Downregulation of a Chitin Deacetylase-Like Protein in Response toBaculovirus Infection and Its Application for
Improving Baculovirus Infectivity�
Agata K. Jakubowska,1,2 Silvia Caccia,1 Karl H. Gordon,3 Juan Ferre,1 and Salvador Herrero1*Department of Genetics, University of Valencia, Dr. Moliner 50, 46100 Burjassot, Spain1; Department of
Biological Control and Quarantine, Institute of Plant Protection, Miczurina 20, Poznan 60318,Poland2; and CSIRO Entomology, P.O. Box 1700, Canberra ACT 2601, Australia3
Received 2 September 2009/Accepted 9 December 2009
Several expressed sequence tags (ESTs) with homology to chitin deacetylase-like protein (CDA) wereselected from a group of Helicoverpa armigera genes whose expression changed after infection with H. armigerasingle nucleopolyhedrovirus (HearNPV). Some of these ESTs coded for a midgut protein containing a chitindeacetylase domain (CDAD). The expressed protein, HaCDA5a, did not show chitin deacetylase activity, but itshowed a strong affinity for binding to chitin. Sequence analysis showed the lack of any chitin binding domain,described for all currently known peritrophic membrane (PM) proteins. HaCDA5a has previously beendetected in the H. armigera PM. Such localization, together with its downregulation after pathogen infection,led us to hypothesize that this protein might be responsible for the homeostasis of the PM structure and that,by reduction of its expression, the insect may reduce PM permeability, decreasing the entrance of baculovirus.To test this hypothesis, we constructed a recombinant nucleopolyhedrovirus to express HaCDA5a in insect cellsand tested its influence on PM permeability as well as the influence of HaCDA5a expression on the perfor-mance of the baculovirus. The experiments showed that HaCDA5a increased PM permeability, in a concen-tration-dependent manner. Bioassays on Spodoptera frugiperda and Spodoptera exigua larvae revealed that NPVexpressing HaCDA5a was more infective than its parental virus. However, no difference in virulence wasobserved when the viruses were injected intrahemocoelically. These findings support the downregulation of amidgut-specific CDA-like protein as a possible mechanism used by H. armigera to reduce susceptibility tobaculovirus by decreasing PM permeability.
Baculoviruses are a naturally occurring group of large dou-ble-stranded DNA viruses that are specific to arthropods andhave potential for widespread use for insect pest management.It has already been proven that they can effectively replacechemical insecticides in the field, for example, in the case ofHelicoverpa armigera single nucleopolyhedrovirus (HearNPV)sprayed on cotton fields in Australia (9) and China (38) tocontrol one of the most widely spread polyphagous pests (10).Baculoviruses occur naturally, are nonpathogenic to humans orother vertebrates, and are relatively host specific, and no im-pact on nontarget organisms has been reported to date. Thesecharacteristics make them environmentally safe insecticides.Despite the environmental advantages of baculoviruses, theiruse as biocontrol agents is limited, mainly due to their slowaction compared to that of other pesticides. Naturally occur-ring baculoviruses, although highly infectious, have adapted totheir hosts during their evolution, therefore killing the hostsrelatively slowly and achieving maximum viral propagation. Ittakes up to 10 days for the virus to stop insect feeding or to killthe infected larvae (34). For this reason, reduction in the timeof killing has been the main focus of research to improvebaculovirus performance, and several strategies have beenused, such as coapplying synergistic chemicals or using genetic
engineering to introducing foreign genes coding for toxins,hormones, or enzymes into their genomes (18, 19). The strat-egy of acquiring foreign genes has been used by viruses them-selves. Most large cytoplasmic and nuclear DNA viruses havebeen shown to capture, by horizontal gene transfer, host genesrelated to ubiquitin signaling, defense against apoptosis, andimmune responses (20). The average baculovirus genome con-tains more than 100 open reading frames (ORFs) encodingpredicted proteins of more than 50 amino acids. Phylogeneticanalyses suggest that during evolution, several baculovirusgenes, such as the inhibitor of apoptosis (iap) and ecdysteroidUDP-glucosyltransferase (egt) genes, were acquired from theirinsect hosts by horizontal gene transfer (17). Access to therecently available genome of Bombyx mori enabled a survey ofB. mori NPV (BmNPV) genes that might have been acquiredfrom the host. The survey identified 35 insect homologs po-tentially encoded by 37 baculoviruses (22). Knockout studies ofinsect homologs in baculoviruses have shown that some hosthomologs are essential for complete in vivo pathogenicity (22).Their functions are maintained or modified in order to controlhost physiology and cell signaling pathways for better virusmultiplication and vertical transmission in nature.
To identify host genes whose expression could be advanta-geous for the baculovirus to increase its insecticidal character-istics, we checked the change in expression of host genes inresponse to baculovirus infection. DNA microarray experi-ments revealed a set of H. armigera midgut genes that were up-and downregulated due to infection with HearNPV (unpub-
* Corresponding author. Mailing address: Department of Genetics,University of Valencia, Dr. Moliner 50, 46-100 Burjassot, Spain.Phone: 34-96-1646003. Fax: 34-96-3983029. E-mail: [email protected].
lished data). Among them, several expressed sequence tags(ESTs) coding for a chitin deacetylase-like protein (CDA)were found to be downregulated after virus infection, suggest-ing its possible role in the response to the infection. CDAshave been isolated from various fungi and bacteria, and theirbiological functions include softening of the insect cuticle toallow easier mycelial penetration (in the case of fungi) andevasion of lysozyme action (in the case of bacteria). Theyconvert chitin, a �-1,4-linked N-acetylglucosamine polymer,into its deacetylated form, chitosan, a natural glucosaminepolymer (42). Recently, CDAs were also identified in insectsand appear to constitute one of two major classes of proteinsrecovered from the peritrophic membrane (PM) (4, 30). PMlining the insect midgut represents a major lepidopteran phys-ical barrier against baculovirus infection (14, 35, 46). It consistsof chitin and glycoproteins, and its physical role is to protectmidgut epithelial cells from food particles, digestive enzymes,and pathogens. It also has a biochemical function, such as theinactivation of ingested toxins and enzyme recycling (3). Dis-ruption of the link between chitin and the protein structure ofthe PM affects its functions in digestion and also leads to thecollapse of the midgut defense against pathogens.
