1 Department of Biochemistry and Microbiology, University of Victoria, PO Box 3055, Victoria, BC, Canada V8W 3P6
2 Microtek Research and Development Ltd, 6761 Kirkpatrick Crescent, Saanichton, BC, Canada V8M 1Z8
Correspondence
William W. Kay
wkay{at}uvic.ca
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AY445520.
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INTRODUCTION |
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The aim of this study was to identify host-recognized protein antigens from F. psychrophilum as potential vaccine candidates. Here we describe the use of convalescent sera from the host, rainbow trout, and proteomics technologies to identify a 20 kDa protein antigen, FspA. We report the expression of this 20 kDa antigen as a fusion protein in Escherichia coli. Further investigation is required to assess whether FspA will elicit a protective response in rainbow trout, as part of a recombinant vaccine against F. psychrophilum.
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METHODS |
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Generation of antisera.
Rainbow trout convalescent serum was obtained from rainbow trout that survived challenge with F. psychrophilum (259-93). Rainbow trout (15 g) were injected with live F. psychrophilum (2·4x104 cells) from a 24 h MAT broth culture. Pooled rainbow trout serum from 45 surviving fish was obtained 5 weeks post-challenge. Sera were also obtained from unexposed fish injected with saline for use as a negative control.
SDS-PAGE.
Protein analyses of whole-cell lysates and inclusion body samples were carried out by SDS-PAGE according to the method of Laemmli (1970) as modified by Ames (1974)
. Samples were resolved in 12 % polyacrylamide separating gels with 5 % stacking gels. Molecular mass was estimated according to the apparent molecular mass of prestained protein markers. SDS-PAGE gels were washed for 15 min in distilled H2O and stained with GelCode (Pierce), a Coomassie G-250 based stain.
Immunoblotting.
Bacterial cell proteins separated by SDS-PAGE were transferred onto nitrocellulose membranes by electroblotting at 50 mA per gel for 1 h in a semidry transblot apparatus (LKB Multiphor II, Pharmacia) as described by Towbin et al. (1979). The membrane-immobilized immunogenic proteins were detected using rainbow trout serum raised against F. psychrophilum. Primary fish antibody (1/40 dilution) was incubated with the membrane overnight at 4 °C. The primary fish antibody was detected by rabbit anti-salmon Ig polyclonal antibody (Immuno-Precise Antibodies) followed by a goat anti-rabbit IgG (1/4000 dilution) conjugated to alkaline phosphatase (Caltag Labs) as outlined previously (Collinson et al., 1991
). Negative controls were carried out using naïve sera as the primary antibody. The immunoreactive bands were visualized using 5-bromo-4-chloro-3-indoyl phosphate and 4-nitro blue tetrazolium chloride (Sigma) as previously described (Müller et al., 1989
).
Triton X-114 phase partitioning of F. psychrophilum.
Phase partitioning of F. psychrophilum cells was carried out by the method of Cunningham et al. (1988) with the following modifications. Wet cell pellets were suspended in Triton X-114 (Sigma) solubilization buffer (1 % Triton X-114/10 mM Tris pH 8/5mM EDTA pH 8) to 20 mg ml1. The hydrophobic fraction was solubilized by rotating the cell suspension in a tube at 4 °C for 23 h. The suspension was centrifuged at 4 °C (5 min, 12 100 g) and the clear supernatant (detergent-soluble fraction) transferred to a new tube, leaving behind the insoluble cold pellet. The suspension was then incubated at 37 °C for 15 min to precipitate the detergent and the phases separated by centrifugation at room temperature (15 min, 12 100 g). This resulted in a top aqueous phase, a lower detergent phase and a translucent warm pellet. The aqueous and detergent phases were transferred to new tubes and washed three times each as follows: to the aqueous phase, Triton X-114 was added to a final concentration of 1 % and to the detergent phase, an equal volume of TE (10 mM Tris/5 mM EDTA pH 8) was added. Both Triton X-114 suspensions were dissolved by incubating on ice for 10 min and incubated at 37 °C for 10 min for phase partitioning prior to centrifugation at room temperature (15 min, 12 100 g) to separate the phases. The washed detergent and aqueous phases were transferred to new tubes. Acetone precipitations were performed to remove detergent from the Triton phase and to concentrate proteins in the aqueous phase: ten volumes of cold acetone was added to the sample, which was mixed by vortexing briefly and incubated at 20 °C overnight. The precipitated protein mixture was centrifuged (17 400 g, 15 min, 4 °C) and the protein pellet air-dried.
