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Stage-dependent transcriptional changes and characterization of paramyosin of the bovine lungworm Dictyocaulus viviparus
Parasitology International 58 (2009) 334–340
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Stage-dependent transcriptional changes and characterization of paramyosin of the bovine lungworm Dictyocaulus viviparus C. Strube ⁎, S. Buschbaum, G. von Samson-Himmelstjerna, T. Schnieder Institute for Parasitology, University of Veterinary Medicine Hannover, Buenteweg 17, 30559 Hannover, Germany
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Article history: Received 11 May 2009 Received in revised form 2 July 2009 Accepted 3 July 2009 Available online 13 July 2009 Keywords: Dictyocaulus viviparus Lungworm Paramyosin Introns Real time PCR Relative quantification
a b s t r a c t The bovine lungworm Dictyocaulus viviparus is of major economic importance in cattle farming in the temperate zones. The invertebrate protein paramyosin is one of the main components of muscle thick filaments but can also exhibit immunomodulatory functions. It represents a promising vaccine candidate in parasitic helminths. In this study, D. viviparus paramyosin (DvPmy) was characterized on the transcriptional as well as genomic level. The identified genomic sequence comprises 19 introns compared to only 10 introns in the Caenorhabditis elegans orthologue. Quantitative real time PCR transcriptional analysis revealed paramyosin transcription throughout the whole parasite's life cycle with the highest transcription rate in the agile moving first-stage larvae and the lowest in motionless hypobiosis induced third stage larvae. Recombinantly expressed DvPmy was found to bind collagen and IgG. Thereby the present study is the first showing that nematode paramyosin has the capability for immunomodulation and thus may be involved in host immune defence. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The bovine lungworm Dictyocaulus viviparus is one of the most important parasites in cattle in the temperate zones. The parasite causes production losses due to illness (parasitic bronchitis) or even death of infected animals. Paramyosin exclusively occurs in invertebrate organisms among a variety of groups including annelids, molluscs, crustaceans, insects, cestodes, trematodes and nematodes. It is predominantly located in muscles as a major structural component assembling with myosin and core proteins to form the macromolecular thick filaments [1]. In nematodes, the two major muscle groups, the body wall and pharyngeal muscles, are organized as striated muscles with sarcomers. Additional muscle groups like the anal–intestinal or sex-specific muscles exhibit smooth muscle properties. Ardizzi and Epstein [2] found myosin heavy chains C and D restricted to the pharyngeal muscles, whereas myosin heavy chains A and B as well as paramyosin were localized in all muscle cells of the free-living nematode Caenorhabditis elegans. In schistosomes, paramyosin was observed also in a non-filamentous form in the tegument and the post-acetabular gland suggesting that it may be a secretory product, thus becoming a
Abbreviations: cds, coding sequence; DvPmy, Dictyocaulus viviparus paramyosin; qPCR, quantitative real time PCR. ⁎ Corresponding author. Tel.: +49 511 953 8796; fax: +49 511 953 8555. E-mail address: [email protected] (C. Strube). 1383-5769/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.parint.2009.07.003
target for protective immunity [3–5]. However, investigations of Schmidt et al. [6] did not reveal any evidence for an extramuscular occurrence of paramyosin. Nevertheless, it is one of the immunogens proposed as a vaccine candidate against a number of helminths including Schistosoma mansoni [7,8], S. japonicum [9,10], Taenia solium [11] and Brugia malayi [12]. The explanation for this immunoprotective potential may be the immunomodulatory functions found for paramyosin: Loukas et al. [13] showed Fc-receptor binding activity of S. mansoni and S. japonicum paramyosin. As a consequence, host immunoglobulins are adsorbed to the tegument of the schistosomes, masking them against immune recognition. Further IgG-binding properties of paramyosin were described for Paragonimus westermani [14], Taenia crassiceps [15] and Rhipicephalus microplus [16]. Moreover, paramyosin of S. mansoni, P. westermani, T. solium, T. saginata, the cattle tick R. microplus and the mussel Mytilus edulis exhibit collagen-binding activity [14,16–18]. This property is of particular importance because T. solium as well as S. mansoni paramyosin inhibit the complement component C1, presumably by binding to the collagen-like stalk regions of the subcomponent C1q [17]. Further studies have shown that paramyosin inhibits the complement membrane attack complex by binding to the complement proteins C8 and C9 [5,19,20]. In the present study paramyosin of D. viviparus (DvPmy) was characterized on a molecular and transcriptional level. Furthermore, bovine IgG- and collagen-binding ability of recombinantly expressed DvPmy was investigated to examine whether lungworm paramyosin might also play a role in the defence against host immune mechanisms.
C. Strube et al. / Parasitology International 58 (2009) 334–340
2. Materials and methods 2.1. Parasite material for cDNA and genomic characterization First-stage D. viviparus larvae (L1, strain HannoverDv2000) were isolated from the faeces of experimentally infected calves using the Baermann method (Baermann 1917). The larvae were incubated in 20–40 ml of tap water at room temperature for about 10 days, until development to third stage larvae (L3) was completed. To produce adult parasites, helminth-free male Holstein–Friesian calves at the age of five months were infected with 3300 L3. The calves were euthanized 28 days post infection (p.i.) and the worms were collected from the lungs as described by Wood et al. (1995). The worms were washed five times in a 200 ml saline solution (0.9%), transferred to sterile tubes, and frozen at −75 °C. 2.2. Full-length cDNA To amplify full-length cDNA of lungworm paramyosin (DvPmy), primers were designed spanning the whole cDNA sequence obtained from overlapping fragments from previous RACE experiments (accession number AY552027, [21]). The PCR was carried out using L3 cDNA generated with the SMART™ PCR cDNA Synthesis Kit (Clontech) as template. After cloning and sequencing the obtained nucleotide sequences were aligned to lungworm paramyosin from previous work (see above) for sequence verification by using the Align Plus 4.0 software (Scientific and Educational Software). 2.3. Genomic characterization Gene-specific primer pairs for genomic characterization were designed using the PrimerSelect program of the Lasergene software (DNASTAR, version 5.06) based on the DvPmy cDNA sequence (accession no. AY552027). Genomic DNA was isolated from 10 pooled adult male and female worms using the NucleoSpin® Tissue Kit (Macherey-Nagel) following the manufacturer's instructions. PCR setup was as follows: 1 µl of genomic DNA as template was added to 18 µl deionized H2O, 2.5 µl of 10× buffer, 1.5 µl of MgCl2 (25 mM), 1 µl of deoxyribonucleotide triphosphates (2 mM each), 0.5 µl of gene-specific forward and reverse primer (50 µM each), respectively, and 0.125 µl HOT FIREpol® Polymerase (5 U/µl; Solis Biodyne). PCR cycling (35 cycles) was performed using the following temperature profile: initial denaturing at 95 °C for 15 min, denaturing at 95 °C for 1 min, annealing primers at the optimal annealing temperature for the given primer pair (calculated by the primer design software) for 1 min, extending primers at 72 °C for 1 min, and final extension at 72 °C for 10 min. The full genomic sequence of DvPmy was obtained from overlapping gene fragments. Exon–intron boundaries were located by comparison with the full-length DvPmy cDNA sequence using the program Align Plus 4.0 (Scientific and Educational Software). The exon–intron boundaries of DvPmy were compared to those of the unspliced premessenger RNA of the C. elegans unc-15 paramyosin gene (available at http://www.wormbase.org) to analyze similarities and differences between these two organisms. 2.4. Protein expression in Escherichia coli For recombinant protein expression of DvPmy the coding region was amplified by PCR using L3-cDNA as template. Primers included BamHI and NotI restriction sites, respectively, which were used for subsequent ligation in the pET-41a(+) vector (Novagen). The correct coding sequence and open reading frame was confirmed by sequencing (SEQLAB Sequence laboratories). The expression plasmid was transformed in BL21 Star™ DE3 One Shot chemically competent E. coli (Invitrogen) according to a protocol based on the TSS (transformation
