Journal of Medical Microbiology (2014), 63, 602–609
DOI 10.1099/jmm.0.060095-0
Characterization and antigenicity of recombinant
Campylobacter jejuni flagellar capping protein FliD
Hung-Yueh Yeh, Kelli L. Hiett, John E. Line and Bruce S. Seal
Correspondence
[email protected]
US Department of Agriculture, Agricultural Research Service, Richard B. Russell Agricultural
Research Center, Poultry Microbiological Safety Research Unit, 950 College Station Road,
Athens, GA 30605-2720, USA
Received 8 March 2013
Accepted 18 January 2014
Campylobacter jejuni, a flagellated, spiral-rod, Gram-negative bacterium, is the leading pathogen
of human acute bacterial gastroenteritis worldwide, and chickens are regarded as a major
reservoir of this micro-organism. Bacterial flagella, composed of more than 35 proteins, play
important roles in colonization and adhesion to the mucosal surface of chicken caeca. In this
study, the flagellar capping protein, FliD, encoded by the fliD gene, from the Campylobacter
jenuni D1-39 isolate was expressed and characterized, and its antigenicity determined. The fliD
gene comprised 1929 nt, potentially encoding a 642 aa peptide with a calculated molecular mass
of 69.6 kDa. This gene was PCR amplified and overexpressed in Escherichia coli. The
recombinant FliD protein was purified by cobalt-chelating affinity chromatography and confirmed
by nucleotide sequencing of the expression plasmid, SDS-PAGE analysis, His tag detection and
matrix-assisted laser desorption/ionization time of flight mass spectrometry. The immunoblot data
showed that the purified recombinant FliD protein reacted strongly to sera from broiler chickens
older than 4 weeks, indicating that anti-FliD antibody may be prevalent in the poultry population.
These results provide a rationale for further evaluation of the FliD protein as a vaccine candidate
for broiler chickens to improve food safety for poultry.
Hung-Yueh Yeh
INTRODUCTION
It is estimated that more than 400 million cases of human
campylobacteriosis occur worldwide (2 million cases in the
USA) (Scallan et al., 2011; WHO, 2009). The economic loss
caused by this disease has been estimated at US$1.7 billion
dollars annually in the USA (Hoffmann et al., 2012). The
major bacterial aetiological agent is Gram-negative Campylobacter jejuni, which is a commensal in the chicken
intestinal microbiota (Hermans et al., 2012b; European
Food Safety Authority, 2010). Risk assessment studies have
demonstrated that a reduction in the number of Campylobacter on poultry carcasses or shedding in poultry farms
results in a reduction of human campylobacteriosis cases
(Rosenquist et al., 2003; Messens et al., 2007). Therefore,
many strategies for reduction of this micro-organism in
broiler chickens have been investigated (e.g. Bahrndorff
et al., 2013; Lin, 2009; de Zoete et al., 2007; Hermans et al.,
2011, 2012a; Line et al., 2008; Robyn et al., 2013; Wagenaar
et al., 2006). One of these strategies is through vaccination,
and, prior to exposure, chicken anti-Campylobacter antibodies may reduce bacterial colonization and counts from
the chicken intestinal tract (Cawthraw & Newell, 2010;
Abbreviations: HRP, horseradish peroxidase; MALDI-TOF MS, matrixassisted laser desorption/ionization time of flight mass spectrometry.
The GenBank/EMBL/DDBJ accession number for the Campylobacter
jejuni fliD gene sequence determined in this study is KC618388.
602
Layton et al., 2011; Shoaf-Sweeney et al., 2008; Sahin et al.,
2003).
Because C. jejuni flagella have been studied extensively,
we first selected flagellar proteins as targets for potential
vaccine development. Bacterial flagella are organelles required for their movement towards or away from various
environments (Berg, 2003; Macnab, 2003). Like Escherichia
coli and Salmonella flagella, the flagellum of C. jejuni
consists of three distinct structural regions (composed
of more than 35 proteins): (i) the basal body anchored
in the cytoplasm and inner membrane; (ii) the curved
hook region extended from periplasmic space to the
outer surface; and (iii) extracellular filaments (reviewed
by Gilbreath et al., 2011; Lertsethtakarn et al., 2011).