Two dimensional (2D) gel electrophoresis.
High-resolution 2D SDS-PAGE was performed using the ISO-DALT multiple 2D system (Anderson & Anderson, 1978a, b
). Protein samples were solubilized in 30 µl urea mix (9 M urea, 4 %, v/v, NP-40, 2 %, v/v, Pharmalyte 310 ampholines, 2 %, w/v, DTT) and loaded onto pre-focused tube gels containing pH range 3-10 ampholines (Pharmalyte 310, Amersham Pharmacia). First-dimension IEF was conducted at 800 V for 18 h (14 400 V h). Following electrophoresis, the tube gels were equilibrated for 15 min at room temperature in equilibration buffer and immediately mounted onto 1016·5 % gradient SDS-PAGE slab gels with the acidic end positioned to the left. Electrophoresis was performed at 4 °C at 1 A, until the dye front was about 1 cm from the bottom of the gel (
5 h). Alternatively, mini-2D gels were run using the Mini-Protean II tube cell module (Bio-Rad) as follows. Capillary IEF tube gels were pre-focused by running at 200 V for 10 min, 300 V for 15 min and 400 V for a further 15 min. Tube gels were run at 500 V for 10 min followed by 750 V for 3·5 h. Mini-slab gels were run at 10 mA through the stacking gel (5 %) and 20 mA through the separating gel (12 %) until the dye front reached the bottom of the gel (
90 min). After electrophoresis, gels were fixed and stained with colloidal Coomassie brilliant blue G-250 or electroblotted onto nitrocellulose membranes for immunoblot analysis.
Staining of 2D gels with Coomassie brilliant blue G-250.
Gels were agitated gently in fixative (50 %, v/v, ethanol, 3 %, v/v, orthophosphoric acid) for 14 days at room temperature, washed 3x30 min in distilled H2O and allowed to equilibrate in Neuhoff's solution (16 %, w/v, ammonium sulphate, 25 %, v/v, methanol, 5 %, v/v, orthophosphoric acid; Neuhoff et al., 1988) for 1 h with gentle agitation. Coomassie brilliant blue G-250 (1 g) (EM Science) was added to the Neuhoff's solution and staining continued for 35 days. Digital images of Coomassie-stained gels were captured by scanning at 300 d.p.i. using a colour scanner (UMAX Astra 3400). Protein spots of interest were then cored from the gel for further analysis.
Reduction, alkylation and tryptic digests of 2D gel spots.
Protein spots were excised from 2D gels using a 4 mm plastic straw and either transferred to 1·5 ml microcentrifuge tubes (previously autoclaved and rinsed with 50 % methanol to remove any contaminants) for digestion or to 96-well sterile tissue culture plates (one spot per well in 10 µl 20 %, w/v, ammonium sulphate; Neuhoff et al., 1988) for storage at 20 °C. For analysis by mass spectrometry, 2D gel protein spots were destained (50 %, v/v, methanol/5 %, v/v, acetic acid), reduced with 10 mM dithiothreitol and alkylated with 100 mM iodoacetamide as described by Kinter & Sherman (2000)
. The carboxyamidomethylated protein spots were digested overnight at 37 °C with 20 ng ml1 modified, sequence-grade, porcine trypsin according to the manufacturer's directions (Promega). Peptides were extracted from the gel pieces using a series of elutions with 50 % (v/v) acetonitrile/5 % (v/v) formic acid. The resulting pooled eluates were each reduced to a final volume of 20 µl in a Speed Vac Concentrator (Savant) and processed for mass spectrometry.
Nanospray tandem mass spectrometry (MS/MS) and peptide sequencing.