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and storage solution) method (first described by Chung et al. [22]). The cells were grown in Terrific Broth medium and overnight recombinant protein expression was induced with 0.1 mM IPTG. 2.5. Recombinant protein extraction To extract the GST/His-fusion protein, cells from 1500 ml culture were harvested at 4 °C by centrifugation at 5400 g for 25 min. The pellet was suspended in 5 ml lysis/binding buffer (20 mM NaH2PO4, 500 mM NaCl, 20 mM imidazole, pH 7.4) and 10 mg/ml lysozyme (Sigma), 25 µl of 1 mg/ml DNase I (Sigma) as well as 25 µl of 40× complete solution (proteinase inhibitor; Roche). This suspension was incubated for 30 min at room temperature with gentle shaking. Afterwards 4 mg/ml of deoxycholic acid was added followed by incubation at 37 °C until the solution became viscous. Then another 25 µl of 1 mg/ml DNase I (Sigma) was added and the solution was gently shaken until liquefaction. The resulting cell lysate was centrifuged at 3200 g for 30 min. The clear supernatant was collected and analyzed via 8% SDS-PAGE followed by Western Blot. 2.6. Protein purification and immunoblotting The GST/His-fused DvPmy was purified by affinity chromatography using two serially connected HisTrap HP 1 ml columns (GE Healthcare) operated by an ÄKTA FPLC system (GE Healthcare) according to the manufacturer's protocol. To remove contaminating E. coli proteins as well as His/GST-tags alone from the affinity purified protein solution, gel-filtration chromatography on a HiLoad 16/60 Superdex-200 prepgrade column (GE Healthcare) was performed. SDS-PAGE was used to determine the purification efficiency. To confirm the purified protein as paramyosin, immunoblots with monoclonal antibodies were carried out. These antibodies were 1:1000 diluted anti-paramyosin (antigen species: C. elegans, Developmental Studies Hybridoma Bank) and 1:1000 diluted anti-GST (Sigma), respectively. 2.7. Maldi-TOF analysis To confirm the appropriate bands as recombinantly expressed DvPmy, they were cut out of the polyacrylamide gel and analyzed via matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) performed at the Seqlab Sequence Laboratories. The provided peptide mass data were identified using Matrix Science's Mascot MS/MS Ion Search (http://www.matrixscience.com/search_form_select.html) searching against the MSDB (Mass Spec Database) protein database. The following modifications were made in the input mask: the peptide tolerance was changed to 50 ppm, the MS/MS tolerance to 0.5 kDa, and the peptide charge to 1+. The selected instrument was MALDI-TOF-TOF. 2.8. IgG-binding assay IgG-binding activity of DvPmy was evaluated by an enzyme-linked immunosorbent assay (ELISA). Each well of a 96-well Immobilizer Amino microtiter plate (Nunc) was coated with either 0.25 µg recombinant DvPmy or GST or bovine serum albumin (BSA; Sigma), diluted in 100 µl phosphate-buffered saline (PBS) by overnight incubation at 4 °C. The plates were washed three times with PBS containing 0.05% Tween 20 (PBST) followed by protein-free blocking of free binding sites on a polymer basis (Roti®-Block; Roth) for 1 h at 37 °C. Concentrations of 0–30 µg bovine IgG (Sigma) diluted in PBST were added to the wells and incubated for 1 h at 37 °C. The plates were then washed three times with PBST and incubated with peroxidaseconjugated anti-bovine IgG1 (Serotec) diluted 1:10,000 in PBST for 1 h at 37 °C. Following three washes with PBST, antibody reactions were visualized by adding o-phenylenediamine (OPD) substrate consisting of 10 mg OPD in 25 ml citrate phosphate buffer (pH 5) and 10 µl H2O2 (30%). After 10 min colour development in the dark the reaction was
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stopped with 2.5 M H2SO4, and the optical density (OD) was determined at 490 nm using the ELx800 Universal Microplate recorder (Bio-Tek). All experiments were performed in triplicate. 2.9. Collagen-binding assay To determine the possible collagen-binding properties of DvPmy, a binding assay was performed in an ELISA format following Ferrer et al. [18]. Microplate wells (Immobilizer Amino microtiter plates; Nunc) were coated with 0.125, 0.25, 0.5, 1.0, 2.0, 4.0, and 8.0 µg of calf skin collagen type I (Sigma) and incubated overnight at 4 °C. Unbound collagen was removed by washing the wells three times with PBST. Afterwards the plates were blocked for 1 h at 37 °C using Roti®-Block (Roth) followed by three washes with PBST. After washing, 0.5 µg of recombinant DvPmy was added to each well. The same amounts of GST and BSA were used as controls. The plates were incubated for 1 h at 37 °C. After washing, rabbit anti-GST antibodies (GE Healthcare) diluted 1:5000 in PBST were added and incubated for 1 h at room temperature. Peroxidase-conjugated antirabbit IgG (Sigma) diluted 1:10,000 in PBST was added and incubated for 1 h at 37 °C. Visualization of the reactions run as triplicates and plate reading were performed as described in the section above. 2.10. Phylogenetic analysis Phylogenetic and molecular evolutionary analyses of helminth and arthropod paramyosin amino acid sequences were conducted in MEGA 4.0 (Tamura et al. 2007) available at http://www.megasoftware.net/. The phylogenetic tree was constructed by bootstrap test of phylogeny (1000 replicates) using the Neighbor-Joining method. For these analyses, the different DvPmy isoform 1 cDNA sequence (accession no. AY552027) was conceptually translated and compared with the paramyosin amino acid sequence of other helminths as well as arthropods. Paramyosin sequence comparison of these organisms was performed with CLUSTAL W 1.83 (http://www.ebi.ac.uk/clustalw/) via multiple sequence alignment. 2.11. Parasite material and first-strand cDNA synthesis for quantitative real time PCR Different lungworm stages were used in quantitative real time PCR (qPCR). These stages were: eggs; L1; L3; male, female and mixed preadults (L5); male and female adults; hypobiosis induced L3 (L3i, chilled for 8 weeks at 4 °C); and hypobiotic L5 (larvae b5 mm, isolated on day 28 p.i.) of the strain HannoverDv2000. Recovery of these stages, mRNA isolation, and first-strand cDNA synthesis was carried out as described previously [23]. For each lungworm stage, three independent mRNA isolations were performed. 2.12. Quantitative real time PCR (qPCR) Quantitative real time PCR (qPCR) was performed to determine the DvPmy transcription level in 10 different lungworm stages. According to Strube et al. [23] the housekeeping genes ß-tubulin and ef-1 alpha were chosen to correct for variations of mRNA amounts and cDNA synthesis efficiency. Primers and TaqMan™-MGB-probes for ß-tubulin and ef-1 alpha were adapted from the above mentioned study. For paramyosin, primer and probe design was done using the Primer Express software (Applied Biosystems). Sequences were: DvPmy for 5′-CACCCGTCTCGAGGATAAAATT-3′, DvPmy rev 5′-AGACGATCAGTAAGAGCGATAAGCT-3′, and DvPmy-MGB 5′-FAMACCGAATCGAACGCGAACG-MGBNFQ-3′. Probes were purchased from Applied Biosystems and primers from Invitrogen. First-strand cDNA-preparations generated from three different mRNA-isolations of the different lungworm stages were used as template. In addition,
10-fold serial dilutions of cloned cDNA ranging from 107 to 101 copies per sample were used as templates to generate standard curves to calculate copy numbers of each gene on each plate. The reactions were set up using the Brilliant® QPCR Master Mix (Stratagene). Thermal cycling conditions were: 10 min at 95 °C followed by 40 cycles of 20 s at 95 °C, 20 s at 55 °C and 30 s at 72 °C. The microtiter plates contained duplicates of each cDNA sample as well as serially diluted plasmid standards to generate standard curves and a no-template control. To ensure repeatability of the experiments, each run was replicated. Thus, the number of reactions of each lungworm stage was n = 12. Experiments and data analysis were performed using the Mx3005 Multiplex Quantitative PCR System (Stratagene). qPCR data mining and calculation of amplification efficiency corrected relative quantities were performed with the qBasePlus 1.0 software (Biogazelle).
