-stained bacteria resuspended in nonparasitized plasma could be easily attached to nonparasitized hemocytes (inset in Fig. 1A). However, parasitized hemocytes incubated with parasitized plasma significantly lost this binding ability. To clarify the source of inhibitory factor(s), parasitized or nonparasitized hemolymph was separated into hemocyte and plasma. Bacteria did not bind well to parasitized hemocytes
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even in the presence of nonparasitized plasma (Fig. 1A). Also, parasitized plasma significantly inhibited the ability of nonparasitized hemocytes to bind bacteria. Inhibitory factor(s) of parasitized plasma was further analyzed for dosimetric effect (Fig. 1B). When nonparasitized plasma was mixed with parasitized plasma in different ratios, the efficacy of nonparasitized hemocytes to bind bacteria decreased with increase in proportion of parasitized plasma. This suggests that parasitized plasma possesses inhibitory factor (s) to prevent hemocytes to bind bacteria. The plasma factor(s) was heat-labile because nonparasitized hemocytes overlaid with heat-treated nonparasitized plasma lost the bacterial binding ability just like the inhibition induced by parasitized plasma (Fig. 1C). 3.2. cDNA cloning and sequence of CpBV-lectin Heat-labile bacterial binding factor(s) in nonparasitized plasma and inhibitory effect of parasitized plasma on the hemocytes to bind bacteria suggest a hypothesis that there is an
Fig. 2. Gene structure of Cotesia plutellae bracovirus (CpBV) lectin (NCBI accession No. AY461733). (A) DNA sequence and predicted amino acid sequence of CpBV-lectin. Initiation and termination codons are shown in black box. The degenerate primers (forward and reverse) were designed from the consensus regions of Cotesia-associated spp. lectin genes. Gene-specific primers (5′-1, 5′-2, 3′-1 and 3′-2) used for 5′ and 3′ RACEs are indicated by underlines. RT-FP and RT-RP primers used for the real-time quantitative RT-PCR. The signal peptide is indicated by bold letters. Intron sequence is written in small and italic letter. (B) Intron/exon structure and restriction map, where one intron was positioned by dotted lines between cDNA and genomic DNA (‘gDNA’). Open reading frame (ORF, black bar) and untranslated region (UTR, white bar) are also denoted.
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inhibitory factor to interrupt nonself recognition of hemocytes in parasitized plasma. As an inhibitory candidate in nonself recognition, viral lectin genes have been reported in other Cotesiaassociated polydnaviruses (Glatz et al., 2003). Here, we cloned the viral lectin gene from C. plutellae-parasitized P. xylostella. CpBV-lectin mRNA was amplified by RT-PCR using degenerate primers (forward and reverse in Fig. 2A), which were designed from the consensus residues of other lectin genes from Cotesia spp. An approximately 260 bp cDNA amplicon was obtained and showed high homology with that of other Cotesia lectin genes. Four nested gene-specific primers were designed and produced 5′-RACE product of approximately 200 bp, and 3′RACE product of approximately 230 bp. The complete cDNA of the CpBV-lectin gene was 674 bp long. The cDNA has an ORF of 474 nucleotides encoding the entire CpBV-lectin of 157 amino acids (Fig. 3A). Fifteen amino acid residues at the Nterminus were predicted as a signal peptide by SignalP program (http://www.cbs.dtu.dk/services/SignalP/). The molecular weight of the CpBV-lectin from the predicted amino acid sequence, after removal of the putative signal peptide, was 17,270 Da. A potential N-linked glycosylation site is located at Asn-113, judged by NXS or NXT sequence, where X is any amino acid except proline. PCR using genomic DNA of C. plutellae bracovirus produced longer product than the expected size of cDNA, indicating an intron in ORF (156 bp) (Fig. 2B). The junctions between exons and intron showed a consensus eukaryotic splicing signal (‘GT-AG rule’). CpBV-lectin showed high sequence homologies with other bracovirus lectins based on predicted amino acid sequence; for example, 80% homology with C. ruficrus bracovirus lectin (‘Crf111’ in Fig. 3). CpBV-lectin also shared homology with insect and mammalian lectins (Fig. 3B). Whole sequence except the predicted signal peptide represents a single C-type lectin (CTL) with a single carbohydrate recognition domain (CRD, residues 17–139 after signal peptide cleavage) like other polydnaviral lectins, whereas lepidopteran lectins contains two CRDs (Fig. 3B).