Tryptic peptides were desalted using glass capillary needles (Protana) packed with C18 resin (Perceptive POROS R2, 50 µm bead) and were extracted into sample needles using 1·0 µl 50 % (v/v) methanol/1 % (v/v) formic acid. Nanospray electrospray ionization was used to introduce ions into a PE-SCIEX Q-STRi quadrupole time-of-flight mass spectrometer (Q-TOF) (Applied Biosystems). Data were managed with Bioanalyst Software (PE-SCIEX) and peptides sequenced by following Kinter's nine-step strategy for interpretation of product ion spectra (Kinter & Sherman, 2000). Peptide fragmentation data searching was performed using the Mascot MS/MS Ions Search algorithm (Matrix Science; http://www.matrixscience.com/).
Determination of the fspA gene sequence.
Based on peptide sequences obtained from MS analysis, pairs of degenerate oligonucleotide primers were designed to amplify a segment of the fspA gene. Use of the following primers amplified a gene segment: AAY GTD GCH GGW ACH GTD GG (NVAGTVG forward, 324-fold degeneracy); GCN GCN AAY GAY TTY GA (AANDFE reverse, 128-fold degeneracy). Each 50 µl reaction contained the following: 20 mM Tris/HCl, pH 8·4; 50 mM KCl; 1·5 mM MgCl2; 0·2 mM deoxyribonucleoside; 50 pmol of each degenerate primer; 1 U Taq DNA polymerase (Roche); 500 ng template DNA. Amplification was performed as follows: 2 min at 94 °C; 30 cycles of 30 s at 94 °C, 30 s at 52 °C, and 1 min at 73 °C; followed by 5 min at 72 °C.
Uneven PCR was carried out to amplify the unknown 5' end of the fspA gene, according to the method of Chen & Wu (1997) with minor modifications. Each 50 µl reaction contained the following: 20 mM Tris/HCl, pH 8·4; 50 mM KCl; 1·5 mM MgCl2; 0·2 mM deoxyribonucleoside; 12·5 pmol specific primer; 2·5 pmol decamer random primer; 1 U Taq DNA polymerase (Roche). The template in the first set of cycles (round 1) was 50 ng genomic DNA, amplified with primer 1 (GCT ACC TTC AAT ACC TAC TGT CAT G) and the random primer GTT TCG CTC C. One microlitre of the final amplified reaction mix from round 1 was used as the template for round 2 of reactions, using a nested primer (primer 2, GCC AAC CAA CTT TTG GAG AAA AAT CGA AAC C) and the same random primer. The cycling reactions were performed as described by Chen & Wu (1997)
with changes in the specific primer annealing temperatures. Annealing temperatures in round 1 were cycled between 48/50 °C and 42/45 °C. Round 2 annealing temperatures tested were 54 °C and 45 °C. PCR products were cloned into pGEM-T Easy (Promega) according to the manufacturer's instructions and sequenced.
Automated DNA sequencing and sequence analysis.
Plasmids submitted for DNA sequencing were purified using a Qiaprep Spin MiniPrep Kit (Qiagen) according to the manufacturer's instructions. Pure double-stranded plasmids were submitted to the Centre for Biomedical Research, University of Victoria, Victoria, BC, Canada, and automated dideoxynucleotide sequencing was conducted using a NEN Global IR2 DNA Sequencer (LI-COR) using dye-labelled primers. DNA traces were visually examined for errors and ambiguous regions, and aligned using ContigExpress from Vector NTi Suite 7 (InforMax) for derivation of the final consensus sequence. Coding predictions were made to identify ORFs using the National Center for Biotechnology Information (NCBI) program ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Sequences were subjected to BLAST 2 (Altschul et al., 1997) and FASTA 3 (Pearson, 1998
) analysis to determine if any similar sequences were known. Multiple sequence alignments were performed using AlignX from Vector NTi Suite 7 (InforMax). Translated DNA sequences were analysed using software to predict N-terminal signal sequences (iPSORT; Bannai et al., 2002
) and signal peptide cleavage sites (SignalP; Nielsen et al., 1997
).
PCR amplification of fspA'.