The major components of the extracellular filaments are
synthesized as flagellin monomers, which require another
protein, called hook-associated protein 2 or flagellar capping
protein (encoded by the fliD gene), to cap the monomers
into the filament at the distal end (Blair & Dutcher, 1992;
Homma et al., 1984; Ikeda et al., 1987, 1993; Tasteyre et al.,
2001b). The flagellar capping protein has been implicated
as being involved with adherence and colonization of
Clostridium difficile and Helicobacter pylori in the intestinal
mucosa (Kim et al., 1999; Tasteyre et al., 2001a). However,
although the fliD gene is found in the C. jejuni genome
(Parkhill et al., 2000), the role of this gene product in C.
jejuni pathogenesis has not been investigated.
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060095
Printed in Great Britain
Recombinant Campylobacter jejuni FliD
In this report, we describe the expression and characterization of the fliD gene product of the C. jejuni D1-39
isolate. The deduced amino acid sequence of the FliD
protein (with a calculated molecular mass of 69.7 kDa)
from the nucleotide sequence was highly homologous to
the flagellar capping protein of C. jejuni. In addition, the
recombinant FliD protein was tested to see whether it was
antigenic using broiler chicken sera.
METHODS
Bacterial strains, culture conditions and genomic DNA isolation.
The C. jejuni D1-39 isolate from chicken faeces in Georgia, USA, was
propagated in Mueller–Hinton agar plates at 42 uC for 48 h in a
microaerobic environment (5 % O2, 10 % CO2 and 85 % N2) as described previously (Hiett et al., 2008). Competent E. coli for propagation
and expression of recombinant DNA were cultured according to the
manufacturer’s instructions (Lucigen). C. jejuni genomic DNA was
isolated using a DNeasy Blood & Tissue kit in a QIAcube automation
system (Qiagen) according to the manufacturer’s instructions. The
genomic DNA in 10 mM Tris/HCl (pH 8.0) was stored in a 280 uC
freezer.
Expression of C. jejuni FliD, flagellar capping protein, in the E.
coli expression system. The fliD gene was PCR amplified from the
genomic DNA, ligated in a pRham expression vector and transformed
in competent E. cloni cells (Lucigen) according to the manufacturer’s
instructions, as described previously (Yeh et al., 2013). The nucleotide
sequence between the insert and expression vector junctions of the
recombinant gene was verified by DNA sequencing (see below) to
have the start and stop codons at the 59 and 39 end junctions,
respectively. In screening, several colonies were randomly selected for
cultures in the absence and presence of 0.2 % L-rhamnose. The total
cultures from both conditions were harvested and solubilized in 26
Laemmli sample buffer (Bio-Rad Laboratories), and proteins were
separated in 10–20 % SDS-PAGE gels (see below). The positive clones
were stored in 20 % glycerol for further study. The primers used for
PCR amplification were: fliD-F (59-GAAGGAGATATACATATGGCATTTGGTAGTCTATCTAGTTTAGGATTT-39) and fliD-R (59-GTGATGGTGGTGATGATGATTATTAGAATTGTTTGCCGCATTAATCAT-39).
Purification of recombinant FliD protein. For large-scale produc-
tion of FliD, 10 ml seed culture from an overnight culture of one
colony was added to 500 ml Luria–Bertani broth containing kanamycin (30 mg ml21) and incubated at 37 uC. After the OD600 reached 0.4,
L-rhamnose was added to the culture at a final concentration of 0.2 %.
After incubation at 37 uC for 16–18 h, the cells were harvested for
protein purification by cobalt-chelated affinity chromatography, as
described previously (Yeh & Klesius, 2011, 2012; Yeh et al., 2013).
