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Applied and Environmental Microbiology, November 2008, p. 6867-6875, Vol. 74, No. 22
0099-2240/08/$08.00+0     doi:10.1128/AEM.01097-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Identification of Campylobacter jejuni Proteins Recognized by Maternal Antibodies of Chickens ^,

Kari D. Shoaf-Sweeney,¹ Charles L. Larson,¹ Xiaoting Tang,² and Michael E. Konkel^1*

School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4234,¹ Department of Chemistry, Proteomics Core, LBB2, Washington State University, Pullman, Washington 99164-4630²

Received 15 May 2008/ Accepted 10 September 2008

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Campylobacter jejuni is one of the leading bacterial causes of food-borne gastroenteritis. Infection with C. jejuni is frequently acquired through the consumption of undercooked poultry or foods cross-contaminated with raw poultry. Given the importance of poultry as a reservoir for Campylobacter organisms, investigators have performed studies to understand the protective role of maternal antibodies in the ecology of Campylobacter colonization of poultry. In a previous study, chicks with maternal antibodies generated against the S3B strain of C. jejuni provided protection against Campylobacter colonization (O. Sahin, N. Luo, S. Huang, and Q. Zhang, Appl. Environ. Microbiol. 69:5372-5379, 2003). We obtained serum samples, collectively referred to as the C. jejuni S3B-SPF sera, from the previous study. These sera were determined to contain maternal antibodies that reacted against C. jejuni whole-cell lysates as judged by enzyme-linked immunosorbent assay. The antigens recognized by the C. jejuni S3B-SPF antibodies were identified by immunoblot analysis, coupled with mass spectrometry, of C. jejuni outer membrane protein extracts. This approach led to the identification of C. jejuni proteins recognized by the maternal antibodies, including the flagellin proteins and CadF adhesin. In vitro assays revealed that the C. jejuni S3B-SPF sera retarded the motility of the C. jejuni S3B homologous strain but did not retard the motility of a heterologous strain of C. jejuni (81-176). This finding provides a possible mechanism explaining why maternal antibodies confer enhanced protection against challenge with a homologous strain compared to a heterologous strain. Collectively, this study provides a list of C. jejuni proteins against which protective antibodies are generated in hens and passed to chicks.

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Campylobacter jejuni, a gram-negative, spiral-shaped, motile bacterium, is one of the leading bacterial causes of food-borne gastroenteritis worldwide. Campylobacteriosis is a self-limiting infection, characterized by a rapid onset of fever, severe abdominal cramps, and diarrhea that may include blood and leukocytes. Ingestion of as few as 500 organisms may result in C. jejuni infection, with symptoms becoming apparent within 1 to 7 days after consumption of the contaminated food or liquid (36). More severe complications can result from C. jejuni infections. For example, C. jejuni has been implicated in postinfection sequelae such as irritable bowel syndrome and Guillain-Barré syndrome, which is a common cause of acute neuromuscular paralysis (40, 46).

Epidemiological studies have shown a link between the consumption of undercooked poultry or other products that have come into contact with undercooked or raw poultry. C. jejuni colonizes the ceca of chickens at densities of 10⁸ CFU per gram of cecal contents or greater without causing disease (1, 38). Day-old chicks can become colonized with C. jejuni when experimentally inoculated, but colonization of chickens with C. jejuni under commercial conditions does not occur until after 2 to 3 weeks of age (2, 38, 41, 47). After C. jejuni colonizes a few birds in a flock, it rapidly spreads throughout the flock (7, 41). Once colonized, C. jejuni can remain present throughout the bird's life span (38, 47).

Dramatic changes in the levels of antibodies against C. jejuni occur throughout the lifetime of a broiler chicken. In general, the level of maternal antibodies detected in the sera of chicks remains high for 3 to 4 days after hatching, after which it gradually decreases to undetectable levels at 2 to 3 weeks of age (38). Interestingly, C. jejuni colonization of chickens coincides with the decrease (absence) of antibodies reactive against the bacterium. Once a chicken is colonized with C. jejuni, antibodies against the bacterium are generated (28, 39). Although these antibodies may not clear an established population of Campylobacter bacteria, a decrease in the number of C. jejuni organisms colonizing the intestinal tract has been observed (30, 37). Researchers have hypothesized that the presence of these antibodies results in a decrease in the microbial load (37, 38). Also, antibodies generated against C. jejuni prior to exposure greatly reduce the bacterium's ability to colonize chickens (50).