In this work, H. armigera EST sequence analyses allowed theidentification and full-length sequencing of three different CDA-like proteins, revealing that only one of them (HaCDA5a) wasdownregulated during the initial stages of baculovirus infectionin larvae. HaCDA5a has been recombinantly expressed in in-sect cells, and its influence on PM permeability was checked.Given the natural ability of baculovirus to acquire insect hostgenes in order to improve survival and prevalence, we alsoanalyzed the effects of CDA expression on the performance ofbaculovirus in insect bioassays. The results revealed that ex-pression of CDA-like proteins by baculovirus may increase itsinfectivity and speed of kill and thus be applied for better pestcontrol.
MATERIALS AND METHODS
Insects and viruses. H. armigera, Spodoptera frugiperda, and Spodoptera exigualarvae were reared on an artificial diet at 25°C, 70% humidity, and a 16-h/8-hphotoperiod, as described by Shorey and Hale (36), Smits et al. (37), and Zhanget al. (48), respectively. The insect cell line used in this study was Sf21 from S.frugiperda (43). Sf21 cells were cultured at 27°C in Grace’s medium (Gibco-Invitrogen Corp., NY) supplemented with 10% fetal bovine serum (FBS).
Viruses used in this study included wild-type HearNPV isolate C1 (47),SeMNPV isolate US1 (11), and a bacmid-derived virus, AcBac�CC, based onAutographa californica MNPV (21).
DNA microarray analysis of baculovirus-exposed insects. Based on initialDNA microarray analysis, midgut genes whose expression changed significantlyupon virus infection were identified. PCR products derived from a cDNA libraryfrom the H. armigera larva midgut, containing around 4,000 ESTs, were used forthe microarray printing (1). For each biological replication, around 20 fourth-instar larvae (molted within the last 10 h) were starved overnight and then fed 5�l of a 5% sucrose-10% methylene blue (MB) solution containing 2.5 � 106
occlusion bodies (OBs) from HearNPV. After 30 min, larvae that consumed thewhole drop were selected and used for the subsequent steps. Larvae were trans-ferred to a standard insect diet and maintained individually until tissue collec-tion. Midguts of healthy and virus-treated larvae were collected (at 8 h postin-fection [p.i.]) and pooled. Total RNA was extracted using a Perfect RNApurification kit (Eppendorf, North Ryde, Australia) following the manufacturer’sspecifications. For each sample, 50 �g of total RNA was reverse transcribed andthe resulting cDNA sample was split in half for Cy3 and Cy5 labeling. cDNAlabeling, microarray hybridization, and data capturing and analysis were per-formed following standard protocols. Three biological replications were per-formed for each group of control and virus-treated larvae.
Semiquantitative and real-time quantitative RT-PCR. The presence andabundance of mRNAs from different CDAs in various larval tissues were firstestimated by semiquantitative reverse transcription-PCR (RT-PCR). For thispurpose, total RNA was isolated from the anterior, middle, and posterior part ofthe midgut, from the hindgut, from the fat body, and from Malpighian tubules offourth-instar H. armigera larvae. RNA was isolated using Tripure (Roche Diag-nostics, Mannheim, Germany) and reverse transcribed using oligo(dT) primerswith SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA). For semi-quantitative PCR, 5 �l of a 1:50 dilution of cDNA, synthesized from 1 �g of totalRNA, was used for amplification with specific primer sets. The amplificationreactions were carried out in an Eppendorf thermocycler, using 25 cycles of 94°C(30 s), 50°C (30 s), and 72°C (1 min) and a final extension step of 5 min at 72°C.
Real-time quantitative RT-PCR (qPCR) was used to determine the changes inexpression of the Hacda5a gene in virus-infected larvae. Third-instar larvae wereinfected orally with 1 � 106 HearNPV OBs, by diet contamination. Larvae thatconsumed the entire disk were transferred to fresh diet and maintained individ-ually until tissue collection. Midguts from infected and control larvae werecollected at 6, 24, and 48 h p.i. Total RNA was isolated and cDNA synthesizedas described above. qPCR was carried out in an ABI Prism 7000 thermocyclerfrom Applied Biosystems. All reactions were performed using Power SYBRgreen PCR master mix (Applied Biosystems, Foster City, CA) in a total reactionvolume of 25 �l. Five-microliter cDNA templates were added to each reactionmix. Forward and reverse primers designed using Primer Express software (Ap-plied Biosystems, Foster City, CA) were added to a final concentration of 0.3 nM.The reactions were performed in triplicate. All primers used are available uponrequest. The 2��CT method (25) was used to calculate relative changes in geneexpression determined from qPCR experiments. The data are presented as foldchanges in target gene expression in infected tissue normalized to the internalcontrol gene (ATP synthase) and relative to the noninfected tissue control.