Briefly, the bacterial cells were collected by centrifugation. Cell pellets
were resuspended in 10 mM Tris/HCl (pH 8.0), followed by homogenization in Lysing Matrix B beads (MP Biomedicals) with a FastPrep24 instrument (MP Biomedicals) according to the manufacturer’s
instructions. The crude lysates containing inclusion bodies were
separated from the beads by low-speed centrifugation. The resulting
lysates were centrifuged at 10 000 g for 30 min to harvest inclusion
bodies. These inclusion bodies were dissolved in 10 mM Tris/HCl
(pH 8.0) containing 6 M urea solution, followed by incubation with
cobalt-chelated agarose beads (Thermo Scientific) on an end-over-end
rotator at room temperature. After 1 h incubation, the beads were
washed extensively with 10 mM Tris/HCl/urea solution containing
20 mM imidazole. The bound FliD protein on the beads was eluted
with the same solution as for the washing step but containing 250 mM
http://jmm.sgmjournals.org
imidazole. The eluted protein was dialysed against a series of decreasing
concentrations of urea in 10 mM Tris/HCl buffer (pH 8.0). The
purified protein was stored at 280 uC.
DNA sequencing and bioinformatics. DNA sequencing in both
directions was carried out at the US Department of Agriculture,
Agricultural Research Service, Genomics and Bioinformatics Research
Unit (Stoneville, MS, USA). Sequence chromatograms were edited,
trimmed to remove vector sequence and analysed using the Phred
(Ewing & Green, 1998; Ewing et al., 1998) and Lucy (Li & Chou,
2004) programs. Amino acid sequences were deduced from the
nucleotide sequences using the ExPASy Translate tool (Gasteiger et al.,
2003). Sequence alignment was performed using MUSCLE (Edgar, 2004;
http://www.ebi.ac.uk/Tools/msa/muscle/). Phylogenetic analysis was
carried out using MEGA version 5.2.1 (Tamura et al., 2011) based on
the MUSCLE alignment results.
Collection of chicken sera. Sera from broiler chickens older than 4
weeks of age were withdrawn from the wing vein. Blood was clotted
by incubation at 37 uC for 1 h, followed at 4 uC overnight. Sera
were collected by low-speed centrifugation, aliquoted and stored at
280 uC. All broilers for serum collection were clinically healthy, and
their bacteriological status was not checked.
The experimental uses of broiler chickens were approved by the
Institutional Animal Care and Use Committee, Richard B. Russell Agricultural Research Center, Agricultural Research Service, US Department of Agriculture, Athens, GA (IACUC no. PMSRU-08-2013-A).
SDS-PAGE and immunoblotting. Proteins were solubilized in
26 Laemmli sample buffer (Bio-Rad Laboratories) and separated
in 10–20 % SDS/Tris/HCl polyacrylamide pre-cast gels (Bio-Rad
Laboratories). After electrophoresis, the proteins were stained with
Bio-Safe Coomassie G-250 stain (Bio-Rad Laboratories) according to
the manufacturer’s instructions.
For immunoblotting, proteins in the SDS-PAGE gels were electrotransferred onto Immobilon membranes in Towbin buffer (Towbin
et al., 1979). The membranes were blocked with a solution of 10 mM
Tris/HCl (pH 8.0), 0.5 M NaCl, 0.5 % Tween 20 and 5 % skimmed
milk at room temperature for 2 h, followed by incubation with broiler
chicken sera (1 : 500 dilution). To detect serum IgG, the membranes
were incubated with goat anti-chicken IgG antibody conjugated with
horseradish peroxidase (HRP; KPL) and 3,39,5,59-tetramethylbenzidine peroxidase substrate (KPL). The membranes were washed
extensively after each antibody incubation. The 66His tag fused to
the recombinant protein was detected with a Nickel HisDetector
Western blot kit (KPL) according to the manufacturer’s instructions.
Images were recorded and processed with an AlphaImager HP System
(ProteinSimple) and its associated software.