3. Results 3.1. D. viviparus paramyosin characterization on nucleotide and protein level PCR experiments revealed three full-length cDNA sequence isoforms consisting of 3207 bp each (without poly(A)+ tail). The coding region is preceded by a 5′ untranslated region (UTR) of 85 bp, and followed by 490 bp representing the 3′ UTR. The bases 3190–3195 within the 3′ UTR represent the polyadenylation signal 5′-AAUAAA-3′ leading to the covalent linkage of a poly(A)+ tail downstream nucleotide position 3207. The coding sequence (cds) has 2631 bp encoding 876 deduced amino acids. The first ATG codon (bases 86–88) fulfils the requirement for a favorable context for initiation of translation as there is a purine (A) in position − 3 [24]. The three DvPmy isoforms slightly differ in their nucleotide composition due to 24 nucleotide substitutions affecting 22 codons with the consequence of 10 amino acid substitutions. According to the amino acid isofunctionality scale by Gindilis et al. [25], there are three isofunctional amino acid substitutions only. These are valine ↔ alanine (codon 200 and 785) as well as arginine ↔ histidine (codon 776), whose isofunctionality ranged on a weak and moderate level, respectively. The bovine lungworm paramyosin has a theoretical molecular weight of 101 kDa and an isoelectric point of 5.2. The genomic paramyosin sequence spans 11,558 bp and consists of 20 exons and 19 introns. Of these, 18 splicing site pairs follow the GT– AG rule, whereas intron 9 has a non-canonical GC–AG junction. The C. elegans paramyosin premessenger RNA contains 11 exons and 10 introns. Sequence comparison with D. viviparus revealed that these two organisms share 7 intron insertion sites while the remaining 3 intron insertion sites of C. elegans and 12 of D. viviparus, respectively, are specific for the particular nematode. Table 1 shows this sequence comparison in more detail. SDS-PAGE analysis of the FPLC purified protein solutions resulted in bands of approximately 135 kDa corresponding to the calculated molecular weight of the GST/His-DvPmy fusion protein. In immunoblots these bands were specifically detected by antibodies and confirmed as paramyosin by Maldi-TOF analysis.
3.2. IgG-binding assay The binding affinity of recombinant DvPmy was tested in an ELISA. As shown in Fig. 1, IgG-binding of DvPmy is dose-dependent over a range of 0.25–30 µg. As GST alone showed no binding activity, the paramyosin part of the DvPmy fusion protein must be responsible for IgG-binding. Besides GST the BSA control did not exhibit any IgGbinding activity.
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Table 1 Intron–exon comparison of D. viviparus and C. elegans paramyosin. D. viviparus
C. elegans
Exon no.
Size (bp)
Intron no.
Size (bp)
Exon no.
Size (bp)
Intron no.
Size (bp)
1 2
211 57
1 2
293 238
3 4 5 6 7 8 9 10 11 12 13
254 79 60 81 105 162 237 148 116 102 90
3 4 5 6 7 8 9 10 11 12 13
1153 3618 53 655 669 71 54 378 99 58 101
1 2 3 4 5 6
231 57 87 111 56 220
1 2 3 4 5 6
1136 46 69 47 1071 307
7
105
7
122
14 15 16 17 18 19 20 bpa bpa cdsb Intron number per kbc cds Intron size (bp) per kb cds
207 198 138 128 160 156 517 3206 2631 7.2
14 15 16 17 18 19
172 327 77 52 73 265
8 9
932 130
8 9
49 49
10 11
777 691 3397 2649 3.8
10
274
3195
8406
3170
1197
D. viviparus and C. elegans introns in the same line are inserted at the same position when the sequences of these two nematodes are aligned. Empty lines stand for the lack of a corresponding intron insertion sites in one of the two species. a Base pairs. b Coding sequence. c Kilobase.
Fig. 2. Collagen-binding activity of recombinant D. viviparus paramyosin. Microtiter plates coated with different concentrations of collagen type I (0.125–8.0 µg) were incubated with 0.5 µg DvPmy. GST and BSA were used as controls. Data are expressed as means ± SD of triplicate experiments.
3.3. Collagen-binding assay The collagen-binding assay showed the property of DvPmy to bind collagen which is depicted in Fig. 2. Collagen binding was dosedependent over a 0.125–4.0 µg range. When 8.0 µg collagen was incubated with DvPmy, the OD490 value reached a plateau. The control proteins GST and BSA showed no significant collagen binding. 3.4. Phylogenetic analysis Of the sequences examined in the phylogenetic analysis, DvPmy was most closely related to paramyosin of Ancylostoma caninum followed by C. elegans. The phylogenetic tree is given in Fig. 3. 3.5. Quantitative real time PCR (qPCR)
Fig. 1. IgG-binding assay with recombinant D. viviparus paramyosin. Microtiter plates were coated with 0.25 µg DvPmy per well followed by incubation with different concentrations of bovine IgG (2.5–30 µg). Data are expressed as means ± SD of triplicate experiments.
The qPCR amplification efficiencies and squared correlation coefficients (R2) of the target sequence DvPmy and the reference genes were as follows: 79.0% (amplification efficiency), − 3.96 (slope), and 0.996 (R2) for β-tubulin; 68.0%, − 4.44, and 0.986 for ef1α as well as 94.3%, −3.47, and 0.960 for DvPmy. After raw qPCR data correction for specific amplification efficiencies, the DvPmy transcription profile was normalized to the reference genes β-tubulin and ef1α. The DvPmy transcription profile was compiled via amplification efficiency corrected relative quantification using eggs as calibrator, whose transcription rate was set to 1. The highest DvPmy transcription was observed in the L1 (3.572 copies relating to 1 copy in eggs) stages followed by eggs. The lowest transcription rate occurs in chilled L3i stages with a relative transcript amount of 0.004. Compared to female stages (0.677 in preadult L5 and 0.362 in adults) the male stages have lower relative transcription levels (0.273 in preadult L5 and 0.065 in adults). Fig. 4 pictures a graphical overview about the DvPmy transcription levels including standard error of the mean.
