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Division of Health Sciences, School of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, WA 6150, Australia
Correspondence
David J. Hampson
d.hampson{at}murdoch.edu.au
Received 13 October 2006
Accepted 16 March 2007
67 kDa protein in whole-cell preparations of WesB. Sera from mice vaccinated with formalin-treated whole-cell proteins of WesB reacted with the recombinant protein. These results indicate that ClpX is both conserved and immunogenic and hence might be useful as a subunit vaccine component for Brachyspira spp. infections. Sera from humans with no known exposure to B. pilosicoli reacted with the recombinant ClpX protein, indicating that it is unlikely to be useful as a reagent for serological detection of Brachyspira spp. infections.  |
INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONREFERENCES |
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Intestinal spirochaetosis is characterized by the end-on attachment of large numbers of spirochaetal cells to the luminal surface of the large intestine epithelium, to form a ‘false brush border’ (Harland & Lee, 1967; Mikosza & Hampson, 2001). The basis of the observed attachment is unknown, but it has been suggested that spirochaetal outer-membrane proteins may have an important role in this interaction (Trott et al., 2001). To date, relatively few outer-membrane proteins of Brachyspira pilosicoli or other Brachyspira species have been characterized. Recently, Trott et al. (2003) identified a membrane-bound 61 kDa clip protease (ClpX) homologue in porcine Brachyspira pilosicoli strain 95/1000 by screening a Brachyspira pilosicoli genome library using a monoclonal antibody generated against a Brachyspira pilosicoli membrane vesicle preparation. They described the ClpX protein as an inner-membrane protein of Brachyspira pilosicoli but with a low-density presentation in the outer membrane that accounted for the reactivity of the protein with the monoclonal antibody. ClpX ATPase is a member of a group of molecular chaperones involved in the conformational integrity of proteins under conditions of normal growth as well as under stress (Gottesman et al., 1993; Skinner & Trempy, 2001).
In the current study, the distribution of the clpX gene amongst a large collection of Brachyspira spp. strains was determined by PCR analysis and the whole gene was sequenced in 20 strains of Brachyspira species. The clpX gene was expressed in Escherichia coli and the purified recombinant protein was used to immunize mice and to evaluate its reactivity with sera from mice and humans. The long-term aim of the study was to investigate recombinant ClpX as a potential subunit vaccine component for Brachyspira infections and/or as a reagent for Brachyspira-specific serological assays.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Spirochaetal reference strains and culture conditions. A total of 101 Brachyspira species strains were used, comprising 35 strains of Brachyspira pilosicoli (from humans, pigs, dogs and chickens), 24 strains of Brachyspira hyodysenteriae, 17 strains of Brachyspira innocens, 16 strains of Brachyspira intermedia, six strains of Brachyspira murdochii, two strains of Brachyspira aalborgi and one strain of Brachyspira alvinipulli. The strains were obtained as frozen stocks from the culture collection held at the Reference Centre for Intestinal Spirochaetes, Murdoch University, Australia. The cells were thawed and plated onto trypticase soy agar (BBL) containing 5 % (v/v) defibrinated ovine blood. The plates were incubated for 7–10 days at 37 °C in anaerobic conditions generated using anaerobic Gaspak Plus sachets (BBL). The purity of each culture was examined by phase-contrast microscopy and pure cells were propagated in 10 ml and then 100 ml Kunkle's pre-reduced anaerobic broth, containing 2 % (v/v) fetal bovine serum and 1 % (v/v) ethanolic cholesterol solution (Kunkle et al., 1986).
Preparation of genomic DNA from Brachyspira spp. strains. Approximately 2x109 cells from mid-exponential phase spirochaetal growth were used to prepare chromosomal DNA using a DNeasy tissue kit according to the manufacturer's instructions (Qiagen). RNA was removed by adding 20 µl RNase A (100 mg ml–1) to each sample, followed by incubation at room temperature for 2 min. DNA concentrations were calculated by measuring the A260 of the solution in a Lambda 25 UV/Vis spectrometer using UV WINLAB software (PerkinElmer).
Primer design and specification.
Five sets of primers (one set for gene distribution analysis, three sets for sequencing and one set for cloning) were designed to be complementary to the internal and external regions of the clpX gene (Table 1
). The oligonucleotides were designed using PRIMER DESIGNER software (Education and Scientific Software), SEQED version 1.0.3 (Applied Biosystems) and the AMPLIFY program version 1.2 (University of Wisconsin). Primers were purchased from GeneWorks.