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Fig. 4. Viral lectin gene in Cotesia plutellae bracovirus (CpBV) genome. (A) Southern hybridization (right box) of the viral lectin against CpBV genome (left box). The DNA segments were separated on 5% agarose gel for 24 h and visualized under UV after ethidium bromide staining. Numbering segments follows alphabetic order from anode to cathode. (B) RT-PCR of the CpBV-lectin was performed with RNA extracts from nonparasitized (‘NP’) or parasitized (‘P’) Plutella xylostella. Primers used ‘Lec-FP’ and ‘Lec-RP’ as described in Materials and methods (Section 2.8). ‘M’ indicates molecular weight marker. β-Actin gene was used as an internal control of RT-PCR.
3.3. Encoding viral lectin gene in CpBV and its expression in parasitized P. xylostella By a Southern hybridization analysis, CpBV-lectin gene was confirmed to be present in the CpBV genome (Fig. 4A). Two segments (‘B’ and ‘I’) of CpBV genome (Kim, 2006) were hybridized by CpBV-lectin probe. RT-PCR analysis showed that CpBV-lectin gene was expressed only in the parasitized P. xylostella (Fig. 4B). A temporal expression pattern of CpBV-lectin in parasitized P. xylostella was analyzed by real-time quantitative RT-PCR using gene-specific primers (‘RT-FP’ and ‘RT-RP’ in
Fig. 3). The estimated fluorescence levels of CpBV-lectin expression were normalized by concomitant fluorescence levels produced by β-actin expression as an endogenous reference. Based on the normalized expression level at 7 days post-parasitization as a calibrator because of the lowest detectable expression level, relative CpBV-lectin expression values of all other samples were calculated and compared (Table 1). The expression of the CpBV-lectin gene was detected at the first day after parasitization and its level increased up to 4 days, and then decreased.
Fig. 3. Structural analysis of putative amino acid sequence of Cotesia plutellae bracovirus (CpBV) lectin. (A) Sequence comparison of CpBV-lectin with those of Manduca sexta (MsIML-2), Hyphantria cunea (Hdd15), Periplaneta americana (LPSBP), Mus musculus (MmMBP-A), Homo sapiens (HsMBP-2) and other Cotesia spp. (CrV3, Crf111, and Cky811). Conserved regions are black-boxed. Gaps were introduced to optimize alignment by a Clustal W program with a parameter set of gap penalty (10.00), gap length penalty (0.20), delay divergent seqs (30%), and DNA transition weight (0.50). (B) Their phylogenetic tree of the selected lectins and comparison of their carbohydrate recognition domain (‘CRD’) regions. Numbers on the phylogenetic tree indicate the percentage of similarity index on each branch analyzed by a Clustal W program. CrV3 (NCBI accession No. AY234855); Crf111 (BAC55179); Cky811 (BAC55180); MsIML-2 (AAF91316); Hdd15 (AAD09286); LPSBP (P26305); MmMBP-A (LNMSMA); HSMBP-2 (AAC31937).
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Table 1 Relative quantification of Cotesia plutellae bracovirus (CpBV) lectin gene expression in parasitized host (Plutella xylostella) by real-time quantitative RT-PCR a using the comparative CT method Days after parasitization
Viral lectin (average CT)
Actin (average CT)
ΔCT (CT(Viral
1 2 3 4 5 6 7 8
25.6 ± 0.9 23.0 ± 2.1 22.7 ± 1.5 20.5 ± 2.2 23.5 ± 1.2 24.4 ± 1.6 25.3 ± 2.0 24.5 ± 1.6
26.0 ± 0.6 25.8 ± 0.7 26.6 ± 1.0 26.1 ± 0.9 27.1 ± 0.8 25.8 ± 1.1 25.7 ± 1.9 24.7 ± 1.9
−1.5 ± 0.5 −2.9 ± 1.6 −3.9 ± 0.7 −5.5 ± 1.5 −3.6 ± 0.7 −1.4 ± 0.8 −0.3 ± 0.3 −0.4 ± 0.1
lectin) − CT(Actin))
ΔΔCT b (ΔCT − ΔCT(P7))
2− ΔΔCT
− 1.1 ± 0.5 − 2.5 ± 1.6 − 3.6 ± 0.7 − 5.2 ± 1.5 − 3.2 ± 0.7 − 1.0 ± 0.8 0.0 ± 0.3 − 0.1 ± 0.1
2.3 ± 0.7 9.0 ± 10.2 12.9 ± 6.8 52.8 ± 55.6 10.1 ± 4.9 2.3 ± 1.4 1.0 ± 0.2 1.1 ± 0.0
Amplified products are shown below table, where the products with post-parasitization are compared in quantity with reference actin products. a The primers used for amplifying viral lectin and actin are described in Materials and methods. b Deviation from ΔCT at 7 days after parasitization (P7).