Maternal antibodies in young chickens are known to confer partial protection against C. jejuni colonization. More specifically, Sahin et al. (37) performed experiments to determine the protective role of anti-Campylobacter maternal antibodies. For these experiments, the investigators obtained fertile eggs from specific-pathogen-free (SPF) White Leghorn hens and allowed the eggs to hatch in order to establish SPF flocks free of Campylobacter. At 15 weeks of age, the birds were divided into two flocks. One flock was then challenged at 22 weeks of age with C. jejuni S3B (flock A), and the other flock remained uninfected to serve as a negative control (flock B). Two weeks after the oral challenge with C. jejuni, eggs were collected from both groups and tested for the levels of Campylobacter-specific antibodies by enzyme-linked immunosorbent assays (ELISA). When the Campylobacter-specific antibodies reached a significant level in flock A (approximately 4 weeks after oral inoculation), hatchlings were obtained from both flocks. The young hatchlings from flock A tested positive for Campylobacter-specific maternal antibodies, whereas flock B tested negative. Using these two groups of young chicks, challenge experiments were performed with homologous (S3B) and heterologous (21190) strains of C. jejuni. The experiments revealed that the Campylobacter strain-specific maternal antibodies delayed the onset of colonization and reduced the rate of horizontal spread of C. jejuni compared to the case for the chicks without Campylobacter specific antibodies. This protection by the Campylobacter-specific maternal antibodies was observed with the C. jejuni S3B strain and extended to the chicks challenged with the C. jejuni 21190 heterologous strain. Also performed were complement-dependent bactericidal assays with sera obtained from 2-day old SPF White Leghorn chicks; interestingly, the Campylobacter-specific maternal antibodies were effective in killing the homologous strain of C. jejuni in the presence of complement but had no effect on the heterologous 21190 strain. We obtained the sera from the 2-day old SPF White Leghorn chicks that contained the Campylobacter-specific maternal antibodies for use in this study. These sera were designated the C. jejuni S3B-SPF sera.

We focused this study on the identification of C. jejuni membrane-associated proteins recognized by maternal antibodies, as the antibodies passed from hens to chicks are partially protective against Campylobacter colonization of chicks. More specifically, immunoblot analysis was performed with the C. jejuni S3B-SPF sera, and the reactive C. jejuni proteins were identified by tandem mass spectrometry. We report a list C. jejuni proteins recognized by maternal antibodies, which furthers our understanding of the poultry immune response to C. jejuni.

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Bacterial cultures and chicken sera.
The Campylobacter jejuni S3B strain, isolated from a chicken, was kindly provided by Q. Zhang (Iowa State University). The C. jejuni 81-176 strain was isolated from an individual with diarrhea containing blood and leukocytes (16). C. jejuni S3B and 81-176 were cultured on Mueller-Hinton (MH) agar plates containing 5% citrate-buffered bovine blood (MH-blood) under microaerobic conditions (5% O₂, 10% CO₂, and 85% N₂) at 37°C. The bacteria were subcultured to a fresh MH-blood plate every 48 h.

The generation of the sera is described in detail elsewhere (37). Briefly, SPF eggs from White Leghorn chickens were obtained from a supplier and hatched in isolation. The chickens were examined for the absence of C. jejuni colonization by cloacal swabs, bred at 22 weeks of age, and after an additional 2 weeks inoculated with the C. jejuni S3B strain. Fertilized eggs were collected from the inoculated hens and hatched in isolation. In total, blood was collected from nine SPF White Leghorn chickens at 2 days of age. The serum was harvested from each blood sample and stored at –20°C. We obtained 25 to 100 µl of each serum sample. Throughout this paper we will refer to these serum samples as the C. jejuni S3B-SPF sera.

Preparation of C. jejuni OMPs.
Outer membrane proteins (OMPs) were prepared using N-lauroyl sarcosine as previously described by de Melo and Pechère (6) with slight modifications. Briefly, bacteria were grown overnight in MH broth with shaking at 37°C under microaerobic conditions. The bacterial cells were harvested and suspended in 10 mM phosphate buffer (pH 7.4) containing 1 mM phenylmethylsulfonyl fluoride (Sigma, St. Louis, MO). The bacterial cell suspensions were sonicated five times (30 s each) with a 30-s cooling period on ice between each pulse with a Branson Sonifier cell disruptor (model 250; Branson Sonic Power Co., Danbury, CT). Cell debris was removed by centrifugation at 6,000 x g for 10 min. The crude membranes were obtained by centrifugation at 100,000 x g at 4°C for 2 h. The resulting pellets were suspended in 10 mM Tris (pH 7.5), and the protein concentration in each sample was determined using the bicinchoninic acid assay as outlined in the manufacturer's instructions (Pierce, Rockford, IL). N-Lauroyl sarcosine (Sigma) was added to the crude extracts at a protein-to-detergent ratio of 1:4 total (wt/wt). The samples were incubated at room temperature with gentle rocking for 30 min. The OMPs were obtained by centrifugation at 100,000 x g at 15°C for 2 h. The pellets were washed with 50 mM Tris (pH 7.5), suspended in the same buffer, and stored at –20°C. The protein concentration in the OMP extracts was determined by bicinchoninic acid assay.