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Thermocycling conditions consisted of an initial template denaturation for 5 min at 94 °C, followed by 30–35 cycles of denaturation at 94 °C for 30 s, annealing at 57 °C for 30 s and primer extension at 72 °C for 1–1.5 min. The final cycle had the extension time increased to 7 min to complete synthesis of all strands. Amplified products were separated by electrophoresis in 1.2 % (w/v) agarose in 1x TAE buffer [40 mM Tris/acetate (pH 8.0), 1 mM EDTA], stained by immersion in a 1 µg ethidium bromide ml–1 solution and viewed over UV light.
Distribution analysis.
PCR was used to amplify the 878 bp internal portion of the clpX gene between nt 369 and 1246 from all 101 Brachyspira species strains using primer pair CLPX.369.DF1 and CLPX.1246.DR1 (Table 1
).
Sequencing reactions.
The full clpX gene was sequenced from 12 strains of Brachyspira pilosicoli, three strains of Brachyspira innocens, two strains each of Brachyspira hyodysenteriae and of Brachyspira intermedia and one strain of Brachyspira aalborgi. Initially, an external primer set, CLPX.–140.SF1 and CLPX.+63.SR3 (Table 1), amplifying 1863 bp that included the coding region of clpX, was applied to chromosomal DNA from 20 Brachyspira spp. strains in 50 µl PCRs. The PCR products were purified using an UltraClean PCR Clean-up kit (Mo Bio Laboratories). The purified PCR products then were sequenced using the three sequencing primer sets (Table 1) using an ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Mix (PE Applied Biosystems) and an ABI 373A DNA sequencer (PE Applied Biosystems), according to the manufacturer's specifications.
Phylogenetic analysis. Sequence data were edited using SEQED version 1.0.3 (PE Applied Biosystems) and compared with the sequence of the clpX gene of porcine Brachyspira pilosicoli strain 95/1000 strain (GenBank accession no. AY466377). The nucleotide and translated protein sequences were aligned using CLUSTALX and CLUSTALW multiple alignment software to obtain a similarity matrix of the 20 different strains of Brachyspira spp. A neighbour-joining phylogenetic tree based on a distance matrix was drawn using the BIOEDIT Sequence Alignment Editor (North Carolina State University) and MEGA 3.1 (Kumar et al., 2004).
In silico analysis.
The clpX gene was analysed using BLASTP (Altschul et al., 1997), reverse-position-specific BLAST (RPS-BLAST; Marchler-Bauer et al., 2002) and CDART (Marchler-Bauer et al., 2005) to find protein domain similarities and functional architecture. SIGNALP (EMBOSS) (Bendtsen et al., 2004), PSORTB (Gardy et al., 2005), TMPRED (Hofmann & Stoffel, 1993), PENCE (http://www.cs.ualberta.ca/bioinfo/PA/Subcellular) and CELLO (http:/cello.life.nctu.edu.tw) were used to predict subcellular localization. The EMBOSS programs IEP and GEECEE also were used to identify the isoelectric point and the molecular mass of the ClpX protein and to calculate the GC content of the clpX gene.
Preparation of recombinant histidine-tagged ClpX.
The entire clpX gene encoding 595 aa was amplified from the genomic DNA of Brachyspira pilosicoli strain WesB using primers CLPX.4.CF1 and CLPX.1785.CR1 (Table 1) using the optimized conditions described above. The amplified product was restricted with EcoRI and BamHI (New England Biolabs) in a double digest according to the manufacturer's instructions. The restricted insert DNA was purified using an UltraClean PCR Clean-up kit and cloned into an Xpress Protein Expression System (Invitrogen) according to the manufacturer's instructions. Recombinant histidine (His6)-tagged ClpX protein was expressed in E. coli BL21 Star (DE3)pLys (Invitrogen) in 2x YT medium supplemented with 100 µg ampicillin ml–1, 1 mM IPTG and 1 % (w/v) glucose. Affinity chromatography using a Ni-NTA column (Qiagen) was applied to purify the recombinant His6–ClpX protein under denaturing conditions according to the manufacturer's instructions. The purified protein was dialysed against distilled water to remove denaturing agent and salts. The dialysed protein was then lyophilized and resuspended in PBS. Quantification of recombinant His6–ClpX protein was carried out by electrophoresis of serial dilutions of the protein and BSA and lysozyme standards (100, 250, 500 and 1000 ng) on an SDS-polyacrylamide gel and capturing the gel image using a densitometer (proxPRESS Proteomic Imaging System; PerkinElmer Life Sciences). The image was analysed using the PROTEOME 1D ANALYSER version 1.10 (PerkinElmer Life Sciences) to calculate protein concentration.