3.4. Expression levels of CpBV-lectin protein in parasitized P. xylostella To raise polyclonal antibody of CpBV-lectin, its ORF excluding a signal peptide was cloned into pBAD-TOPO bacterial expression system. The recombinant CpBV-lectin was expected as 20,240 Da in molecular weight because it contained 140 residues of the viral lectin and 28 residues of enterokinase recognition, V5 epitope, and polyhistidine sites. As expected, the expressed and purified recombinant viral lectin was detected at just above 20 kDa and confirmed by immunoblotting using V5 antibody (Fig. 5A). This purified protein was used to raise antibody, which was then used to analyze CpBV-lectin protein levels in the parasitized P. xylostella (Fig. 5B). The CpBV-lectin protein was detected as early as 12 h after parasitization and maintained up to 2 days post-parasitization. Unlike mRNA levels measured by quantitative RT-PCR, the CpBV-lectin protein did not appear after 48 h post-parasitization.
old eggs (data not shown). Quantitative analysis of encapsulation response against early (0 h) and late (24 h) parasitized eggs showed a complete inhibition of the recognition efficacy of hemocytes against late parasitized eggs. 4. Discussion Endoparasitoids can manipulate host immune capacity to defend themselves and accommodate their development (Schmidt et al., 2001). Especially in some endoparasitoids with symbiotic polydnavirus like C. plutellae, host immunosuppression can be induced by the help of viral products (Li and Webb, 1994; Summers and Dib-Hajj, 1995; Asgari et al., 1997). Generally, hemocytes mediate and express various immune responses after their nonself recognition (Gillespie et al., 1997). Thus, if the
3.5. Inhibitory effect of CpBV-lectin on hemocyte responses As shown above, plasma from parasitized P. xylostella inhibited hemocyte and bacterial association. To demonstrate this inhibition caused by CpBV-lectin, bacteria were preincubated with purified recombinant CpBV-lectin and incubated with nonparasitized hemocytes (Fig. 6A). As expected, untreated bacteria were easily bound on hemocytes, but bacteria treated with CpBV-lectin did not well attach to the hemocytes. To examine if CpBV-lectin protects C. plutellae eggs from hemocytic encapsulation of P. xylostella, encapsulation analyses were performed (Fig. 6B). A preliminary in vitro encapsulation assay showed that hemocytes of P. xylostella exhibited in vitro encapsulation against agarose beads (P-60, Biorad, Hercules, CA, USA) (data not shown). When parasitoid eggs were incubated with hemocyte suspension, newly laid eggs (‘0 h’) were encapsulated by the hemocytes, compared to inhibition of encapsulation response against 24 h old eggs. Immunostaining using CpBV-lectin antibody showed strong positive reaction on surface of 24 h old eggs, but not on newly laid eggs. For a control, pre-immune serum was used for the primary antibody and did not detect any CpBV-lectin on 24 h
Fig. 5. A recombinant viral lectin of Cotesia plutellae bracovirus (CpBV). (A) The CpBV-lectin was produced using a bacterial expression system and purified by NiNTA column against His-tag. The protein was separated on 15% SDS–PAGE and visualized by immunoblotting against V5 antibody. (B) Immunoblot of CpBVlectin from whole body extracts of nonparasitized (‘NP’) and parasitized Plutella xylostella. Each protein extract (10 μg) was run on 15% SDS–PAGE and transferred to a membrane, which were followed by incubation with anti-mouse IgG (1000× dilution) against CpBV-lectin.