To confirm the expression pattern of cda genes in another insect-virus com-bination, Secda5a gene expression was also determined for S. exigua larvaeinfected with SeMNPV. An Secda5 partial gene sequence (EST) was obtainedfrom an S. exigua midgut cDNA library (15). Third-instar larvae were infectedorally with 1 � 106 SeMNPV OBs, by diet contamination, and Secda5 expressionwas calculated as described above.
Sequencing of H. armigera cda genes. An EST sequence from an H. armigeragene for CDA (Hacda5a) was used to design specific primer sets to amplifyoverlapping fragments of the 5� and 3� ends of this gene. 5� and 3� rapidamplification of cDNA ends (RACE)-ready cDNAs were synthesized using aSMART-RACE kit (Clontech, Saint-Germain-en-Laye, France), and 5� and 3�fragments were amplified using two sets of specific primers. The amplified frag-ments were purified and cloned into the pGEM-T Easy vector (Promega, Mad-ison, WI) for subsequent sequencing. Several clones were sequenced for eachcDNA end. Sequences were assembled by using the Seqman program from theDNAstar software package (DNASTAR, Madison, WI). Full sequences for twoother cda genes, Hacda5b and Hacda1, were completed based on the informationfrom the H. armigera genomic sequencing project (12). Alignment of all threeHacda genes was performed using ClustalX (39) and visualized in GeneDoc (28).Predicted amino acid sequences were aligned with other lepidopteran CDAsequences available from the NCBI and ButterflyBase databases (29; www.butterflybase.com). Other insect CDA sequences were omitted in our analysisfor the sake of clarity. Phylogenetic analyses were performed using the neighbor-joining method with 1,000 bootstraps, using the ClustalX (39) and MEGA 3.1(24) programs. Distances were corrected for multiple substitutions according tothe method of Kimura (23).
Recombinant baculovirus production. The full-length Hacda5a ORF was am-plified by PCR from H. armigera midgut cDNA. Primers were designed toinclude the NsiI restriction site to enable further cloning, as well as a polyhisti-dine (6�His) tag upstream of the stop codon. The obtained amplicon was clonedinto the pGEM-T Easy vector for sequence verification. The Hacda5a ORF wasthen excised from pGEM-T Easy with NsiI and cloned into pFBD-ph (16)downstream of the p10 promoter to obtain pFBD-ph-Hacda5a. pFBD-ph con-tains the AcMNPV polyhedrin gene under the control of the polyhedrin pro-moter. In order to generate recombinant viruses, Escherichia coli strainDH10Bac, containing the �cc bacmid (21) and the pMON7124 helper plasmid(26), was transformed with the pFBD-ph and pFBD-ph-Hacda5a plasmids ac-cording to the procedure described for the Bac-to-Bac system (Invitrogen, Carls-bad, CA). Recombinant bacmids were selected based on white-blue screening ofDH10Bac colonies, and the positive clones were confirmed by PCR. BacmidDNA was isolated from bacterial cells according to standard procedure and usedto transfect Sf21 cells, using Insect GeneJuice transfection reagent (Novagen,Darmstadt, Germany). Recombinant �ccph and �ccph-Hacda5a bacmid-derivedviruses were multiplied in an additional round of infection to produce high-titer
stocks for further experiments. Viruses were titrated by end-point dilution assay(EPDA) in Sf21 cells according to the method of Vlak (44), and the titers wereexpressed as the 50% tissue culture infective dose (TCID50).
HaCDA5a expression in Sf21 cells. Sf21 cells were infected with �ccph and�ccph-Hacda5a at a multiplicity of infection (MOI) of 5, and cells and mediumwere collected at 72 h p.i. to analyze protein expression. Cells were lysed inphosphate-buffered saline (PBS)–0.2% Triton X-100. Cell extracts as well as cellculture medium proteins were separated by 12% SDS-PAGE, and the presenceof the recombinant protein was detected using antibodies against the 6�His tag(BD Pharmingen, San Diego, CA). For protein production, Sf21 cells wereseeded in T75 flasks (Nunc, Rochester, NY) and infected according to previouslymentioned conditions.
Chitin binding assay. The chitin binding activity of HaCDA5a was checkedaccording to a previously described method (13). Chitin was regenerated asfollows. Two hundred fifty milligrams of chitin from crab shells (Sigma) wasdissolved in 5 ml of 85% phosphoric acid and incubated for 16 h at 4°C, withstirring. After incubation, the suspension was centrifuged for 5 min at 10,000rpm, and the gelatinous chitin pellet was washed several times with water, untila pH of about 7.0 was obtained. Finally, the chitin pellet was broken in a mortar.For the binding assay, 4 ml of cell culture medium containing recombinantHaCDA5a was incubated with 100 mg (wet weight) of regenerated chitin in thepresence of a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Ger-many) for 4 h at 4°C, with continuous stirring. The mixture was centrifuged, andthe chitin pellet was washed three times with PBS, followed by centrifugation.After the last centrifugation step, chitin pellets were suspended in 0.2 ml of PBS,0.5 M NaCl, 20 mM acetic acid, 6 M urea, and 2% SDS with the addition of 5%�-mercaptoethanol or in 1% calcofluor white (fluorescent brightener 28; Sigma)at room temperature in the presence of the protease inhibitor cocktail. After 15min of incubation, suspensions were centrifuged for 10 min at 10,000 rpm, andthe supernatants were checked for the presence of HaCDA5a released fromchitin by Western analysis.