Matrix-assisted laser desorption/ionization time of flight mass
spectrometry (MALDI-TOF MS). After electrophoresis, the protein
bands corresponding to FliD were excised and sent to the Mass
Spectrometry/Proteomics Shared Facility of the University of Alabama
at Birmingham for identification by MALDI-TOF MS. The protein was
digested in gel with trypsin as described previously (Wang et al., 2011).
RESULTS AND DISCUSSION
The flagellum of C. jejuni is an important factor for this
micro-organism’s adherence and colonization to host cells
(Grant et al., 1993; Hiett et al., 2008; Nachamkin et al.,
1993). Previously, we cloned and expressed a battery
of Campylobacter jejuni flagellar proteins in an E. coli
expression system (Yeh et al., 2013). Among these, we
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H.-Y. Yeh and others
further studied the FliD (flagellar capping protein) protein,
which is located at the distal end of the flagellin and is
essential for assembly of the functional flagella (Blair &
Dutcher, 1992; Kim et al., 1999; Tasteyre et al., 2001a). The
fliD gene of the D1-39 isolate had 1929 nt, potentially
encoding 642 aa with a calculated molecular mass of
69.6 kDa. When the deduced amino acid sequence of
our isolate was compared with its counterpart from other
campylobacters, we noted that the degree of conservation
of the FliD proteins ranged from .95 % (vs Campylobacter
jejuni) to 77 % (vs Campylobacter coli) to ,40 % (vs
Campylobacter lari, Campylobacter showae, Campylobacter
fetus subsp. fetus, Campylobacter curvus and Campylobacter concisus). Phylogenetic analysis showed that the FliD
protein from our D1-39 isolate fell in the well-segregated
clade of C. jejuni (Fig. 1). It is well established that Campylobacter flagella undergo post-translational glycosylation,
which may contribute to antigen specificity (Guerry et al.,
2006; Logan, 2006; Ewing et al., 2009; Howard et al., 2009).
The deduced FliD amino acid sequence of C. jejuni D1-39
Campylobacter jejuni CP001876
Campylobacter jejuni NCTC 11168
0.2
Campylobacter jejuni LMG 23210
FliD G52
Campylobacter jejuni 81116
Campylobacter jejuni RM1221
Campylobacter jejuni 81-176
Campylobacter coli
Campylobacter upsaliensis
Campylobacter lari
Campylobacter fetus
Campylobacter showae
Campylobacter concisus
Campylobacter curvus
Helicobacter pylori B128
Helicobacter pylori Shi470
Helicobacter pylori 98-10
Helicobacter pylori 52
Salmonella enterica
Escherichia coli
Pseudomonas aeruginosa
Vibrio cholerae
Vibrio alginolyticus
Vibrio harveyi
Clostridium difficile
Fig. 1. Phylogenetic analysis of C. jejuni FliD proteins. Amino acid sequences were retrieved from GenBank and aligned with
MUSCLE (Edgar, 2004). The maximum-likelihood method in MEGA version 5.2.1 (Tamura et al., 2011) was carried out to infer the
phylogenetic tree. The GenBank or NCBI Protein sequences of the FliD proteins used for this analysis are as follows: FliD G52,
KC618388; C. jejuni subsp. jejuni LMG 23210, WP_002935293; C. jejuni subsp. jejuni, CP001876; C. jejuni subsp. jejuni
81-176, ZP_02270961; C. jejuni subsp. jejuni NCTC 11168, YP_002343979; C. jejuni subsp. jejuni 81116,
YP_001482085; C. jejuni subsp. jejuni RM1221, YP_178667; Campylobacter coli Z163, EIA42544; Campylobacter
upsaliensis RM3195, ZP_00370083; Campylobacter lari RM2100, YP_002575342; Campylobacter fetus subsp. fetus 82-40,
YP_891302; Campylobacter showae, EKU10495; Campylobacter concisus 13826, YP_001466708; Campylobacter curvus
525.92, YP_001408376; Clostridium difficile QCD-32g58, ZP_01232795; Escherichia coli B7A, ZP_00715153;
Helicobacter pylori Shi470, NC_010698; Helicobacter pylori 98-10, ZP_03439182; Helicobacter pylori B128,
ZP_03437654; Helicobacter pylori 52, CP001680; Pseudomonas aeruginosa PAO1, NC_002516; Salmonella enterica
subsp. enterica serovar Saintpaul str., ZP_02346561; Vibrio alginolyticus 12G01, ZP_01258802; Vibrio cholerae MO10,
ZP_00759609; Vibrio harveyi ATCC BAA-1116, NC_009783. Bar, amino acid substitutions per site.