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4. Discussion The non-vertebrate muscle protein paramyosin has been analyzed in a variety of arthropods and helminths. However, the present characterization of D. viviparus paramyosin (DvPmy) is the first of a parasitic nematode belonging to nematode Clade V [26]. Three fulllength mRNA sequences containing nucleotide substitutions were identified. These span 3207 bp, comprise a Kozak sequence [24], a polyadenylation signal and encode 876 amino acid residues. Comparison of the genomic organization with that of C. elegans paramyosin showed that the number of introns is nearly doubled in D. viviparus, which possesses 19 introns in contrast to 10 introns only in C. elegans. The tendency that C. elegans has less introns than D. viviparus was also observed for other genes like the major sperm protein [27], 60S ribosomal protein L37a and a N-methyltransferase (unpublished results). Investigations on intron–exon structures in C. elegans resulted in an average of 3.6 introns per kilobase coding sequence (kb cds) and an intron size of 1003.8 bp per kb cds [28], which is principally also applicable to C. elegans paramyosin. In contrast, DvPmy has 7.2 introns and 3195 bp intron size per kb cds. Another feature that the first intron tends to be longer than downstream introns [29] holds also true for paramyosin of C. elegans but not for D. viviparus. Although the comparison of only one gene between these two species is not
representative, the described tendencies were also observed for intron–exon comparisons of other bovine lungworm genes with those of C. elegans (unpublished results). Therefore it is questionable whether C. elegans is an appropriate model organism for the genomic organization of Clade V animal parasites. This assumption is supported by the characteristics of A. caninum paramyosin, whose intron–exon structure is similar to that of D. viviparus (unpublished results). Paramyosin is one of the major components of invertebrate muscles and was reported to be present in all types of C. elegans muscle cells [2]. Furthermore, paramyosin is suggested to exhibit non-muscle functions in terms of inhibition of the complement cascade probably by binding to the collagen-like region of the complement component C1q [17] and in terms of immunological defence by binding antibodies [13]. These binding abilities of paramyosin were described for different species of trematodes, cestodes, and arthropodes [13–18]. And indeed, in the present study DvPmy was also found to bind bovine IgG and collagen. This is noteworthy since this result not only confirms functionality of the recombinantly expressed DvPmy but also is the first report of a nematode paramyosin exhibiting these suggested immunomodulatory functions as well. Even if paramyosin is not secreted in nematodes this function may be important for host defence since it has been shown in flies that immunoglobulins can passage through the gut and attain the muscles [30,31]. Furthermore, penetrated antibodies against muscular
Fig. 3. Evolutionary relationships of helminth and arthropod paramyosins. The evolutionary history was inferred using the Neighbor-Joining method. The percentage of replicate trees in which the associated species clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. All positions containing gaps and missing data were eliminated from the dataset (complete deletion option). There were a total of 836 positions in the final dataset. Accession numbers are as follows: Dictyocaulus viviparus isoform 1(AAT36324), Ancylostoma caninum (ABC86903), Caenorhabditis elegans (NP_492085), Onchocerca volvulus (Q02171), Brugia malayi (Q01202), Dirofilaria immitis (P13392), Anisakis simplex (Q9NJA9), Trichinella spiralis (ABO09862), Schistosoma japonicum (AAD29285), S. mansoni (P06198), S. haematobium (BAF62291), Clonorchis sinensis (ABN79674), Paragonimus westermani (AAY44740), Taenia solium (P35418), T. saginata (Q8T305), Echinococcus granulosus (P35417), Rhipicephalus microplus (Q86RN8), Psoroptes ovis (CAJ38271), Chorioptes texanus (ABK54038), Sarcoptes scabiei (Q9BMM8), Blomia tropicalis (Q8MUF6), Aedes aegypti (EAT36995), Drosophila melanogaster (CAA41557), and Apis mellifera (XP_393281).
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mental stages but in different intensities reflecting the variable requirement of muscle activity or developmental growth. Furthermore, collagen- and IgG-binding activity of DvPmy indicates that it might play a role in immunological defence mechanisms against the host suggesting a potential value of DvPmy as a vaccine candidate against Dictyocaulosis. The selection of DvPmy as a vaccine candidate is supported by a study of Kiel et al. [33], in which paramyosin from the closely related sheep gastrointestinal nematode Trichostrongylus colubriformis was identified as an immuno-reactive protein. And indeed, pilot studies to determine the protective potential of recombinant paramyosin against the bovine lungworm D. viviparus show promising results (unpublished) but have to be confirmed in further vaccine trials which are underway.
Acknowledgements The monoclonal paramyosin antibody 5–23 developed by Henry Epstein was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242.
References Fig. 4. Amplification efficiency corrected normalized paramyosin transcription throughout the D. viviparus life cycle.
elements can directly react with the specific antigens thereby interfering with the function and growth of the muscular system with the result of paralysis [30,31]. In this context it is remarkable that the analysis of evolutionary relationships defines DvPmy or rather nematode paramyosin in general, to be more similar to that of arthropods than to that of cestodes and trematodes. Such an antibody passage through barrier epithelia to pharyngeal and body wall muscles might explain the auspicious worm reduction in our vaccine experiments against D. viviparus. Transcriptional analysis of bovine lungworm paramyosin revealed that the highest transcript levels are present in L1. This was expected as the first larval stage is very active because it has to move away from the faeces. Interestingly, the second highest paramyosin transcription was observed in eggs. Within the eggs the parasite undergoes a first developmental differentiation from blastomeres to L1. In consequence, the developing embryos transcribe paramyosin for muscle formation and already developed L1 need this muscle protein for hatching and further movement. The lowest transcript levels were measured in hypobiosis induced L3i which remain curled up without any movement or further development. An increase of paramyosin transcription is detectable in the preadult L5 stages representing a growing and most likely moving stage. Noteworthy, there is a higher transcription rate in preadult and adult females compared to male stages. One explanation might be the larger body size of female individuals requiring more muscle mass. As another reason, at least for adult worms, an additional need for muscles for reproduction may be hypothesized. Adult males need their sex muscles only for copulation. In contrast, female worms require continuous activity of the uterine and vulval muscles for egg-laying. Furthermore, it can be assumed that even ovulation depends on contraction of myoepithelial sheath cells and thus on paramyosin as shown for C. elegans [32]. It can be largely excluded that the higher paramyosin transcript levels in females relate to developing eggs because these were removed by bursting adult females in double distilled water. In conclusion, the results of the present study show that paramyosin of the bovine lungworm is transcribed across all develop-
[1] Epstein HF, Miller III DM, Ortiz I, Berliner GC. Myosin and paramyosin are organized about a newly identified core structure. J Cell Biol 1985;100:904–15. [2] Ardizzi JP, Epstein HF. Immunochemical localization of myosin heavy chain isoforms and paramyosin in developmentally and structurally diverse muscle cell types of the nematode Caenorhabditis elegans. J Cell Biol 1987;105:2763–70. [3] Matsumoto Y, Perry G, Levine RJ, Blanton R, Mahmoud AA, Aikawa M. Paramyosin and actin in schistosomal teguments. Nature 1988;333:76–8. [4] Nara T, Matsumoto N, Janecharut T, Matsuda H, Yamamoto K, Irimura T, et al. Demonstration of the target molecule of a protective IgE antibody in secretory glands of Schistosoma japonicum larvae. Int Immunol 1994;6:963–71. [5] Deng J, Gold D, LoVerde PT, Fishelson Z. Mapping of the complement C9 binding domain in paramyosin of the blood fluke Schistosoma mansoni. Int J Parasitol 2007;37:67–75. [6] Schmidt J, Bodor O, Gohr L, Kunz W. Paramyosin isoforms of Schistosoma mansoni are phosphorylated and localized in a large variety of muscle types. Parasitology 1996;112(Pt 5):459–67. [7] Flanigan TP, King CH, Lett RR, Nanduri J, Mahmoud AA. Induction of resistance to Schistosoma mansoni infection in mice by purified parasite paramyosin. J Clin Invest 1989;83:1010–4. [8] Pearce EJ, James SL, Hieny S, Lanar DE, Sher A. Induction of protective immunity against Schistosoma mansoni by vaccination with schistosome paramyosin (Sm97), a nonsurface parasite antigen. Proc Natl Acad Sci U S A 1988;85:5678–82. [9] Chen H, Nara T, Zeng X, Satoh M, Wu G, Jiang W, et al. Vaccination of domestic pig with recombinant paramyosin against Schistosoma japonicum in China. Vaccine 2000;18:2142–6. [10] Taylor MG, Huggins MC, Shi F, Lin J, Tian E, Ye P, et al. Production and testing of Schistosoma japonicum candidate vaccine antigens in the natural ovine host. Vaccine 1998;16:1290–8. [11] Vazquez-Talavera J, Solis CF, Terrazas LI, Laclette JP. Characterization and protective potential of the immune response to Taenia solium paramyosin in a murine model of cysticercosis. Infect Immun 2001;69:5412–6. [12] Li BW, Zhang S, Curtis KC, Weil GJ. Immune responses to Brugia malayi paramyosin in rodents after DNA vaccination. Vaccine 1999;18:76–81. [13] Loukas A, Jones MK, King LT, Brindley PJ, McManus DP. Receptor for Fc on the surfaces of schistosomes. Infect Immun 2001;69:3646–51. [14] Zhao QP, Moon SU, Na BK, Kim SH, Cho SH, Lee HW, et al. Paragonimus westermani: biochemical and immunological characterizations of paramyosin. Exp Parasitol 2007;115:9–18. [15] Kalinna B, McManus DP. An IgG (Fc gamma)-binding protein of Taenia crassiceps (Cestoda) exhibits sequence homology and antigenic similarity with schistosome paramyosin. Parasitology 1993;106(Pt 3):289–96. [16] Ferreira CA, Barbosa MC, Silveira TC, Valenzuela JG, Vaz Ida Jr S, Masuda A. cDNA cloning, expression and characterization of a Boophilus microplus paramyosin. Parasitology 2002;125:265–74. [17] Laclette JP, Shoemaker CB, Richter D, Arcos L, Pante N, Cohen C, et al. Paramyosin inhibits complement C1. J Immunol 1992;148:124–8. [18] Ferrer E, Moyano E, Benitez L, Gonzalez LM, Bryce D, Foster-Cuevas M, et al. Cloning and characterization of Taenia saginata paramyosin cDNA. Parasitol Res 2003;91:60–7. [19] Deng J, Gold D, LoVerde PT, Fishelson Z. Inhibition of the complement membrane attack complex by Schistosoma mansoni paramyosin. Infect Immun 2003;71:6402–10. [20] Parizade M, Arnon R, Lachmann PJ, Fishelson Z. Functional and antigenic similarities between a 94-kD protein of Schistosoma mansoni (SCIP-1) and human CD59. J Exp Med 1994;179:1625–36.