ELISA.
ELISA were performed to determine the level of C. jejuni-specific immunoglobulin G (IgG) antibodies the C. jejuni S3B-SPF sera. Microtiter plates (Corning Incorporated, Corning, NY) were coated with 100 µl of ovalbumin (negative control), C. jejuni S3B whole-cell lysates (WCLs), or 81-176 WCLs diluted to 10 µg/ml in coating buffer (50 mM Na₂CO₃, 51 mM NaHCO₃, pH 9.6). After incubation at 4°C for 18 h, the coated plates were incubated with 0.5% (wt/vol) bovine serum albumin (BSA) (Sigma) in phosphate buffer (PBS) (0.14 M NaCl, 5 mM Na₂HPO₄·2H₂O, 1.5 mM KH₂PO₄, 19 mM KCl, pH 7.4) at room temperature for 2 h to reduce the nonspecific binding of antibodies. The C. jejuni S3B-SPF serum samples were diluted 1:200 in PBS containing 0.5% BSA, and 100 µl of each sample was added to wells in triplicate and incubated for 2 h at room temperature. The plates were rinsed three times with wash buffer (0.15 M NaCl, 0.1% [vol/vol] Tween 20, pH 7.4), and 100 µl of rabbit anti-chicken IgG conjugated to peroxidase (1:1,000; Sigma) diluted in PBS containing 0.5% (wt/vol) BSA and 0.1% (vol/vol) Tween 20 was added to the wells. After 1 h of incubation at room temperature, the plates were rinsed two times with wash buffer and two times with PBS. Tetramethybenzidine substrate (Pierce-Endogen) was added to the wells, and the reaction was stopped with 0.18 N H₂SO₄ after 10 min of development. Absorbances at 490 nm (A₄₉₀) were determined with an ELx808 Ultra microplate reader (BioTek Instruments, Inc., Winooski, VT). The absorbances obtained using the chicken sera incubated with ovalbumin were subtracted from the appropriate serum sample values to remove background signal. Student's t test was performed on A₄₉₀ values to determine statistical significance between sample groups (P < 0.005). Nine sera were collected from chickens not colonized with C. jejuni and used to calculate the negative cutoff using Student's t distribution. Samples with absorbance values greater than the negative cutoff value were considered positive for C. jejuni-specific antibodies (8).

SDS-PAGE and immunoblot analysis.
Bacterial OMPs (0.5 µg/µl) were solubilized in single-strength electrophoresis sample buffer and boiled for 5 min. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 12.5% polyacrylamide minigels as previously described by Laemmli (18). Separated proteins were either stained with Coomassie brilliant blue R250 (CBB-R250) or transferred to polyvinylidene fluoride (PVDF) membranes (Immobilon P; Millipore Corporation, Bedford, MA). Membranes were blocked in PBS containing 0.05% (vol/vol) Tween 20 (PBS-T) with 20% fetal bovine serum and incubated with the C. jejuni S3B-SPF sera (1:200 dilution) overnight at 4°C. Subsequently, blots were washed three times with PBS-T. Bound antibodies were detected with rabbit anti-chicken peroxidase-conjugated IgG (1:1,000 dilution; Sigma). CadF was detected using a goat anti-CadF specific serum (no. 461) coupled with a rabbit anti-goat peroxidase-conjugated IgG (1:1,000; Sigma). The FlaA and FlaB flagellin proteins were detected using a rabbit anti-C. jejuni flagellin-specific serum with goat anti-rabbit peroxidase-conjugated IgG (1:1,000; Sigma). The blots were washed three times with PBS-T and developed with 4-chloro-1-naphthol peroxidase chromogenic substrate (Thermo Scientific, Rockford, IL) as outlined by the manufacturer.