604
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Journal of Medical Microbiology 63
Recombinant Campylobacter jejuni FliD
was subjected to analysis by the NetNGlyc 1.0 Server
(http://www.cbs.dtu.dk/services/NetNGlyc/) to predict
whether it had potential N-glycosylation sites. This protein
had nine potential N-glycosylation sites at Asn43, Asn104,
Asn131, Asn152, Asn287, Asn417, Asn514, Asn521 and Asn548.
However, it seems that the consensus sequon for bacterial
N-glycosylation requires a negatively charged side chain
at position 22 upstream of the Asn site (Kowarik et al.,
2006). The sequence, therefore, is extended at the N
terminus to (D/E)XNX(S/T), where X is any amino acid but
not proline (Scott et al., 2011). Based on this information,
we re-examined the FliD amino acid sequence, and found
three potential N-glycosylation sites at Asn43, Asn152 and
Asn548. In addition, O-glycosylation is also present in
bacteria (Abu-Qarn et al., 2008; Lees-Miller et al., 2013).
Analysis of the FliD amino acid sequence by the NetOGlyc
4.0 program (http://www.cbs.dtu.dk/services/NetOGlyc/)
(Steentoft et al., 2013) revealed seven potential Oglycosylation sites at Thr44, Thr232, Ser233, Ser234, Ser250,
Thr325 and Thr597. The flagellar proteins including FliD
were not identified in the N-linked glycosylation study
reported by Scott et al. (2011). This may be due to the fact
that glycosylation in C. jejuni flagella is O-linked (Thibault
et al., 2001). Whether the FliD capping protein is N- or Olinked needs further investigation.
To characterize FliD, it was expressed in and purified
from recombinant E. coli cells. As depicted in Fig. 2(a),
the recombinant FliD protein was overexpressed in the
presence of 0.2 % of L-rhamnose (Fig. 2a, lane U vs lane I),
and had a relative mobility of relevant size and position in
the SDS-PAGE. Because a 66His tag sequence was added
during the expression vector construction, the FliD protein
was purified with cobalt-chelating resins to high homogeneity (Fig. 2a, lanes E1–E3). To further confirm the
purified FliD protein fused with a 66His tag, this protein
was electrotransferred to Immobilon membranes. As illustrated in Fig. 2(b), the FliD protein was one major dense
band detected by nickel-nitrilotriacetic acid conjugated to
HRP on the blot, suggesting that this purified recombinant
FliD is a fusion protein. Furthermore, to ensure that the
band was the FliD protein, the band was excised from
the Coomassie-stained SDS-PAGE gels and subjected to
MALDI-TOF/TOF analysis. The MASCOT score was 415, and
the MS spectra of the peptide fragments with about 16 %
coverage of the sequence were identified as the C. jejuni
FliD protein after searching in the deposited database (Fig.
3). Together with the results of SDS-PAGE analysis, His tag
detection and MALDI-TOF analysis, we concluded that
this overexpressed recombinant protein is the C. jejuni FliD
protein.