PM permeability assays. Changes in permeability of the PM to methylene blue(MB) due to HaCDA5a exposure were measured with an Ussing chamber(CHM8; World Precision Instruments, Stevenage, United Kingdom). Prelimi-nary experiments showed a significant loss of HaCDA5a protein when it waspurified through HiTrap chelating HP columns. For this reason, permeabilityassays were performed with cell culture medium, with or without HaCDA5a,partially purified by removal of budded viruses (BVs) by 1 h of centrifugation at20,000 � g at 4°C, and then concentrated by incubation on dry sucrose in adialysis tube.
S. frugiperda PMs were isolated from actively feeding last-instar larvae. Briefly,each larva was anesthetized on ice for 2 min, and the midgut was then extractedand opened longitudinally to expose the PM. The PM was isolated, carefully laidon a large mesh cotton film, cut lengthwise, and gently rinsed with PBS to removethe food content. A properly cut portion of the cotton film with the attached PMwas mounted on the flow chamber, with the result of a 12.6-mm2 portion of PMexposed to the two compartments. The compartment with the ectoperitrophicside (the ectoperitrophic compartment) was filled with 450 �l of PBS, and theone with the endoperitrophic side (the endoperitrophic compartment) was filledwith 450 �l of a 0.2-mg/ml MB solution in PBS. Prior to the start of the assay, theintegrity of isolated PMs was checked by measuring the flux of MB throughthe PM after the first 30 min of incubation at room temperature. Next, 100 �l ofthe buffer solution in both compartments was replaced with an equivalent vol-ume of cell culture medium containing HaCDA5a from Sf21 cells infected with�ccph-Hacda5a. Culture medium from Sf21 cells infected with �ccph was usedas a control. After 1 h of incubation, the solutions in both compartments wererecovered, and the concentration of MB was calculated based on the absorbancemeasured at 661 nm. The MB flux was expressed as �g of dye (calculated bymeans of a calibration curve after subtracting the flux at 30 min from the finalflux) that passed through the 12.6-mm2 portion of mounted PM in 1 h. Threeindependent replicates were performed for each data collection.
Biological activity assays. The effect of heterologous expression of HaCDA5aon baculovirus performance was determined both ex vivo (with Sf21 cells) and invivo (with S. frugiperda and S. exigua larvae).
Sf21 cells were infected for 1 h with �ccph and �ccph-Hacda5a at an MOI of1. After infection, cells were washed and incubated in fresh medium. At 0, 24, 48,72, and 96 h p.i., a 10-�l aliquot of medium was harvested and the quantity ofBVs in each sample was determined by an end-point dilution assay on Sf21 cells.Experiments were duplicated for each time point and each virus.
For oral in vivo assays, fourth-instar S. frugiperda and S. exigua larvae were feddiet disks contaminated with 5 � 104 OBs of a virus. �ccph and �ccph-Hacda5aOBs were purified from cell culture according to standard procedure. Briefly, thecells were incubated in 0.5% SDS for 30 min at 25°C and then washed by a series
of washes in water and 0.5 M NaCl. Finally, the OBs were pelleted through acushion of 30% (wt/wt) sucrose for 30 min at 9,000 rpm. To discard any effect onthe number of nucleocapsids in the OBs due to the expression of Hacda5aprotein, the numbers of DNA copies in OBs from both viruses (as an estimationof nucleocapsid numbers) were compared by means of qPCR, using specificprimers for the DNA polymerase gene.
Larvae that consumed the entire disk were transferred to fresh diet andmaintained individually until death or pupation at 27°C. Larval mortality wasrecorded every 12 h. For each virus and insect species, 20 to 48 larvae weretested, and the bioassays were conducted in duplicate. The mean time to deathwas calculated for each virus and compared using two-way analysis of variance(ANOVA) (GraphPad Prism) (27).
For in vivo intrahemocoelic infection assays, fourth-instar S. frugiperda and S.exigua larvae were injected with 10 �l of the corresponding suspension of BVs ata concentration of 107 TCID50/ml in Grace’s cell culture medium containingphenol red to monitor the injections. At least 20 larvae were used for each virus,and the assay was conducted in duplicate for each insect species. Larval mortalitywas recorded every 12 h. The mean time to death was calculated for eachvirus-insect combination and compared using two-way ANOVA (GraphPadPrism) (27). The bioassays were conducted at 27°C.
A second experiment was performed with third-, fourth-, and fifth-instar S.exigua larvae. Larvae (32 to 48 for each instar) were fed diet disks contaminatedwith 1 � 103 OBs (L3 larvae), 5 � 103 OBs (L4 larvae), and 1 � 104 OBs (L5larvae). Larvae that consumed the entire disk were transferred to fresh diet andmaintained individually until death or pupation. Bioassays were conducted induplicate. Mortality was recorded every 12 h, with the exception of L5 larvae,where the mortality was recorded every 24 h. The percentage of mortality wascalculated for each instar, and the results were analyzed as before. The bioassayswere conducted at 25°C.
Nucleotide sequence accession numbers. Sequences were deposited inGenBank under the following accession numbers: GQ411189 (Hacda1),GQ411190 (Hacda5a), and GQ411191 (Hacda5b).
RESULTS
Identification and expression analysis of chitin deacetylase-like proteins from H. armigera midgut. Among the midgutgenes that were differentially expressed after baculovirusingestion, 8 ESTs with homology to cda genes were de-tected. These ESTs were downregulated, with an averagechange in the expression ratio of around threefold. In con-trast, another 3 ESTs with homology to cda genes did notshow changes in gene expression after baculovirus ingestion.Sequence analysis revealed that the eight downregulatedESTs belonged to the same gene (Hacda5a), while amongthe other three ESTs, two belonged to a second cda gene(Hacda5b) and the other belonged to a third one (Hacda1).These results prompted us to study the role of CDA proteinsin the response to baculovirus.