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[21] Strube C, Schnieder T, von Samson-Himmelstjerna G. Differential gene expression in hypobiosis-induced and non-induced third-stage larvae of the bovine lungworm Dictyocaulus viviparus. Int J Parasitol 2007;37:221–31. [22] Chung CT, Niemela SL, Miller RH. One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc Natl Acad Sci U S A 1989;86:2172–5. [23] Strube C, Buschbaum S, Wolken S, Schnieder T. Evaluation of reference genes for quantitative real-time PCR to investigate protein disulfide isomerase transcription pattern in the bovine lungworm Dictyocaulus viviparus. Gene 2008;425:36–43. [24] Kozak M. An analysis of 5′-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res 1987;15:8125–48. [25] Gindilis V, Goltsman E, Verlinsky Y. Evolutionary classification of homeodomains. J Assist Reprod Genet 1998;15:349–57. [26] Blaxter M, De Lay P, Garey JR, Liu LX, Scheldeman P, Vierstraete A, et al. A molecular evolutionary framework for the phylum nematoda. Nature 1998;392. [27] Strube C, Buschbaum S, Schnieder T. Molecular characterization and real-time PCR transcriptional analysis of Dictyocaulus viviparus major sperm proteins. Parasitol Res 2009;104:543–51.
[28] Deutsch M, Long M. Intron–exon structures of eukaryotic model organisms. Nucleic Acids Res 1999;27:3219–28. [29] Bradnam KR, Korf I. Longer first introns are a general property of eukaryotic gene structure. PLoS ONE 2008;3:e3093. [30] Schlein Y, Sira DT, Jacobson RL. The passage of serum immunoglobulins through the gut of Sarcophaga falculata, Pand. Ann Trop Med Parasitol 1976;70:227–30. [31] Schlein Y, Lewis CT. Lesions in haematophagous flies after feeding on rabbits immunized with fly tissues. Physiol Entomol 1976;1:55–9. [32] Ono K, Yu R, Ono S. Structural components of the nonstriated contractile apparatuses in the Caenorhabditis elegans gonadal myoepithelial sheath and their essential roles for ovulation. Dev Dyn 2007;236:1093–105. [33] Kiel M, Josh P, Jones A, Windon R, Hunt P, Kongsuwan K. Identification of immunoreactive proteins from a sheep gastrointestinal nematode, Trichostrongylus colubriformis, using two-dimensional electrophoresis and mass spectrometry. Int J Parasitol 2007;37:1419–29.
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Parasitology International j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p a r i n t
Stage-dependent transcriptional changes and characterization of paramyosin of the bovine lungworm Dictyocaulus viviparus C. Strube ⁎, S. Buschbaum, G. von Samson-Himmelstjerna, T. Schnieder Institute for Parasitology, University of Veterinary Medicine Hannover, Buenteweg 17, 30559 Hannover, Germany
a r t i c l e
i n f o
Article history: Received 11 May 2009 Received in revised form 2 July 2009 Accepted 3 July 2009 Available online 13 July 2009 Keywords: Dictyocaulus viviparus Lungworm Paramyosin Introns Real time PCR Relative quantification
a b s t r a c t The bovine lungworm Dictyocaulus viviparus is of major economic importance in cattle farming in the temperate zones. The invertebrate protein paramyosin is one of the main components of muscle thick filaments but can also exhibit immunomodulatory functions. It represents a promising vaccine candidate in parasitic helminths. In this study, D. viviparus paramyosin (DvPmy) was characterized on the transcriptional as well as genomic level. The identified genomic sequence comprises 19 introns compared to only 10 introns in the Caenorhabditis elegans orthologue. Quantitative real time PCR transcriptional analysis revealed paramyosin transcription throughout the whole parasite's life cycle with the highest transcription rate in the agile moving first-stage larvae and the lowest in motionless hypobiosis induced third stage larvae. Recombinantly expressed DvPmy was found to bind collagen and IgG. Thereby the present study is the first showing that nematode paramyosin has the capability for immunomodulation and thus may be involved in host immune defence. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The bovine lungworm Dictyocaulus viviparus is one of the most important parasites in cattle in the temperate zones. The parasite causes production losses due to illness (parasitic bronchitis) or even death of infected animals. Paramyosin exclusively occurs in invertebrate organisms among a variety of groups including annelids, molluscs, crustaceans, insects, cestodes, trematodes and nematodes. It is predominantly located in muscles as a major structural component assembling with myosin and core proteins to form the macromolecular thick filaments [1]. In nematodes, the two major muscle groups, the body wall and pharyngeal muscles, are organized as striated muscles with sarcomers. Additional muscle groups like the anal–intestinal or sex-specific muscles exhibit smooth muscle properties. Ardizzi and Epstein [2] found myosin heavy chains C and D restricted to the pharyngeal muscles, whereas myosin heavy chains A and B as well as paramyosin were localized in all muscle cells of the free-living nematode Caenorhabditis elegans. In schistosomes, paramyosin was observed also in a non-filamentous form in the tegument and the post-acetabular gland suggesting that it may be a secretory product, thus becoming a
Abbreviations: cds, coding sequence; DvPmy, Dictyocaulus viviparus paramyosin; qPCR, quantitative real time PCR. ⁎ Corresponding author. Tel.: +49 511 953 8796; fax: +49 511 953 8555. E-mail address: [email protected] (C. Strube). 1383-5769/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.parint.2009.07.003
target for protective immunity [3–5]. However, investigations of Schmidt et al. [6] did not reveal any evidence for an extramuscular occurrence of paramyosin. Nevertheless, it is one of the immunogens proposed as a vaccine candidate against a number of helminths including Schistosoma mansoni [7,8], S. japonicum [9,10], Taenia solium [11] and Brugia malayi [12]. The explanation for this immunoprotective potential may be the immunomodulatory functions found for paramyosin: Loukas et al. [13] showed Fc-receptor binding activity of S. mansoni and S. japonicum paramyosin. As a consequence, host immunoglobulins are adsorbed to the tegument of the schistosomes, masking them against immune recognition. Further IgG-binding properties of paramyosin were described for Paragonimus westermani [14], Taenia crassiceps [15] and Rhipicephalus microplus [16]. Moreover, paramyosin of S. mansoni, P. westermani, T. solium, T. saginata, the cattle tick R. microplus and the mussel Mytilus edulis exhibit collagen-binding activity [14,16–18]. This property is of particular importance because T. solium as well as S. mansoni paramyosin inhibit the complement component C1, presumably by binding to the collagen-like stalk regions of the subcomponent C1q [17]. Further studies have shown that paramyosin inhibits the complement membrane attack complex by binding to the complement proteins C8 and C9 [5,19,20]. In the present study paramyosin of D. viviparus (DvPmy) was characterized on a molecular and transcriptional level. Furthermore, bovine IgG- and collagen-binding ability of recombinantly expressed DvPmy was investigated to examine whether lungworm paramyosin might also play a role in the defence against host immune mechanisms.