Because the FliD protein locates at the tip of the flagellum,
we wanted to determine whether the FliD protein was
antigenic for broiler chickens. Sera from 18 broilers older
than 4 weeks were collected and evaluated by immunoblot
assay. All broiler sera reacted strongly to the FliD protein,
indicating that this anti-FliD antibody may be prevalent in
the poultry population (Fig. 4). This may be due to broiler
http://jmm.sgmjournals.org
(a)
U
I
E1 E2 E3 MW
(b)
E1 M E2
Fig. 2. Purification of recombinant C. jejuni flagellar cap protein,
FliD. Proteins were separated by SDS-PAGE, stained with
Bio-Safe Coomassie G-250 stain and documented with an
AlphaImager HP System. (a) Lanes: U, cells harbouring the FliD
insert not induced by L-rhamnose; I, cells harbouring the insert
induced by 0.2 % of L-rhamnose; E1–E3, FliD protein eluted
from the cobalt-linked agarose resin with 250 mM imidazole
sequentially; lane MW, molecular mass markers (kDa) of 230, 150,
100, 80, 60, 50, 40, 30, 25, 20, 15 and 10 (from top to bottom of
the gel). (b) Detection of a 6¾His tag fused to the recombinant FliD
protein. After SDS-PAGE, the purified protein was transferred to
Immobilon membranes and detected using a Nickel HisDetector
Western blot kit. Lanes: MW, molecular mass markers, as in (a);
E1 and E2, first and second eluate, respectively, of the FliD protein.
chickens naturally harbouring this micro-organism and
the FliD protein being expressed during colonization in
the chicken caeca. Because the chickens were older than 4
weeks, we ruled out the possibility of maternal antibodies,
which diminish within 2 weeks of age (Sahin et al., 2001;
Wyszyńska et al., 2004), being involved in the reaction to
this protein. As the Campylobacter status of the aforementioned broilers was undetermined, attempts were made to
test broilers with no prior Campylobacter exposure (theoretically Campylobacter-negative sera). Specific-pathogenfree chickens were raised according to common industrial
standards, tested and determined to be culture negative;
however, further testing of their faeces using real-time
PCR (Meinersmann et al., 1997) resulted in an amplicon,
suggesting that the chickens had been exposed previously to
Campylobacter. As depicted in Fig. 5, the serum from a
culture-negative chicken reacted strongly to the FliD protein
(Fig. 5, lane 1). Several negative controls such as no serum,
no HRP-conjugated anti-chicken antibody or no serum and
anti-chicken antibody in the reactions revealed no band
on the blot (Fig. 5, lanes 2–4, respectively). These results
indicated that Campylobacter antibody was present in this
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H.-Y. Yeh and others
Fig. 3. Analysis of the recombinant C. jejuni FliD protein by MS peptide mapping and sequencing analysis. The protein band
stained with Coomassie blue was excised from the SDS-PAGE gel and digested with trypsin. The resulting fragments were
concentrated and eluted onto an anchor chip target for analysis on a Bruker Autoflex III MALDI-TOF/TOF instrument. The MS
spectral data were used to search against the protein databases using the MASCOT software. The MASCOT score was 415, and
the mass spectra of these peptide fragments with 16 % coverage of the sequence were identified as C. jejuni FliD protein. The
matched peptides corresponding to the amino acid sequence are indicated in bold red italic (GenBank accession no.
KC618388).
culture-negative (PCR-positive) broiler, possibly as a result
of the numbers of this micro-organism being at levels below
the limit of detection using culture methods, colonization
with ‘viable non-culturable’ Campylobacter (de Boer et al.,
2013; Bullman et al., 2012; Rollins & Colwell, 1986; Yang
et al., 2004) or previous exposure to Campylobacter (Hiett
et al., 2013).
Because our analysis of the FliD proteins from various
micro-organisms showed that the C. jejuni FliD amino
acid sequence shared 26 and 27 % identity with those of
Escherichia coli and Salmonella, respectively, the possibility
that broiler antibody to this recombinant FliD protein may
1 M
2 M
3 M
Although previous studies have demonstrated that a battery
of whole-cell lysate proteins of C. jejuni were identified
by chicken maternal antibodies (Sahin et al., 2001; ShoafSweeney et al., 2008), this extracellular FliD protein was
not detected by the maternal antibodies for several possible
1
M
2 M
3 M 4 M
5 M
4 M
Fig. 4. Reactivities of broiler chicken sera to the purified
recombinant C. jejuni FliD protein analysed by immunoblotting.