In order to confirm the downregulation of Hacda5a ex-pression, qPCR was performed on total midgut cDNA fromH. armigera third-instar larvae infected with HearNPV, us-ing primers specific for Hacda5a. Experiments showed a4.8-fold decrease in expression of Hacda5a at 6 h p.i. and a2.6-fold decrease at 24 h p.i. At 48 h p.i., Hacda5a expres-sion had returned to the regular level (Fig. 1A). Downregu-lation of the cda gene in the first hours after baculovirusinfection was also observed in S. exigua larvae infected withSeMNPV. Experiments showed a 3.9-fold decrease in ex-pression of Secda5a at 6 h p.i. At later times p.i. (24 and 48 hp.i.), Secda5a expression in the midguts of infected larvaedid not significantly differ from the expression in the mid-guts of control larvae (Fig. 1A).
Because Hacda5a expression was first detected in the ESTmicroarray of midgut tissues and confirmed by qPCR to occurin the larval midgut as well, the tissue specificity of expression
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of Hacda5a and the two additional genes coding for chitindeacetylase-like proteins (HaCDA5b and HaCDA1) was de-termined. Among the tissues tested, Hacda5a was expressedmainly in the gut (in the anterior, middle, and posterior partsof the midgut and in the hindgut), with comparatively verylittle expression in the Malpighian tubules and fat body.Hacda5b and Hacda1 were expressed mainly in the fat bodyand Malpighian tubules, although the former also showed lim-ited expression in the midgut (Fig. 1B).
Sequence analysis of Hacda genes. A partial sequence ofHacda5a (EST) was used to design two sets of primers foramplification, cloning, and sequencing of 5� and 3� cDNA endsof this gene. The full sequence has been obtained and depos-ited in the NCBI GenBank under accession numberGQ411190. The full assembled sequence of Hacda5a cDNAwas 1,277 bp long and contained an ORF of 1,173 bp, followedby an AT-rich untranslated region (UTR) and three putativepolyadenylation signals (AATAAA), located 5, 17, and 22 bpupstream of the poly(A) tail. The protein encoded by this ORFcontains 391 amino acid residues, with a 17-amino-acid puta-tive signal peptide. The predicted molecular mass of the se-creted protein without the signal peptide is around 42 kDa. Asearch of the Conserved Domain Database (NCBI) identifieda putative polysaccharide deacetylase domain in amino acidresidues 63 to 193. Fifteen cysteine residues are present alongthe HaCDA5a sequence; however, their distribution does notresemble that for any peritrophin motif described for knownPM proteins.
In a comparison with the other two chitin deacetylase-likeproteins identified in H. armigera, HaCDA5a shows a highamino acid sequence similarity (77%) to HaCDA5b and asimilarity of only 35% to HaCDA1. All three HaCDAs con-tain a putative chitin deacetylase domain (CDAD), whileonly one of them, HaCDA1, codes additionally for a puta-tive chitin binding domain (ChBD) and a lipoprotein recep-tor class A domain (LDLa). HaCDA1 is significantly longerthan HaCDA5a and HaCDA5b and probably belongs to adistant group of deacetylases (Fig. 2).
A phylogenetic analysis was performed with all availablechitin deacetylase-like protein sequences from the Lepidop-tera, most of them from Bombyx mori. Lepidopteran CDAs
form three main branches that correlate with their domaincontents (Fig. 3). Two of the branches are further subdivided,according to Campbell et al. (4), rendering five CDA groupsaltogether (groups I to V). HaCDA5a and HaCDA5b form aclade, together with Trichoplusia ni CDA, S. frugiperda CDA,Mamestra configurata CDA, Epiphyas postvittana CDA, andthree B. mori CDAs, and belong to CDA group V, whosemembers contain the CDAD but not ChBD or LDLa (Fig. 3).HaCDA1 is clearly distant from the other two HaCDAs and,together with Agrotis ipsilon CDA and one B. mori CDA(Bmb030914), falls into CDA group I, whose members containall three domains (CDAD, ChBD, and LDLa).
According to Dixit et al. (7) and Campbell et al. (4), insectCDAs are divided into five major classes, classes I to V. ClassesI and II contain three recognized domains, CDAD, ChBD, andLdLa. Class III and IV CDAs contain CDAD and ChBD, andclass V CDAs contain only CDAD. To prevent nomenclatureconfusion, we propose to follow these divisions and to nameCDAs from a given insect species with the Arabic numeralcorresponding to the CDA class to which they belong followedby a lowercase letter if more than one CDA from a particularclass is identified. For this reason, we have named the CDAsfound in H. armigera HaCDA1, HaCDA5a, and HaCDA5b(see Fig. 3 for the phylogenetic class grouping).
Heterologous expression of HaCDA5a in insect cell culture.In order to study the possible role of HaCDA5a in baculovirusinfection, a recombinant �cc bacmid expressing 6�His-taggedHaCDA5a was constructed. �cc is a deletion mutant of Ac-bacmid in which the chitinase and v-cathepsin ORFs have beenremoved, and it was chosen due to the lack of possible inter-ference of chitinase activity.
HaCDA5a was successfully expressed in Sf21 cells, and itsexpression was monitored by Western analysis using anti-6�His antibody. HaCDA5a expression was detected already at24 h p.i. (not shown), but the maximum yield was observed at72 h p.i. HaCDA5a was present mainly in the cell extracts, butpart of it was also secreted into the cell culture medium (Fig.4A). The mobility of HaCDA5a in 12% SDS-PAGE gels wasestimated to be around that of a 42-kDa protein, as predictedfrom the amino acid sequence without the signal peptide.