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2. Materials and methods 2.1. Parasite material for cDNA and genomic characterization First-stage D. viviparus larvae (L1, strain HannoverDv2000) were isolated from the faeces of experimentally infected calves using the Baermann method (Baermann 1917). The larvae were incubated in 20–40 ml of tap water at room temperature for about 10 days, until development to third stage larvae (L3) was completed. To produce adult parasites, helminth-free male Holstein–Friesian calves at the age of five months were infected with 3300 L3. The calves were euthanized 28 days post infection (p.i.) and the worms were collected from the lungs as described by Wood et al. (1995). The worms were washed five times in a 200 ml saline solution (0.9%), transferred to sterile tubes, and frozen at −75 °C. 2.2. Full-length cDNA To amplify full-length cDNA of lungworm paramyosin (DvPmy), primers were designed spanning the whole cDNA sequence obtained from overlapping fragments from previous RACE experiments (accession number AY552027, [21]). The PCR was carried out using L3 cDNA generated with the SMART™ PCR cDNA Synthesis Kit (Clontech) as template. After cloning and sequencing the obtained nucleotide sequences were aligned to lungworm paramyosin from previous work (see above) for sequence verification by using the Align Plus 4.0 software (Scientific and Educational Software). 2.3. Genomic characterization Gene-specific primer pairs for genomic characterization were designed using the PrimerSelect program of the Lasergene software (DNASTAR, version 5.06) based on the DvPmy cDNA sequence (accession no. AY552027). Genomic DNA was isolated from 10 pooled adult male and female worms using the NucleoSpin® Tissue Kit (Macherey-Nagel) following the manufacturer's instructions. PCR setup was as follows: 1 µl of genomic DNA as template was added to 18 µl deionized H2O, 2.5 µl of 10× buffer, 1.5 µl of MgCl2 (25 mM), 1 µl of deoxyribonucleotide triphosphates (2 mM each), 0.5 µl of gene-specific forward and reverse primer (50 µM each), respectively, and 0.125 µl HOT FIREpol® Polymerase (5 U/µl; Solis Biodyne). PCR cycling (35 cycles) was performed using the following temperature profile: initial denaturing at 95 °C for 15 min, denaturing at 95 °C for 1 min, annealing primers at the optimal annealing temperature for the given primer pair (calculated by the primer design software) for 1 min, extending primers at 72 °C for 1 min, and final extension at 72 °C for 10 min. The full genomic sequence of DvPmy was obtained from overlapping gene fragments. Exon–intron boundaries were located by comparison with the full-length DvPmy cDNA sequence using the program Align Plus 4.0 (Scientific and Educational Software). The exon–intron boundaries of DvPmy were compared to those of the unspliced premessenger RNA of the C. elegans unc-15 paramyosin gene (available at http://www.wormbase.org) to analyze similarities and differences between these two organisms. 2.4. Protein expression in Escherichia coli For recombinant protein expression of DvPmy the coding region was amplified by PCR using L3-cDNA as template. Primers included BamHI and NotI restriction sites, respectively, which were used for subsequent ligation in the pET-41a(+) vector (Novagen). The correct coding sequence and open reading frame was confirmed by sequencing (SEQLAB Sequence laboratories). The expression plasmid was transformed in BL21 Star™ DE3 One Shot chemically competent E. coli (Invitrogen) according to a protocol based on the TSS (transformation
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and storage solution) method (first described by Chung et al. [22]). The cells were grown in Terrific Broth medium and overnight recombinant protein expression was induced with 0.1 mM IPTG. 2.5. Recombinant protein extraction To extract the GST/His-fusion protein, cells from 1500 ml culture were harvested at 4 °C by centrifugation at 5400 g for 25 min. The pellet was suspended in 5 ml lysis/binding buffer (20 mM NaH2PO4, 500 mM NaCl, 20 mM imidazole, pH 7.4) and 10 mg/ml lysozyme (Sigma), 25 µl of 1 mg/ml DNase I (Sigma) as well as 25 µl of 40× complete solution (proteinase inhibitor; Roche). This suspension was incubated for 30 min at room temperature with gentle shaking. Afterwards 4 mg/ml of deoxycholic acid was added followed by incubation at 37 °C until the solution became viscous. Then another 25 µl of 1 mg/ml DNase I (Sigma) was added and the solution was gently shaken until liquefaction. The resulting cell lysate was centrifuged at 3200 g for 30 min. The clear supernatant was collected and analyzed via 8% SDS-PAGE followed by Western Blot. 2.6. Protein purification and immunoblotting The GST/His-fused DvPmy was purified by affinity chromatography using two serially connected HisTrap HP 1 ml columns (GE Healthcare) operated by an ÄKTA FPLC system (GE Healthcare) according to the manufacturer's protocol. To remove contaminating E. coli proteins as well as His/GST-tags alone from the affinity purified protein solution, gel-filtration chromatography on a HiLoad 16/60 Superdex-200 prepgrade column (GE Healthcare) was performed. SDS-PAGE was used to determine the purification efficiency. To confirm the purified protein as paramyosin, immunoblots with monoclonal antibodies were carried out. These antibodies were 1:1000 diluted anti-paramyosin (antigen species: C. elegans, Developmental Studies Hybridoma Bank) and 1:1000 diluted anti-GST (Sigma), respectively. 2.7. Maldi-TOF analysis To confirm the appropriate bands as recombinantly expressed DvPmy, they were cut out of the polyacrylamide gel and analyzed via matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) performed at the Seqlab Sequence Laboratories. The provided peptide mass data were identified using Matrix Science's Mascot MS/MS Ion Search (http://www.matrixscience.com/search_form_select.html) searching against the MSDB (Mass Spec Database) protein database. The following modifications were made in the input mask: the peptide tolerance was changed to 50 ppm, the MS/MS tolerance to 0.5 kDa, and the peptide charge to 1+. The selected instrument was MALDI-TOF-TOF. 2.8. IgG-binding assay IgG-binding activity of DvPmy was evaluated by an enzyme-linked immunosorbent assay (ELISA). Each well of a 96-well Immobilizer Amino microtiter plate (Nunc) was coated with either 0.25 µg recombinant DvPmy or GST or bovine serum albumin (BSA; Sigma), diluted in 100 µl phosphate-buffered saline (PBS) by overnight incubation at 4 °C. The plates were washed three times with PBS containing 0.05% Tween 20 (PBST) followed by protein-free blocking of free binding sites on a polymer basis (Roti®-Block; Roth) for 1 h at 37 °C. Concentrations of 0–30 µg bovine IgG (Sigma) diluted in PBST were added to the wells and incubated for 1 h at 37 °C. The plates were then washed three times with PBST and incubated with peroxidaseconjugated anti-bovine IgG1 (Serotec) diluted 1:10,000 in PBST for 1 h at 37 °C. Following three washes with PBST, antibody reactions were visualized by adding o-phenylenediamine (OPD) substrate consisting of 10 mg OPD in 25 ml citrate phosphate buffer (pH 5) and 10 µl H2O2 (30%). After 10 min colour development in the dark the reaction was