After SDS-PAGE, the protein was transferred to PVDF membranes, followed by probing with broiler chicken sera. Lanes: 1–4,
sera from four different chickens; M, molecular mass markers (see
Fig. 2).
606
have been elicited by other micro-organisms cannot be
ruled out. Further studies on broiler immune response to
this protein are undergoing.
Fig. 5. Reactivities of broiler chicken sera to purified recombinant
C. jejuni FliD protein analysed by immunoblotting. After SDSPAGE, the protein was transferred to PVDF membranes, followed
by probing with broiler chicken sera. Lanes: 1, serum from a
culture-negative, PCR-positive chicken with HRP-conjugated antichicken antibody; 2, chicken serum but no anti-chicken antibody;
3, anti-chicken antibody but no chicken serum; 4, no chicken
serum or anti-chicken antibody; 5, serum from a broiler with
unknown bacteriological status; M, molecular mass markers (see
Fig. 2).
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Journal of Medical Microbiology 63
Recombinant Campylobacter jejuni FliD
reasons. First, the stoichiometry of FliD is about five,
which is relatively small compared with 20 000 filaments
per flagellum (Berg, 2003; Macnab, 2003). Therefore, the
amount of FliD protein in the lysate preparation may be
too low to be detected by the antibodies or may produce
a faint reactive band that is difficult to detect. Second, it
is possible that the C. jejuni S3B strain used in the study
of Shoaf-Sweeney et al. (2008) might not contain the fliD
gene, and therefore no FliD protein is produced to
stimulate an immune response in chickens. Many studies
have demonstrated that humoral immunity on the mucosal
surface in the intestine prevents C. jejuni colonization
in chicken caeca (Buckley et al., 2010; Cawthraw & Newell,
2010; Clark et al., 2012; Layton et al., 2011; Stern et al.,
1990; Wyszyńska et al., 2004). The results in this study
demonstrated that all chickens tested had anti-FliD antibodies in their sera. Whether this antigen elicits mucosal
responses in chicken intestinal tracts that may potentially
be protective is yet to be determined.
In summary, the fliD gene of C. jejuni was successfully
amplified and expressed in an E. coli expression system and
the recombinant FliD protein was purified to homogeneity.
This recombinant protein was confirmed by SDS-PAGE
analysis, His tag detection and MALDI-TOF MS analysis.
Sera from broiler chickens were used to test the antigenicity
of the recombinant FliD protein, and the results of antibody studies suggested that anti-FliD antibody may be
prevalent in the poultry population. These results also
provide a rationale for further evaluation of this protein as
a vaccine target for chickens and as a tool for investigating
host–C. jejuni interactions.
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ACKNOWLEDGEMENTS
Ewing, B., Hillier, L., Wendl, M. C. & Green, P. (1998). Base-calling
We are grateful to Susan Q. Brooks, Johnna K. Garrish and Latoya T.
Wiggins of Poultry Microbiological Safety Research Unit, Agricultural
Research Service, US Department of Agriculture, Athens, GA, USA,
for the excellent technical support. We also thank Dr Brian E.
Scheffler and his Bioinformatics Group at the USDA ARS Genomics
and Bioinformatics Research Unit in Stoneville, MS, USA, for
sequencing and bioinformatics, and Dr James A. Mobley and his
team at the Bioanalytical and Mass Spectrometry Shared Facility of
the University of Alabama at Birmingham, AL, USA, for MALDI-TOF
analysis. We are grateful to Dr Holly Sellers of Poultry Diagnostic &
Research Center of the University of Georgia, GA, USA, for chicken
sera. This study was supported by the USDA Agricultural Research
Service CRIS project no. 6612-32000-060-00, and the US Poultry &
Egg Association project no. 679. Mention of trade names or
commercial products in this paper is solely for the purpose of
providing specific information and does not imply recommendation
or endorsement by the US Department of Agriculture. The US
Department of Agriculture is an equal opportunity provider and
employer.
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