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endoparasitoids interrupt the ability of hemocyte to recognize pathogen, they can effectively induce their host to be in an immunosuppressive state. For the analysis of nonself recognition, we used a binding assay between hemocytes and bacteria. In this assay, most hemocytes in nonparasitized P. xylostella exhibited strong affinity to the bacteria. Most hemocytes fixed on the slide glass during incubation were granular cells and plasmatocytes in cell morphology. These two cells possess phagocytotic activity in
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P. xylostella (Ibrahim and Kim, 2008), suggesting their binding capacities to bacteria. Moreover, granular cells have been known to strongly adhere to culture plate, which is usually used for separating granular cells from other hemocytes by a short incubation in culture plate (Pech and Strand, 1996). Even though our assay was not direct measurement of nonself recognition of the hemocytes, C. plutellae-parasitized P. xylostella hemocytes clearly lost their binding efficacy to bacteria. The inhibitory factors by parasitization were present in both hemocytes and plasma because the reciprocal compensation with nonparasitized hemocytes or plasma did not completely recover the suppressed efficacy of the hemocytes to bind bacteria. Several hemocyte-inhibitory factors have been isolated in other endoparasitoid systems. VHv1.1, derived from Campoletis sonorensis ichnovirus, targets granular cells and reduces host egg encapsulation by 50% (Li and Webb, 1994). In Pseudoplusia includens parasitized by Microplitis demolitor, MdBV directly induces cell death of granular cells (Strand and Pech, 1995). CrV1 is a viral product of C. rubecula and has been regarded as a hemocyte-inhibitory factor by mediating cytoskeleton breakdown (Asgari et al., 1996). In this study, we had interest in the inhibitory factor(s) present in the parasitized plasma. Without parasitization, plasma seemed to mediate pathogen recognition because bacteria did not bind to hemocytes in heat-treated plasma. This suggests a presence of opsonin in the plasma that mediates the association of coupling pathogen and hemocytes. Insects can recognize several pathogens according to their general molecular characteristics such as peptidoglycan against Gram-positive bacteria, lipopolysaccharides against Gram-negative bacteria, and β-1,3-glucan against fungi (Janeway, 1989; Hoffmann et al., 1999). To perform this recognition function, pattern recognition proteins (PRPs) have been proposed and identified in insect plasma (Yu et al., 2002). Among these PRPs, C-type lectins have been proved to function as an opsonin to induce phenoloxidase activation, hemocyte nodule formation, and encapsulation (Koizumi et al., 1999; Yu and Kanost, 1999, 2000, 2004). Interestingly, some polydnaviruses possess C-type lectin in their genome, which is expressed during parasitization (Glatz et al., Fig. 6. Immunosuppressive effects of Cotesia plutellae bracovirus (CpBV) lectin. (A) Inhibitory activity of recombinant CpBV-lectin on the bacterial binding capacity of nonparasitized hemocytes of Plutella xylostella. Escherichia coli were stained with Safranin-O and incubated with CpBV-lectin (10 ng protein plus 105 bacterial cells in 1 mL Grace's medium). The unbound lectin was washed off by three times washings with fresh Grace's medium. Ten μl of the bacterial preparation was overlaid on the hemocytes (≈ 100 cells) and incubated for 30 min at room temperature. After three times washings, the bound hemocytes were counted. (B) Encapsulation assay of C. plutellae eggs (‘E’) by hemocytes (‘H’) of P. xylostella. Newly laid (‘0 h’) and 24 h old eggs were collected from the parasitized larvae and washed with TBS to clear off any plasma. For encapsulation assay, the washed eggs were incubated with hemocyte suspension for 4 h and washed with TBS. Arrow indicates aggregated hemocytes encapsulating the parasitoid egg. For immunostaining assay, the washed eggs were incubated with antibody of CpBV-lectin for 1 h and followed by incubation in secondary antibody conjugated with alkaline phosphatase. For a control, pre-immune serum was used for the primary antibody. Scale bar represents 100 μm. In both graph data, each treatment consisted of three measurements and different letters above standard deviation bars indicate significant difference between means at Type I error = 0.05 (LSD test).
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2003). But, its clear inhibitory implication in immune recognition has not been proved. Here, we hypothesized that an inhibitory factor in the plasma of C. plutellae-parasitized P. xylostella is CpBV-lectin, which interrupts normal opsonin role of host lectin presumably by its structural similarity. This was supported indirectly by heat experiment of nonparasitized plasma and by the fact that the capacity of the nonparasitized hemocytes to bind bacteria decreased as the proportion of parasitized plasma increased in the overlaying plasma volume. With these speculation and indirect evidence, we began to clone viral lectin from the parasitized P. xylostella. With both directional RACEs, a complete cDNA of lectin gene was identified after primary RT-PCR using consensus regions of several viral lectin genes. A Southern hybridization indicated that this CpBV-lectin gene was located in the CpBV genome and RTPCR analysis showed that its expression was only detected in the parasitized P. xylostella. Southern hybridization of CpBV-lectin in two independent viral segments suggests a presence of another related gene or by segment nesting