Liquid chromatography matrix-assisted laser desorption ionization-time of flight (LC/MALDI/TOF-TOF) and data analysis.
OMP extracts were trichloroacetic acid precipitated and washed three times with acetone. The dried pellets were resuspended in 25 µl 8 M urea-100 mM NH₄HCO₃, and the pH was adjusted to 7.5 to 8.0 with NH₄HCO₃. Proteins were reduced with dithiothreitol at a final concentration of 5 mM at 37°C for 30 min and then alkylated with iodoactemide at a final concentration of 25 mM at 37°C for 30 min in the dark. The solution was diluted four times with 100 mM NH₄HCO₃, and 1 µg trypsin (sequence grade; Promega, Madison, WI) was added for overnight digestion at 37°C. The digest solution was concentrated with a speed vacuum to a final volume of 20 to 30 µl. The LC MALDI plate was prepared using the Tempo LC MALDI system (Applied Biosystems, Foster City, CA). Five microliters of digest solution was loaded onto the analytical column (Chromolith CapROD RP-18e, 100 µm by 150 mm; Merck KGaA, Darmstadt, Germany) by the autosampler and separated at a flow rate of 2 µl/min using the following gradient: 5% B for 0 to 2 min, 5 to 20% B for 2 to 25 min, 20 to 60% B for 25 to 50 min, 95% B for 50 to 60 min, and 0% B for 60 to 70 min. Mobile phase A was 0.1% trifluoroacetic acid (TFA) in 2% acetonitrile, and mobile phase B was 0.1% TFA in 95% acetonitrile. A 5-mg/ml concentration of MALDI matrix, -cyano-4-hydroxycinnamic acid, was prepared in a solution of 50% acetonitrile, 0.1% TFA, and 5 mM ammonium monophosphate and delivered at a flow rate of 2 µl/min. The LC effluent and matrix solution were mixed via an Upchurch T connector, and the mixtures were then spotted on a blank MALDI plate (123 by 81 mm) every 4 s during the 50-min LC gradient. The mass spectrometry (MS) and MS/MS spectra were acquired with a 4800 MALDI/TOF-TOF mass spectrometer (Applied Biosystems, Foster City, CA). One thousand laser shots were used for each reflector MS spectrum, and 2,500 laser shots were collected for each MS/MS spectrum. The precursor peaks with S/N values of >40 were selected for MS/MS experiments, and the 25 strongest precursors were allowed for MS/MS per spot, with the weakest precursor submitted first. Peaks with S/N values of>10 were extracted and searched against the C. jejuni 81-176 database (CJJ81176 downloaded from NCBI, 1,758 open reading frames) using ProteinPilot software (version 2.0.1, revision 67476; Applied Biosystems, Foster City, CA). Search parameters were set as follows: enzyme, trypsin; Cys alkylation, iodoacetamide; special factor, urea denaturation; species, none; and ID focus, biological modification. The protein confidence threshold cutoff for this report is ProtScore of 2.0 (unused) with at least one peptide with 99% confidence. (See the supplemental material for the complete ProteinPilot results.) Protein subcellular localization was determined by PSORTb (http://www.psort.org/psortb/).

Nano-LC/MS/MS and data analysis.
Nano-LC/MS/MS was performed as described previously by Tang et al. (44) and used to identify the reactive bands as determined by immunoblotting with the C. jejuni S3B-SPF sera. Briefly, bands representing immunoreactive proteins were excised from SDS-12.5% polyacrylamide gels that had been stained with CBB-R250. After band excision, each gel piece was destained with a solution containing 50% methanol and 5% acetic acid. The disulfide bonds within the proteins were dissociated within the gel by performic acid oxidation. The gel was dried, and the proteins were digested with trypsin overnight at 37°C. Nano-LC/MS/MS analysis was done using an electrospray ion trap (Esquire HCT; Bruker Daltonics, Billerica, MA) mass spectrometer coupled with a nano-high-pressure liquid chromatograph. The resulting data were used to perform searches against the C. jejuni 81-176 genome database using the program MASCOT, licensed in house (version 2.1.0; MatrixScience Ltd., London, United Kingdom). Protein hits with probability-based Mowse scores exceeding their thresholds (P < 0.05) were automatically reported. The protein hits were further filtered using more stringent MudPIT scoring and an ion score cutoff of 0.05, which removed all the peptides with expect values (E) of >0.05.

Motility assay.
To evaluate the functional attributes of the anti-Campylobacter maternal antibodies, motility assays were performed as described previously with slight modifications (15). C. jejuni S3B and 81-176 were grown for 24 h on MH-blood plates, harvested by centrifugation at 6,000 x g, and suspended to an optical density at 540 nm of 0.18 in minimal essential medium (Invitrogen) supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT). Bacterial suspensions were then diluted 1:100 in the same medium that contained either sera from C. jejuni uninoculated chickens, pooled C. jejuni S3B-SPF sera, or heat-inactivated C. jejuni S3B-SPF pooled sera. Complement was inactivated through heat treatment at 56°C for 30 min. The bacterial suspensions were mixed, and 10-µl aliquots were spotted onto the surface of semisolid MH medium with 0.4% agar. Motility plates were incubated for 48 h at 37°C under microaerobic conditions.