FIG. 1. Expression of different CDAs in H. armigera larvae. (A) Changes in expression of Hacda5a and Secda5a after baculovirus infection inthe midguts of third-instar H. armigera (L3) and S. exigua (L3) larvae, respectively. Total RNAs were isolated from the midguts of infected larvaeat different time postinfection and then reverse transcribed, and qPCR was performed using specific primers. The results are the means � standarddeviations for three independent infection experiments. Asterisks demark changes in expression that are significantly different from expression inthe control larvae (by the t test). Dotted lines demark onefold changes in expression (no change in expression compared to control, noninfectedlarvae). (B) Expression of CDAs in different H. armigera (L4) tissues by semi-qPCR, using specific primers for Hacda5a, Hacda5b, and Hacda1.A, anterior midgut; M, middle part of the midgut; P, posterior midgut; H, hindgut; FB, fat body; MT, Malpighian tubules.
Chitin binding activity assay. Due to the facts that theHaCDA5a homolog in Trichoplusia ni was found in the PM(13) and that the PM consists of proteins and chitin, the abilityof HaCDA5a to bind chitin was checked. RecombinantHaCDA5a expressed in Sf21 cells and secreted into the cellculture medium exhibited a strong binding affinity for regen-erated chitin. After incubation with an excess of regeneratedchitin, no HaCDA5a protein remained in the cell culture me-dium (Fig. 4B, PBS lane). Most of the protein bound to chitincould be released by treatment with either 6 M urea, 1%calcofluor white, or 2% SDS–5% �-mercaptoethanol (Fig. 4B).Chitin-bound HaCDA5a could not be released by treatmentwith 0.5 M NaCl or 20 mM acetic acid.
Effect of HaCDA5a on PM permeability. According to ourhypothesis that HaCDA5a protein might be responsible forPM structure homeostasis, we decided to determine ifHaCDA5a had any influence on PM permeability. To checkthe influence of HaCDA5a on PM permeability, we in-tended to isolate PMs from H. armigera larvae. But H. ar-migera PMs appeared unsuitable for these experiments due totheir extremely fragile structure. Instead, we used S. frugiperdalarvae, whose isolated PMs have properties that enable them tobe mounted more easily in the experimental chamber.
Incubation of S. frugiperda PM in the presence of HaCDA5aclearly permeabilized the PM, as demonstrated by the increaseof the flux of the MB dye, in a concentration-dependent man-ner (Fig. 5).
Effect of HaCDA5a overexpression on infectivity and viru-lence of baculovirus. Overexpression of HaCDA5a by the re-combinant virus did not influence virus growth in cell culturecompared to that of virus lacking HaCDA5a (Fig. 6).
Due to the effect of HaCDA5a on PM permeability, wetested whether expression of this protein by baculovirus wouldchange its performance in vivo. We compared the virulence of�ccph and �ccph-Hacda5a in two insect species, S. frugiperdaand S. exigua. S. frugiperda was selected for bioassays becausethis species was also used to measure PM permeability afterHaCDA5a treatment. S. frugiperda, however, is known for itslow level of susceptibility to AcMNPV, and this virus is thebasis of the bacmid-derived virus employed here (�cc). For thisreason, we also included S. exigua larvae, which are moresusceptible to AcMNPV, in the bioassays.
Mean time to death was measured for both insect speciesand both viruses. Only larvae that died were included in thecalculations. Calculation of the mean times to death was pos-sible due to equal mortalities that both viruses caused in eachspecies (26 and 28% for �ccph and �ccph-Hacda5a, respec-tively, in S. frugiperda larvae and 95 and 100% for �ccph and�ccph-Hacda5a, respectively, in S. exigua larvae). For bothspecies, we found significant differences in the mean times todeath between both viruses, but only when the viruses wereadministered orally (Fig. 7). S. frugiperda larvae infected per oswith �ccph-Hacda5a died 32 h earlier (18% faster), on aver-age, than larvae infected with the virus lacking HaCDA5a. In
FIG. 2. Alignment of deduced amino acid sequences from H. armigera CDAs. The sequences have been deposited in GenBank under accessionnumbers GQ411189 (HaCDA1), GQ411190 (HaCDA5a), and GQ411191 (HaCDA5b).
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the case of S. exigua, the difference was smaller. The �ccph-Hacda5a virus killed infected larvae 11 h earlier (9% faster),on average, than larvae infected with the virus not expressingHaCDA5a. In contrast, intrahemocoelic infections using BVsshowed a similar speed of kill for both viruses in the two insectspecies.
The doses used in the first experiment gave very lowmortality for S. frugiperda larvae when the virus was admin-istered orally (28 and 26% for �ccph and �ccph-Hacda5a,respectively) and almost 100% mortality in the case of S.exigua L4 larvae. Therefore, the infectivities of �ccph and�ccph-Hacda5a were compared in third-, fourth-, and fifth-instar S. exigua larvae to assess the influence of larval instaron virus infectivity. In all tested instars, we found significantdifferences in mortality caused by both viruses (Fig. 8). Themortalities caused by �ccph and �ccph-Hacda5a were 50 and63.4% for third-instar larvae, 33.8 and 54.1% for fourth-instarlarvae, and 21.9 and 35.4% for fifth-instar larvae, respectively.