Primers used to amplify the 537 bp fspA gene segment (named fspA') and insert restriction sites for cloning purposes were as follows: forward, CGA CGG ATC CGT CTC ATA TGA ACG TGG CAG GTA CAG TGG GTT TTA ACT C; reverse, TCC GAA GCT TAA TTA ATG AAA TTT TTA GAG ATA CCA ACG CTT ACT TGT TTT CCG. The forward primer encoded BamHI and NdeI resriction sites (underlined). The reverse primer encoded HindIII and VspI restriction sites (underlined). Each 50 µl reaction contained the following: 20 mM Tris/HCl, pH 8·4; 50 mM KCl; 1·5 mM MgCl2; 0·2 mM deoxyribonucleoside; 20 pmol primer 1 U Taq DNA polymerase (Roche); and 50 ng template DNA. Amplification was performed as follows: 1 cycle at 94 °C for 2 min, 30 cycles of 94 °C for 30 s, 54 °C for 30 s and 72 °C for 30 s followed by 1 cycle at 72 °C for 5 min.
Creation of fusion construct CFspA' and protein expression.
fspA' (partial sequence, 537 bp encoding 179 aa) was amplified by PCR, digested with EcoRI and BamHI and cloned into the expression vector pETC, a pET21a(+) (Novagen) derived expression vector encoding a 10 kDa, non-specific N-terminal fusion protein, protein C (Microtek International). C-protein fusions readily form inclusion bodies to aid isolation of the expressed product. Induction experiments were carried out in E. coli BL21(DE3). Overnight 37 °C cultures in LBAp or TBAp were used to inoculate fresh LBAp medium. Cultures were grown at 37 °C to an OD600 of 0·60·8, at which point they were induced by the addition of 1 mM IPTG for 2 h. Inclusion bodies were isolated from cells by sonication as follows: an induced culture was centrifuged at 10 000 g for 10 min and resuspended in distilled H2O; the cell suspension was sonicated on ice for 10x15 s using an ultrasonic processor (model W-385, Heat Systems Ultrasonics) and centrifuged (10 000 g, 15 min); pellets were resuspended in distilled H2O and the procedure repeated. The final inclusion body samples were resuspended in distilled H2O and stored at 20 °C.
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RESULTS |
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Identification of the fspA gene sequence by degenerate PCR
To identify the gene sequence encoding FspA, degenerate PCR (dPCR) primers were designed from peptide sequences to amplify a portion of the gene. A reproducible 852 bp product was amplified by dPCR, cloned into pGEM-T Easy and electroporated into E. coli XL-1 Blue. Transformants were selected by blue/white screening and ampicillin resistance. Three positive clones were confirmed by restriction digest analysis and sequenced. The consensus sequence of the dPCR product was translated into amino acid sequence and searched for the presence of the peptide sequences obtained from MS analysis of FspA. All the peptide sequences (Table 1) were found within the translated dPCR product (Fig. 3
), thus confirming that the partial gene sequence obtained by dPCR was derived from the protein of interest excised from the 2D gel. Analysis of the translated sequence revealed 10 stop codons in the C-terminal portion, starting at position 537 bp (179 aa) (Fig. 3
). All of the peptides sequenced (Table 1
) were found upstream of the first stop codon, including the peptide used to design the antisense primer (AANDE). Closer inspection of the dPCR sequence showed that the 3' end was compatible with the degenerate, sense strand primer at the 5' end. Fortunately, another form of the sense primer, which had 324-fold degeneracy, performed as an anti-sense primer, and resulted in sequencing beyond the C-terminus of the protein. The partial protein sequence obtained, from position 1 to the first stop codon (537 bp, 179 aa), was found to have a theoretical mass of 18·8 kDa and pI of 8·70, which was in close enough agreement with the observed mass and pI from 2D gel electrophoresis (
20 kDa, pI >7), and suggested that the vast majority of the gene sequence had been obtained by dPCR.