process reported in ichnoviruses (Xu and Stoltz, 1993; Cui and Webb, 1997). Considering that polydnaviruses do not replicate in the target host tissues, segment nesting can be exploited to increase expression level of a functionally significant gene. However, a current genome study did not show any clear evidence in segment nesting in CpBV (Kim et al., 2007). This suggests a presence of another homologous gene, which needs to be verified. Predicted amino acid sequence of CpBV-lectin contains a signal peptide, suggesting a secretory protein and exhibits high homologies with ORF size and sequence with those of Cotesia spp.: 162 residues in C. kariyai, 157 residues in C. ruficrus, and 159 residues in C. rubecula (Teramoto and Tanaka, 2003; Glatz et al., 2003). This supports an existence of a novel polydnaviral lectin family in Cotesia-associated bracoviruses (Glatz et al., 2003). Expression of CpBV-lectin was analyzed by a real-time quantitative RT-PCR and showed that it increased during early parasitization, reached a maximal level at 4 days, and then declined. Protein levels of CpBV-lectin showed much transient expression pattern for only 2 days. This transient expression pattern was also found in other analyzed related viral lectins. For examples, maximal transcription of Cky811 gene was found in 6 h after parasitization and declined (Teramoto and Tanaka, 2003), and CrV3 in the parasitized hemolymph showed a maximal level at 6 h after parasitization and disappeared at 24 h (Glatz et al., 2003). The discrepancy between CpBV-lectin mRNA and protein levels was evident through several re-measurements. We speculate that relatively transient protein level may be due to proteolytic degradation after 48 h by teratocytes of C. plutellae because teratocytes, derived from embryonic serosal membrane, are well known to secrete proteolytic enzymes (Strand et al., 1988) and are in fact detected in the hemolymph of P. xylostella with egg hatch of C. plutellae after 36 h post-parasitization (Basio and Kim, 2005). But we do not know physiological significance in transcriptional activation of CpBV-lectin after 48 h from this study. Bacterial binding assay and CpBV-lectin expression pattern suggest that CpBV-lectin plays a role in inducing host immunosuppression by inhibiting nonself recognition of
P. xylostella hemocytes. First of all, we showed that purified CpBV-lectin significantly interfered with association between hemocytes and bacteria. This indicates that the suppression of hemocyte and bacterial association observed in the parasitized plasma treatment may be contributed by CpBV-lectin. To prove this speculation, we performed in vitro encapsulation assay with different ages of parasitoid eggs from the parasitized P. xylostella. Though we did not observe full encapsulation shown in in vivo conditions (Ibrahim and Kim, 2008), in vitro encapsulation assay used in this study showed hemocyte clumpings around beads or early (0 h) parasitoid eggs. Compared to newly laid eggs, 24 h old eggs were not encapsulated. As expected, the encapsulated eggs contained CpBV-lectin on their surface detectable by immunostaining, but the newly laid eggs did not. Based on these results on CpBV-lectin, we propose a working hypothesis how the viral lectin interrupts immune response of parasitized P. xylostella in terms of pathogen recognition. Generally, other viral lectins are surface proteins that are involved in virus entry to target tissues with specific sugar determinants (Sharon and Lis, 2004). However, polydnaviral lectins are expressed and secreted, which result in soluble proteins in the plasma as shown in CrV3 (Glatz et al., 2003). Signal peptide in putative ORF and detection of CpBVlectin proteins on the parasitoid egg surface suggest that CpBVlectin is produced and secreted into plasma. All lepidopteran CTLs identified have two CRDs, which are contrasting with other animal CTLs that contain only a single CRD (Yu et al., 2002). The single CRD of CpBV-lectin shows homology with C-terminal CRD of Hyphantria cunea, which is another host of C. plutellae (Shin et al., 2000; Bae and Kim, 2004), representing a truncated structure of host counterpart. This suggests that CpBV-lectin and other polydnaviral lectins, have structural and sequence similarities to host lectins, enabling to compete with host lectins for binding sites that are pathogen-specific moieties for recognition or involved in induction of immune reactions. This competitive inhibition may lead to immunosuppression of the parasitized P. xylostella in terms of nonself recognition. Acknowledgments This study was supported by the Korea Research Foundation Grant funded by the Korean Government (R0520020000 48802004) and Biogreen 21 program of Rural Development Administration. NM was financially supported by the 2nd stage of BK21. We also appreciate Youngim Song for supplying research materials and valuable encouragement. References Asgari, S., Hellers, M., Schmidt, O., 1996. Host hemocyte inactivation by an insect parasitoid: transient expression of a polydnavirus gene. J. Gen. Virol. 77, 2653–2662. Asgari, S., Schmidt, O., Theopold, U., 1997. A polydnavirus-encoded protein of an endoparasitoid wasp is an immune suppressor. J. Gen. Virol. 78, 3061–3070. Bae, S., Kim, Y., 2004. Host physiological changes due to parasitism of a braconid wasp, Cotesia plutellae, on diamondback moth, Plutella xylostella. Comp. Biochem. Physiol. A 138, 39–44. Basio, N.A., Kim, Y., 2005. A short review of teratocytes and their characters in Cotesia plutellae (Braconidae: Hymenoptera). J. Asia-Pac. Entomol. 8, 211–217.
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