DISCUSSION
In this study, a protein with homology to chitin deacetylaseswas found to be downregulated due to baculovirus infection inthe midguts of H. armigera and S. exigua larvae. Quick down-regulation of CDA as a consequence of baculovirus infectionsuggests an active role of this protein in the insect response tobaculovirus. Our experiments on HaCDA5a influence on PMpermeability have demonstrated, for the first time, an activityof insect CDA-like proteins on the PM. The decrease ofHaCDA5a expression due to baculovirus infection would leadto less HaCDA5a protein present in the PM and thus increasethe PM stiffness, and this may constitute an early mechanism ofprotection from oral infection by baculovirus.
In agreement with this hypothesis, several studies have re-vealed the effect of PM-disrupting agents or enzymes in en-hancing baculovirus infectivity (6, 33, 45, 46). According to ourexpression results at different h p.i., the downregulation occurs
FIG. 3. Unrooted phylogenetic tree obtained from alignment of lepidopteran CDAs. CDA groups I to V and their domain contents areindicated. CDA, chitin deacetylase domain; ChBD, chitin binding domain; LDLa, low-density lipoprotein receptor class A domain. Sequences usedin the alignment were as follows (with GenBank accession numbers in parentheses): H. armigera CDAs HaCDA1, HaCDA5a, and HaCDA5b (thispublication), Agrotis ipsilon CDA (FJ899541), Epiphyas postvittana CDA (EV809282), Mamestra configurata CDA (EU660852), Spodopterafrugiperda CDA (ButterflyBase cluster SFP00364), and Trichoplusia ni CDA (AY966402). Bombyx mori sequences were obtained from SilkDB(http://silkworm.genomics.org.cn), and their accession numbers are listed in the figure. Sequences were aligned using ClustalX, and a neighbor-joining tree with 1,000 replicates was generated. Numbers on the branches indicate neighbor-joining bootstrap percentages. The scale bar indicatesan evolutionary distance of 0.1 amino acid substitution per position in the sequence.
during the first 6 h of infection, while the viral occluded de-rived viruses (ODVs) are still present in the gut lumen, andexpression returns to normal levels once the gut lumen hasbeen cleared of viral particles. Whether downregulation occursas a response to virus penetration into the midgut cells or as aresult of the detection of certain viral elements that couldactivate the response to baculovirus by the midgut epitheliumis unknown.
The character of HaCDA5a activity on the PM remainsunknown. Whether this activity is due to the physical inter-action of HaCDA5a disrupting the PM, to its hypotheticaldeacetylation activity, or to any other enzymatic activity issomething yet to be determined. We were unable to dem-onstrate deacetylase activity of HaCDA5a by measuringchitin deacetylation by native gel electrophoresis (41; datanot shown), confirming previous results with TnPM-P42CDA from T. ni (13). In contrast, Toprak et al. (40), usinga similar methodology, have shown deacetylase activity of a
26-kDa form of the HaCDA5a ortholog from M. configurata(MacoCDA) recombinantly expressed in E. coli.
The proposed model of CDA-like protein expression regula-tion due to baculovirus infection as a protective mechanism wassupported by the increase in virulence and pathogenicity ofHaCDA5a-expressing virus in comparison to the parental virus.The virus expressing HaCDA5a killed both tested species, S.frugiperda and S. exigua, faster than the parent virus, but onlywhen administered orally. The same recombinant virus injectedintrahemocoelically killed the larvae with the same speed as thevirus lacking HaCDA5a, also revealing that this protein does nothave any apparent detrimental effect on virus multiplication innon-midgut cells. Additional experiments with S. exigua larvaerevealed that the virus expressing Hacda5a causes significantlyhigher mortality than the parental virus. These data suggest thatexpression of CDA changes the performance of the virus in vivo,and most likely it is active at the entrance of the virus to midgutepithelial cells while bypassing the PM barrier. We have foundCDA in the polyhedra of recombinant virus by Western analysis(data not shown). Possibly, CDA-associated polyhedra releasethis protein while being dissolved in the alkaline insect midgutlumen, and CDA may act on the PM, changing its permeabilityand thus enabling easier virus entrance into epithelial cells. An-other explanation for the better performance of recombinantvirus expressing HaCDA5a could be the secretion of active pro-tein by epithelial cells back to the ectoperitrophic compartment.Evidence against this hypothesis is the fact that the protein is mostlikely expressed after all the remaining ODVs disappear from themidgut lumen. It is also possible that HaCDA5a protein synthe-sized after infection facilitates virus spread from the epithelialcells to the trachea. Insect tracheas are lined with a protein-chitinlayer (5), and tracheal cells serve as a conduit for the systemicspread of baculovirus infection Midgut epithelial cells are primarytarget cells, but the underlying basal lamina prevents the progenyvirus from directly entering the hemocoel. Passage through thebasal lamina is facilitated by immediate infection of tracheoblasts,the cells that cross the basal lamina and can deliver the virus tothe trachea and to the hemocoel (8).
CDA-like proteins have recently been described by pro-teomic approaches for the midguts of H. armigera (4, 30) andMamestra configurata (40). Also, for T. ni, an HaCDA5a or-tholog was identified in the larval midgut (13). The availabilityof the whole B. mori genome enabled the identification of eightchitin deacetylase homologs in this species. In general, it ap-
FIG. 5. Methylene blue flux across isolated S. frugiperda peritrophicmembrane incubated with increasing amounts of HaCDA5a. The re-sults are the means � standard deviations for three independent in-fection experiments.
FIG. 6. One-step growth curve analysis of �ccph-Hacda5a and�ccph viruses in Sf21 cells infected at an MOI of 1. The results are themeans � standard deviations (error bars) for independent infectionand titration experiments. BV accumulation is shown as the titer,expressed in TCID50 units/ml, calculated for each time point.FIG. 4. Recombinant expression and chitin binding of HaCDA5a.