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stopped with 2.5 M H2SO4, and the optical density (OD) was determined at 490 nm using the ELx800 Universal Microplate recorder (Bio-Tek). All experiments were performed in triplicate. 2.9. Collagen-binding assay To determine the possible collagen-binding properties of DvPmy, a binding assay was performed in an ELISA format following Ferrer et al. [18]. Microplate wells (Immobilizer Amino microtiter plates; Nunc) were coated with 0.125, 0.25, 0.5, 1.0, 2.0, 4.0, and 8.0 µg of calf skin collagen type I (Sigma) and incubated overnight at 4 °C. Unbound collagen was removed by washing the wells three times with PBST. Afterwards the plates were blocked for 1 h at 37 °C using Roti®-Block (Roth) followed by three washes with PBST. After washing, 0.5 µg of recombinant DvPmy was added to each well. The same amounts of GST and BSA were used as controls. The plates were incubated for 1 h at 37 °C. After washing, rabbit anti-GST antibodies (GE Healthcare) diluted 1:5000 in PBST were added and incubated for 1 h at room temperature. Peroxidase-conjugated antirabbit IgG (Sigma) diluted 1:10,000 in PBST was added and incubated for 1 h at 37 °C. Visualization of the reactions run as triplicates and plate reading were performed as described in the section above. 2.10. Phylogenetic analysis Phylogenetic and molecular evolutionary analyses of helminth and arthropod paramyosin amino acid sequences were conducted in MEGA 4.0 (Tamura et al. 2007) available at http://www.megasoftware.net/. The phylogenetic tree was constructed by bootstrap test of phylogeny (1000 replicates) using the Neighbor-Joining method. For these analyses, the different DvPmy isoform 1 cDNA sequence (accession no. AY552027) was conceptually translated and compared with the paramyosin amino acid sequence of other helminths as well as arthropods. Paramyosin sequence comparison of these organisms was performed with CLUSTAL W 1.83 (http://www.ebi.ac.uk/clustalw/) via multiple sequence alignment. 2.11. Parasite material and first-strand cDNA synthesis for quantitative real time PCR Different lungworm stages were used in quantitative real time PCR (qPCR). These stages were: eggs; L1; L3; male, female and mixed preadults (L5); male and female adults; hypobiosis induced L3 (L3i, chilled for 8 weeks at 4 °C); and hypobiotic L5 (larvae b5 mm, isolated on day 28 p.i.) of the strain HannoverDv2000. Recovery of these stages, mRNA isolation, and first-strand cDNA synthesis was carried out as described previously [23]. For each lungworm stage, three independent mRNA isolations were performed. 2.12. Quantitative real time PCR (qPCR) Quantitative real time PCR (qPCR) was performed to determine the DvPmy transcription level in 10 different lungworm stages. According to Strube et al. [23] the housekeeping genes ß-tubulin and ef-1 alpha were chosen to correct for variations of mRNA amounts and cDNA synthesis efficiency. Primers and TaqMan™-MGB-probes for ß-tubulin and ef-1 alpha were adapted from the above mentioned study. For paramyosin, primer and probe design was done using the Primer Express software (Applied Biosystems). Sequences were: DvPmy for 5′-CACCCGTCTCGAGGATAAAATT-3′, DvPmy rev 5′-AGACGATCAGTAAGAGCGATAAGCT-3′, and DvPmy-MGB 5′-FAMACCGAATCGAACGCGAACG-MGBNFQ-3′. Probes were purchased from Applied Biosystems and primers from Invitrogen. First-strand cDNA-preparations generated from three different mRNA-isolations of the different lungworm stages were used as template. In addition,
10-fold serial dilutions of cloned cDNA ranging from 107 to 101 copies per sample were used as templates to generate standard curves to calculate copy numbers of each gene on each plate. The reactions were set up using the Brilliant® QPCR Master Mix (Stratagene). Thermal cycling conditions were: 10 min at 95 °C followed by 40 cycles of 20 s at 95 °C, 20 s at 55 °C and 30 s at 72 °C. The microtiter plates contained duplicates of each cDNA sample as well as serially diluted plasmid standards to generate standard curves and a no-template control. To ensure repeatability of the experiments, each run was replicated. Thus, the number of reactions of each lungworm stage was n = 12. Experiments and data analysis were performed using the Mx3005 Multiplex Quantitative PCR System (Stratagene). qPCR data mining and calculation of amplification efficiency corrected relative quantities were performed with the qBasePlus 1.0 software (Biogazelle).
3. Results 3.1. D. viviparus paramyosin characterization on nucleotide and protein level PCR experiments revealed three full-length cDNA sequence isoforms consisting of 3207 bp each (without poly(A)+ tail). The coding region is preceded by a 5′ untranslated region (UTR) of 85 bp, and followed by 490 bp representing the 3′ UTR. The bases 3190–3195 within the 3′ UTR represent the polyadenylation signal 5′-AAUAAA-3′ leading to the covalent linkage of a poly(A)+ tail downstream nucleotide position 3207. The coding sequence (cds) has 2631 bp encoding 876 deduced amino acids. The first ATG codon (bases 86–88) fulfils the requirement for a favorable context for initiation of translation as there is a purine (A) in position − 3 [24]. The three DvPmy isoforms slightly differ in their nucleotide composition due to 24 nucleotide substitutions affecting 22 codons with the consequence of 10 amino acid substitutions. According to the amino acid isofunctionality scale by Gindilis et al. [25], there are three isofunctional amino acid substitutions only. These are valine ↔ alanine (codon 200 and 785) as well as arginine ↔ histidine (codon 776), whose isofunctionality ranged on a weak and moderate level, respectively. The bovine lungworm paramyosin has a theoretical molecular weight of 101 kDa and an isoelectric point of 5.2. The genomic paramyosin sequence spans 11,558 bp and consists of 20 exons and 19 introns. Of these, 18 splicing site pairs follow the GT– AG rule, whereas intron 9 has a non-canonical GC–AG junction. The C. elegans paramyosin premessenger RNA contains 11 exons and 10 introns. Sequence comparison with D. viviparus revealed that these two organisms share 7 intron insertion sites while the remaining 3 intron insertion sites of C. elegans and 12 of D. viviparus, respectively, are specific for the particular nematode. Table 1 shows this sequence comparison in more detail. SDS-PAGE analysis of the FPLC purified protein solutions resulted in bands of approximately 135 kDa corresponding to the calculated molecular weight of the GST/His-DvPmy fusion protein. In immunoblots these bands were specifically detected by antibodies and confirmed as paramyosin by Maldi-TOF analysis.
3.2. IgG-binding assay The binding affinity of recombinant DvPmy was tested in an ELISA. As shown in Fig. 1, IgG-binding of DvPmy is dose-dependent over a range of 0.25–30 µg. As GST alone showed no binding activity, the paramyosin part of the DvPmy fusion protein must be responsible for IgG-binding. Besides GST the BSA control did not exhibit any IgGbinding activity.
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Table 1 Intron–exon comparison of D. viviparus and C. elegans paramyosin. D. viviparus
C. elegans
Exon no.
Size (bp)
Intron no.
Size (bp)
Exon no.
Size (bp)
Intron no.
Size (bp)
1 2
211 57
1 2
293 238
3 4 5 6 7 8 9 10 11 12 13
254 79 60 81 105 162 237 148 116 102 90
3 4 5 6 7 8 9 10 11 12 13
1153 3618 53 655 669 71 54 378 99 58 101
1 2 3 4 5 6
231 57 87 111 56 220
1 2 3 4 5 6
1136 46 69 47 1071 307
7
105
7
122
14 15 16 17 18 19 20 bpa bpa cdsb Intron number per kbc cds Intron size (bp) per kb cds
207 198 138 128 160 156 517 3206 2631 7.2
14 15 16 17 18 19
172 327 77 52 73 265
8 9
932 130
8 9
49 49
10 11
777 691 3397 2649 3.8
10
274
3195
8406
3170
1197
D. viviparus and C. elegans introns in the same line are inserted at the same position when the sequences of these two nematodes are aligned. Empty lines stand for the lack of a corresponding intron insertion sites in one of the two species. a Base pairs. b Coding sequence. c Kilobase.
Fig. 2. Collagen-binding activity of recombinant D. viviparus paramyosin. Microtiter plates coated with different concentrations of collagen type I (0.125–8.0 µg) were incubated with 0.5 µg DvPmy. GST and BSA were used as controls. Data are expressed as means ± SD of triplicate experiments.
3.3. Collagen-binding assay The collagen-binding assay showed the property of DvPmy to bind collagen which is depicted in Fig. 2. Collagen binding was dosedependent over a 0.125–4.0 µg range. When 8.0 µg collagen was incubated with DvPmy, the OD490 value reached a plateau. The control proteins GST and BSA showed no significant collagen binding. 3.4. Phylogenetic analysis Of the sequences examined in the phylogenetic analysis, DvPmy was most closely related to paramyosin of Ancylostoma caninum followed by C. elegans. The phylogenetic tree is given in Fig. 3. 3.5. Quantitative real time PCR (qPCR)
Fig. 1. IgG-binding assay with recombinant D. viviparus paramyosin. Microtiter plates were coated with 0.25 µg DvPmy per well followed by incubation with different concentrations of bovine IgG (2.5–30 µg). Data are expressed as means ± SD of triplicate experiments.