Preparation of mouse polyclonal antiserum against Brachyspira pilosicoli strain WesB. A whole-cell bacterin was prepared from Brachyspira pilosicoli strain WesB as described previously (Hampson et al., 2000). A total of 108 formalin-treated WesB cells were emulsified in an equal volume of Freund's incomplete adjuvant (Sigma) in a total volume of 100 µl and administered subcutaneously into five 5-week-old male C3H/HeJ mice (Western Australian State Animal Resource Centre) three times at 2-week intervals. Three weeks after the last inoculation, the mice were euthanized by methoxyflurane inhalation, followed by cervical dislocation. Blood was collected by cardiac puncture and the serum was separated and stored at –20 °C until used in Western blot analysis.
Immunization of mice using recombinant His6–ClpX protein. Ten C3H/HeJ male mice of 5 weeks of age were obtained from the Western Australian State Animal Resource Centre and housed in two groups each comprising five mice. The five mice in the vaccinated group were injected subcutaneously twice with a 2-week interval with 100 µg His6–ClpX protein emulsified in an equal volume of Freund's incomplete adjuvant in a total volume of 100 µl. The second group of five mice was left unvaccinated. Three weeks after the last inoculation, all of the mice were euthanized and the blood was collected as described above. Aliquots of 5 µl of the sera from the vaccinated mice were adsorbed by mixing with 5 µl recombinant His6–ClpX protein at 300 ng µl–1 in PBS and incubating at 37 °C on a rotary mixer for 1 h.
Western blot analysis using sera from immunized mice. The mouse sera were analysed by Western blot analysis against both whole-cell protein preparations of Brachyspira pilosicoli and the recombinant His6–ClpX protein. For preparation of whole-cell proteins, cells of Brachyspira pilosicoli strain WesB were suspended in PBS at a concentration of 108 cells ml–1 and sonicated on ice for three cycles of 30 s with a 2 min rest between each cycle. The sonicate was centrifuged at 10 000 g for 10 min and the supernatant was separated for a blot assay (Hampson et al., 2000). The total protein concentration was quantified using a Bio-Rad protein assay kit, according to the manufacturer's instructions. Ten micrograms of cell protein was separated by 12.5 % (w/v) denaturating SDS-PAGE in a Tris/glycine buffer system. The loaded protein was then transferred electrophoretically to a 0.2 µm nitrocellulose membrane. The membrane was blocked with TBS-T (Tris-buffered saline supplemented with 0.05 %, v/v, Tween 20) containing 5 % (w/v) skimmed milk powder for 1 h, followed by three washings with TBS-T. The membrane was assembled into a multi-screen apparatus (Bio-Rad) for multiple serum analysis. Mouse sera were diluted 100-fold in 100 µl TBS-T or 50-fold for the adsorbed sera, added to the membrane through the wells of the multi-screen probe and incubated for 1 h at room temperature with gentle mixing. The membrane was reacted with a 1 : 2500 dilution of goat anti-mouse IgG–alkaline phosphatase conjugate (Sigma) for 1 h at room temperature, followed by three washes with TBS. Immunodetection of proteins was carried out using 10 ml developing buffer [100 mM Tris/HCl (pH 9.5), 100 mM NaCl, 5 mM MgCl2] containing 0.33 mg nitro blue tetrazolium chloride (Sigma) ml–1 and 0.165 mg 5-bromo-4-chloro-3-indolyl phosphate (Sigma) ml–1. For Western blotting using recombinant His6–ClpX protein, 10 µg of the recombinant protein was used. All other procedures were the same as described above.
Western blot immunoreactivity of His6–ClpX protein with human sera. Human sera were also reacted in Western blots with recombinant His6–ClpX protein as described for mouse sera, except that anti-human IgG–alkaline phosphate conjugate was used.
The 33 human sera tested were obtained as part of a previous study (Brook et al., 2001); they comprised 12 samples from migrant individuals from developing countries who, at the time of sampling, were colonized by Brachyspira pilosicoli as determined by faecal culture and PCR, five samples from migrant individuals who were negative for Brachyspira pilosicoli and 16 samples from healthy, culture-negative individuals from Perth, Western Australia.