(A) Detection of 6�His tag from HaCDA5a by Western blot analysisof the medium and cell extract (72 h p.i.) of Sf21 cells infected with the�ccph-HaCDA5a baculovirus or infected with the control �ccph bac-ulovirus. The total amount of protein loaded was 10 �g for the cellextract lanes and 30 �g for the medium lanes. (B) Detection of 6�Histag of HaCDA5a released from chitin after incubation in PBS, 1%calcofluor white (fluorescent brightener 28; Sigma), 0.5 M NaCl, 6 Murea, 2% SDS–5% �-mercaptoethanol (SDS-�ME), or 20 mM aceticacid, using 6�His antibody.
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pears that CDAs constitute a common protein composition inLepidoptera and apparently belong to one of two major pro-tein classes of the PM.
So far, many CDA-like proteins have been identified frominsects, but it is likely that genomic and proteomic data from newspecies will add many more to this list. The largest number (nineCDAs) has been identified in the red flour beetle, Triboliumcastaneum. Expression profiles and double-stranded-RNA-medi-ated downregulation of all nine CDA transcripts revealed spatialand functional specialization among T. castaneum CDAs (2).Clear division into midgut-specific CDAs and non-gut CDAs wasobserved. Similar to lepidopteran CDAs, T. castaneum midgut-specific CDAs lack ChBD, while all non-gut CDAs carry thisdomain. Differences in the distribution of T. castaneum midgut-specific CDAs along the gut suggest that each of the CDAs mayfunction specifically in different parts of the gut, locally changingthe properties of PM, including permeability.
Bombyx mori CDA tissue specificity analysis using the Bom-byx mori Microarray Database (BmMDB) (http://silkworm.swu.edu.cn/microarray/) revealed that group V CDAs aremostly expressed in the midgut, while CDAs from the othergroups (I to IV) are expressed in other tissues, such as silkglands, Malpighian tubules, or hemocytes. HaCDA expressionanalysis by RT-PCR confirmed that the CDA grouping reflectsnot only domain content but also tissue distribution. HaCDA5a,expressed mainly in the midgut, and HaCDA5b, expressed in
the midgut and other tissues, fall into group V, together withBmCDAs that are mostly expressed in the midgut and togetherwith other CDAs from Lepidoptera that have been describedas midgut proteins.
The lack of a typical chitin binding domain in HaCDA5a andother lepidopteran CDAs implies the existence of alternativemodes of interaction with chitin. Apart from CDAs recentlyidentified from PMs of a few insect species, to date most of theknown PM proteins, including mucins, contain multiple C6motifs of six cysteine residues that interact with chitin. Of thethree H. armigera CDAs described here, only HaCDA1 con-tains a C6 motif, but interestingly, this CDA is hardly ex-pressed in the midgut and its level is not regulated due to virusinfection. HaCDA5a, which was demonstrated to bind chitin,contains 15 cysteines along its sequence, but their distributiondoes not resemble the C6 motif. It was proposed that the CDAdomain may serve as a chitin binding domain (13). Alternativenon-cysteine interactions with chitin have been described forarthropods (32). Many cuticular proteins have a single con-served domain, known as the R&R consensus, first reported byRebers and Riddford (31). However, this consensus is notfound in HaCDA5a or other CDA-like proteins from Lepi-doptera. It would be interesting to investigate which CDAepitopes are able to bind chitin and if those as yet undescribedmotifs are multiple and equally distributed, enabling strongbinding, as in the case of the C6 motif.
In summary, we have found the downregulation of a midgut-specific CDA-like protein as a possible mechanism used by H.armigera to reduce the susceptibility to baculovirus by decreas-ing PM permeability. Recombinant baculoviruses expressingthis protein have shown significant increases in the speed of killand in pathogenicity against larvae from different Spodopteraspp., two parameters highly appreciated in baculovirus-basedbiopesticides. We believe that the study of the larval responsewill point out candidate host genes to be used, by either silenc-ing or overexpression, for improvement of the insecticidalcharacteristics of baculovirus.
ACKNOWLEDGMENTS
We are grateful to Cindy Goodman for providing the HzGUT cellline and to Just Vlak for the Sf21 and HzAM1 cell lines. We gratefullyacknowledge Anh Cao and Leon Court from CSIRO-Entomology for
FIG. 7. Mean times to death for fourth-instar S. frugiperda (A) and S. exigua (B) larvae infected orally or by intrahemocoelic injection with�ccph-Hacda5a in comparison to those for larvae infected with �ccph. The results are the means � standard deviations for independent infectionexperiments. Asterisks denote a significant difference (two-way ANOVA; P � 0.05) from infection with �ccph.
FIG. 8. Mortality of S. exigua third-, fourth-, and fifth-instar larvaeorally infected with �ccph-Hacda5a in comparison to �ccph. The re-sults are the means � standard deviations (error bars) for independentinfection experiments. Asterisks denote a significant difference (two-way ANOVA; P � 0.05) compared to infection with �ccph.
their contributions to microarray printing and hybridization. We alsothank Manuela Barneo for her excellent help with insect rearing. Wethank the anonymous reviewers for their constructive suggestions thathelped to improve the manuscript.
This research was supported by the Spanish Ministry of Scienceand Innovation (research contract from the Ramon y Cajal Programand projects AGL2005-07909-C03-03 and AGL2008-05456-C03-03). A.K.J. was supported by a Marie Curie fellowship (MRTN-CT-2006-035850) from the EU.
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