The qPCR amplification efficiencies and squared correlation coefficients (R2) of the target sequence DvPmy and the reference genes were as follows: 79.0% (amplification efficiency), − 3.96 (slope), and 0.996 (R2) for β-tubulin; 68.0%, − 4.44, and 0.986 for ef1α as well as 94.3%, −3.47, and 0.960 for DvPmy. After raw qPCR data correction for specific amplification efficiencies, the DvPmy transcription profile was normalized to the reference genes β-tubulin and ef1α. The DvPmy transcription profile was compiled via amplification efficiency corrected relative quantification using eggs as calibrator, whose transcription rate was set to 1. The highest DvPmy transcription was observed in the L1 (3.572 copies relating to 1 copy in eggs) stages followed by eggs. The lowest transcription rate occurs in chilled L3i stages with a relative transcript amount of 0.004. Compared to female stages (0.677 in preadult L5 and 0.362 in adults) the male stages have lower relative transcription levels (0.273 in preadult L5 and 0.065 in adults). Fig. 4 pictures a graphical overview about the DvPmy transcription levels including standard error of the mean.
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4. Discussion The non-vertebrate muscle protein paramyosin has been analyzed in a variety of arthropods and helminths. However, the present characterization of D. viviparus paramyosin (DvPmy) is the first of a parasitic nematode belonging to nematode Clade V [26]. Three fulllength mRNA sequences containing nucleotide substitutions were identified. These span 3207 bp, comprise a Kozak sequence [24], a polyadenylation signal and encode 876 amino acid residues. Comparison of the genomic organization with that of C. elegans paramyosin showed that the number of introns is nearly doubled in D. viviparus, which possesses 19 introns in contrast to 10 introns only in C. elegans. The tendency that C. elegans has less introns than D. viviparus was also observed for other genes like the major sperm protein [27], 60S ribosomal protein L37a and a N-methyltransferase (unpublished results). Investigations on intron–exon structures in C. elegans resulted in an average of 3.6 introns per kilobase coding sequence (kb cds) and an intron size of 1003.8 bp per kb cds [28], which is principally also applicable to C. elegans paramyosin. In contrast, DvPmy has 7.2 introns and 3195 bp intron size per kb cds. Another feature that the first intron tends to be longer than downstream introns [29] holds also true for paramyosin of C. elegans but not for D. viviparus. Although the comparison of only one gene between these two species is not
representative, the described tendencies were also observed for intron–exon comparisons of other bovine lungworm genes with those of C. elegans (unpublished results). Therefore it is questionable whether C. elegans is an appropriate model organism for the genomic organization of Clade V animal parasites. This assumption is supported by the characteristics of A. caninum paramyosin, whose intron–exon structure is similar to that of D. viviparus (unpublished results). Paramyosin is one of the major components of invertebrate muscles and was reported to be present in all types of C. elegans muscle cells [2]. Furthermore, paramyosin is suggested to exhibit non-muscle functions in terms of inhibition of the complement cascade probably by binding to the collagen-like region of the complement component C1q [17] and in terms of immunological defence by binding antibodies [13]. These binding abilities of paramyosin were described for different species of trematodes, cestodes, and arthropodes [13–18]. And indeed, in the present study DvPmy was also found to bind bovine IgG and collagen. This is noteworthy since this result not only confirms functionality of the recombinantly expressed DvPmy but also is the first report of a nematode paramyosin exhibiting these suggested immunomodulatory functions as well. Even if paramyosin is not secreted in nematodes this function may be important for host defence since it has been shown in flies that immunoglobulins can passage through the gut and attain the muscles [30,31]. Furthermore, penetrated antibodies against muscular
Fig. 3. Evolutionary relationships of helminth and arthropod paramyosins. The evolutionary history was inferred using the Neighbor-Joining method. The percentage of replicate trees in which the associated species clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. All positions containing gaps and missing data were eliminated from the dataset (complete deletion option). There were a total of 836 positions in the final dataset. Accession numbers are as follows: Dictyocaulus viviparus isoform 1(AAT36324), Ancylostoma caninum (ABC86903), Caenorhabditis elegans (NP_492085), Onchocerca volvulus (Q02171), Brugia malayi (Q01202), Dirofilaria immitis (P13392), Anisakis simplex (Q9NJA9), Trichinella spiralis (ABO09862), Schistosoma japonicum (AAD29285), S. mansoni (P06198), S. haematobium (BAF62291), Clonorchis sinensis (ABN79674), Paragonimus westermani (AAY44740), Taenia solium (P35418), T. saginata (Q8T305), Echinococcus granulosus (P35417), Rhipicephalus microplus (Q86RN8), Psoroptes ovis (CAJ38271), Chorioptes texanus (ABK54038), Sarcoptes scabiei (Q9BMM8), Blomia tropicalis (Q8MUF6), Aedes aegypti (EAT36995), Drosophila melanogaster (CAA41557), and Apis mellifera (XP_393281).
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mental stages but in different intensities reflecting the variable requirement of muscle activity or developmental growth. Furthermore, collagen- and IgG-binding activity of DvPmy indicates that it might play a role in immunological defence mechanisms against the host suggesting a potential value of DvPmy as a vaccine candidate against Dictyocaulosis. The selection of DvPmy as a vaccine candidate is supported by a study of Kiel et al. [33], in which paramyosin from the closely related sheep gastrointestinal nematode Trichostrongylus colubriformis was identified as an immuno-reactive protein. And indeed, pilot studies to determine the protective potential of recombinant paramyosin against the bovine lungworm D. viviparus show promising results (unpublished) but have to be confirmed in further vaccine trials which are underway.
Acknowledgements The monoclonal paramyosin antibody 5–23 developed by Henry Epstein was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242.
References Fig. 4. Amplification efficiency corrected normalized paramyosin transcription throughout the D. viviparus life cycle.
elements can directly react with the specific antigens thereby interfering with the function and growth of the muscular system with the result of paralysis [30,31]. In this context it is remarkable that the analysis of evolutionary relationships defines DvPmy or rather nematode paramyosin in general, to be more similar to that of arthropods than to that of cestodes and trematodes. Such an antibody passage through barrier epithelia to pharyngeal and body wall muscles might explain the auspicious worm reduction in our vaccine experiments against D. viviparus. Transcriptional analysis of bovine lungworm paramyosin revealed that the highest transcript levels are present in L1. This was expected as the first larval stage is very active because it has to move away from the faeces. Interestingly, the second highest paramyosin transcription was observed in eggs. Within the eggs the parasite undergoes a first developmental differentiation from blastomeres to L1. In consequence, the developing embryos transcribe paramyosin for muscle formation and already developed L1 need this muscle protein for hatching and further movement. The lowest transcript levels were measured in hypobiosis induced L3i which remain curled up without any movement or further development. An increase of paramyosin transcription is detectable in the preadult L5 stages representing a growing and most likely moving stage. Noteworthy, there is a higher transcription rate in preadult and adult females compared to male stages. One explanation might be the larger body size of female individuals requiring more muscle mass. As another reason, at least for adult worms, an additional need for muscles for reproduction may be hypothesized. Adult males need their sex muscles only for copulation. In contrast, female worms require continuous activity of the uterine and vulval muscles for egg-laying. Furthermore, it can be assumed that even ovulation depends on contraction of myoepithelial sheath cells and thus on paramyosin as shown for C. elegans [32]. It can be largely excluded that the higher paramyosin transcript levels in females relate to developing eggs because these were removed by bursting adult females in double distilled water. In conclusion, the results of the present study show that paramyosin of the bovine lungworm is transcribed across all develop-
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