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Comparative analysis of the clpX gene and protein
The clpX gene in the 20 strains in which it was sequenced was highly conserved, sharing 99.3–99.7 % similarity at the nucleotide level and 99.7–100 % at the amino acid level. The nucleotide sequence for clpX in Brachyspira pilosicoli strain 95/1000 was identical to those in Brachyspira intermedia strains OR2 and OF11, whilst the peptide sequences of clpX in nine strains including Brachyspira pilosicoli strains ‘S. jonesii’, WesB and Q98.0026, Brachyspira innocens strains B256T, Q91-12233 and Q91-1530-1, Brachyspira intermedia strains OR2 and OF11 and Brachyspira hyodysenteriae strain 155.11 were identical to those in Brachyspira pilosicoli strain 95/1000. The other strains had one or two amino acid differences (Table 2) and these occurred throughout the molecule. Five strains had a substitution of asparagine for serine at position 154. The general conservation of the protein across the different strains and species again is advantageous for subunit vaccine development.
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. The sequences started at the ATG start codon and ended at the stop codon. The genes from six of the 12 strains (50 %) of Brachyspira pilosicoli were located in cluster IV, whilst the genes from the others, including 95/1000, were found in the other three clusters, together with those from other Brachyspira spp. The general imperfect match of the dendrogram with the accepted phylogeny of the Brachyspira spp. may have been associated with recent mutational changes in clpX or with horizontal gene transfer and genetic recombination between the species. There was no consistent clustering of Brachyspira pilosicoli strains according to the host species of origin, consistent with strains being able to cross species boundaries and potentially being zoonotic (Hampson et al., 2006b).
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200 amino acids at the N-terminal end of the protein compared with the ClpX protein in these other species and, consequently, the similarities were all in the C-terminal end of the molecule. The only similarity found at the N-terminal end was to the ATP-dependent zinc metallopeptidase of Bacillus halodurans, which had about 8 % amino acid similarity with the N-terminal protein sequence from the ClpX of Brachyspira pilosicoli strain 95/1000. RPS-BLAST analysis identified a 409 aa domain (residues 175–583) that was similar to the ATP-dependent Clp protease ATP-binding subunit clpX of Bacillus halodurans (GenBank accession no. Q9K8F4). The conserved domain architecture retrieval tool (CDART) identified a 105 aa region (residues 274–378) of the ClpX polypeptide that showed functional and architectural similarity to the Ruvb domain of the Thermus thermophilus Hb8 protein (GenBank accession no. 1HQCA).
Results from SIGNALP showed that the Brachyspira ClpX had no signal peptide in the 70 aa of its N-terminal sequence. PSORT predicted ClpX as a bacterial inner-membrane protein, whilst PSORT-B recognized it as a protein with unknown localization in the cell. PENCE and CELLO recognized ClpX as a cytoplasmic protein with high prediction scores. The TMPRED software predicted a possible 24-residue transmembrane helix at aa 396–419. Searches with IEP and GEECEE predicted a molecular mass for ClpX of 67.42 kDa, a pI of 6.74 and a GC content of 28.06 mol%.
These in silico results generally agreed with those of Trott et al. (2003). As the Brachyspira ClpX protein had no signal peptide or obvious transmembrane region, it is likely to be cytoplasmic, but may be associated with the inner membrane. An inner-membrane and possible outer-membrane localization was suggested by Trott et al. (2003), based on partitioning of the molecule in different cell fractions. The general conservation of amino acid sequence at the N-terminal end emphasizes the unique nature of the ClpX proteins in Brachyspira spp., but no clues were found as to the likely function of the unusual N-terminus.
Expression and immunogenicity of the His6–ClpX protein in mice
The native ClpX protein had a predicted molecular mass of 67.42 kDa according to its amino acid sequence and an apparent molecular mass of 71.5 kDa as a His-tagged protein (Fig. 2). In Western blot analysis, the recombinant His6–ClpX protein reacted strongly with mouse sera raised against whole-cell proteins of Brachyspira pilosicoli strain WesB (Fig. 2). Likewise, sera from the mice vaccinated with the recombinant His6–ClpX protein reacted strongly with a 67 kDa band in the whole-cell preparations of Brachyspira pilosicoli strain WesB (Fig. 3). Sera absorbed with recombinant His6–ClpX protein no longer reacted with this band (data not shown). These results are encouraging for future development of Brachyspira ClpX as a vaccine subunit, as they demonstrate that native ClpX stimulates an immune reaction and that the recombinant His-tagged ClpX is immunogenic. Further work is required to evaluate whether vaccination with recombinant ClpX can provide protection against Brachyspira spp. infections.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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