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The FspA sequence is specific to Flavobacterium species
Database searching for similar sequences (performed March 2005) resulted in only two significant matches, which were hypothetical proteins from Flavobacterium johnsoniae (Fig. 4). The two neighbouring F. johnsoniae genes were 72 % identical and encoded hypothetical proteins of 193 aa and 194 aa that shared 66 % identity, and for the purpose of this study were arbitrarily named Fj1 and Fj2 respectively. At the protein level, FspA was found to be 53 % and 56 % similar to Fj1 and Fj2 respectively, and 43 % identical. Alignment of the translated amino acid sequences of the hypothetical proteins and FspA showed overall 81 % similarity and 36 % homology between the three sequences (Fig. 4
). All three Flavobacterium sequences were found to contain a putative N-terminal signal sequence cleavage site (ANA
QK) that occurred at position 18 in Fj1 and Fj2, and at position 19 in FspA.
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DISCUSSION |
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The 20 kDa protein antigen partitioned into the detergent phase following extraction with Triton X-114, indicating that the protein is, at least in part, hydrophobic and most likely membrane associated; although the gene sequence (predicted 36 % hydrophobic amino acids) suggests the protein is hydrophobic as well, but not strongly. In 2D gel electrophoresis, the
20 kDa antigen tended to smear across the basic region of the second-dimension slab gel. The poor resolution of basic proteins in 2D gel electrophoresis is a well-known artifact, and can be indicative of partial insolubility, over/underfocussing or high protein load (Berkelman & Stenstedt, 1998
). Insolubility of basic proteins and their poor resolution can be caused by migration of the reducing agent (dithiothreitol) towards the anode during IEF, resulting in a depletion of reducing agent at the cathode (Pennington et al., 2004
). Despite the poor resolution, we were able to identify the protein by Q-TOF MS analysis, and showed that the samples taken from both the smear and the resolved spot were of the same protein.
The results obtained from dPCR analysis were initially surprising, representing an apparent gene fragment somewhat larger than the expected size for the observed protein. However, these results would be expected were one of the degenerate primers acting as both a sense and an anti-sense primer. This fortunate match led to the sequencing of the protein's C-terminus, leaving only the N-terminal sequence unknown, which was later obtained by uneven PCR.
Database searching with the translated 203 aa FspA sequence resulted in only two significant matches. The two similar sequences found were hypothetical proteins from a closely related fish pathogen F. johnsoniae. The two predicted proteins were 193 and 194 aa in length and are encoded by consecutive genes on the F. johnsoniae chromosome (Agarwal et al., 1997). Database searching revealed that the 194 aa F. johnsoniae protein is 27 % identical and 46 % similar to Ail from Yersinia pseudotuberculosis (Yang et al., 1996
), a 17 kDa outer-membrane protein that mediates adhesion to mammalian cells and contributes to serum resistance. It is therefore possible that the surface-localized FspA is an adhesin and an important pathogenesis factor of F. psychrophilum. A
18 kDa, immunogenic (rabbit) protein was shown to be readily released from F. psychrophilum with aqueous buffers at different pHs and by chelators and was speculated to be an S-layer protein (Massias et al., 2004
). While of similar molecular mass to FspA these proteins appear to differ in their solubilization properties. Two genes representing immunogenic (rabbit) membrane proteins of the related fish pathogen Flavobacterium columnare, a metalloprotease and an oligopeptidase, have also been characterized but are clearly different from FspA based on molecular mass (Xie et al., 2004
).
A 179 aa fragment of FspA could be abundantly expressed in E. coli as an N-terminal fusion protein, CFspA', and retained its immunoreactivity with convalescent serum. Not surprisingly, the antibody reaction with the native protein (Figs 1 and 2b) is apparently stronger than the reaction observed with the C-protein fusion (CFspA') (Fig. 5b
). This may be due to the removal of the N-terminal residues and conformational change following fusion with C-protein.
In summary, a 20 kDa (SDS-PAGE) F. psychrophilum protein recognized strongly by convalescent rainbow trout serum was characterized by 2D gel electrophoresis, immunochemistry, MS, dPCR and uneven PCR, as a 184 aa (19·3 kDa) surface-localized protein (FspA). A 179 aa portion of FspA was overexpressed in E. coli and retained immunoreactivity with convalescent rainbow trout serum. The FspA antigen of F. psychrophilum is currently the only well-characterized prospective subunit vaccine candidate against bacterial cold water disease (rainbow trout fry syndrome).
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ACKNOWLEDGEMENTS |
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Received 20 April 2005;
accepted 14 June 2005.
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