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Chicks hatched from hens colonized with Campylobacter possess anti-C. jejuni maternal antibodies.
Nine serum samples (designated 121, 123, 129, 132, 135, 139, 140, 144, and 147) generated in a previous study were obtained and termed the C. jejuni S3B-SPF sera. These sera were collected from 2-day-old SPF White Leghorn chicks hatched from hens inoculated with the C. jejuni S3B strain. To determine the level of C. jejuni-specific IgG maternal antibodies in each serum, ELISA were performed with wells coated with WCLs prepared from C. jejuni homologous (S3B) and heterologous (81-176) strains (Fig. 1). Nonspecific antibody reactivity was determined by calculating the negative cutoff value of antibody reactivity for the sera harvested from nine chickens not colonized with C. jejuni (control sera). The reactivity of the control sera against the WCL of the C. jejuni S3B strain was less than (P < 0.005) that obtained for the WCL of the C. jejuni 81-176 strain (Fig. 1).

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FIG. 1. Reactivity of the C. jejuni S3B-SPF sera against C. jejuni OMPs extracted from homologous (S3B) and heterologous (81-176) strains determined by ELISA as outlined in Materials and Methods. The C. jejuni S3B-SPF sera were processed from 2-day-old SPF White Leghorn chicks hatched from hens experimentally inoculated with the C. jejuni S3B strain. Numbers on the x axis indicate the identification numbers of the serum samples. Vertical bars represent the arithmetic means, and the error bars represent the standard deviations for triplicate samples. The horizontal lines represent the negative cutoff values determined from nine sera collected from control chickens not colonized with C. jejuni.

Each of the C. jejuni S3B-SPF serum samples contained antibodies that reacted specifically against the WCLs of the C. jejuni S3B and 81-176 strains as judged by ELISA. However, an increase was observed in the reactivity of the C. jejuni S3B-SPF sera against the WCL of the S3B homologous strain (mean A₄₉₀ 0.665) compared to WCL from prepared from the 81-176 heterologous strain (mean A₄₉₀ of 0.463) (P < 0.005). The differences in reactivity with the WCLs suggested either that the C. jejuni S3B-SPF sera contained antibodies that react with antigens unique to the C. jejuni S3B strain or that variations in the amino acid composition of strain-specific antigens occur and contribute to the increase in reactivity of the sera against a specific strain.

Identification of OMPs.
LC/MALDI/TOF-TOF was performed with the total OMP extracts prepared from the C. jejuni 81-176 strain, for which the genome has been sequenced, to ensure that the composition of the preparations was predominantly OMPs and not cytoplasmic proteins (see Table S1 in the supplemental material). The ProteinPilot software was employed as the search engine for protein identification using LC/MALDI/TOF-TOF data. Since the unused ProtScore is a measurement of all the peptide evidence for a protein that is not explained by a higher-ranking protein and is a true indicator of protein evidence, we set the unused score at 2.0 as the threshold cutoff for protein identification with at least one peptide with 99% confidence. With these criteria, 60 proteins were identified with 2,944 MS/MS spectra and searching against the C. jejuni 81-176 database (total of 1,758 open reading frames) (see Table S1 in the supplemental material). Of the 60 proteins identified, approximately 32% were localized in the cytoplasm as determined by PSORTb. Additional analysis of the proteins contained within the OMP extracts revealed that 18% were categorized as having an unknown subcellular location and 50% were identified as extracellular, outer membrane, periplasmic, or inner membrane proteins or designated as having an unknown subcellular location with a signal peptide.

Reactivity of the C. jejuni S3B-SPF sera against the OMPs of homologous and heterologous C. jejuni strains.
To determine the reactivity of the antibodies contained within the C. jejuni S3B-SPF sera, OMP extracts were separated by SDS-PAGE and transferred to PVDF membranes, and immunoblot analysis was performed with the S3B-SPF sera. The C. jejuni S3B-SPF sera produced repeatable banding profiles for the OMP extracts from both the C. jejuni S3B homologous strain and 81-176 heterologous strain as judged by immunoblot analysis (Fig. 2A). The reactive bands in the OMP extracts ranged from 16 to 90 kDa. The representative banding profiles generated against the C. jejuni S3B and 81-176 strains were similar, but some bands were unique to a particular strain.

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FIG. 2. Immunoblots of OMP extracts of the C. jejuni homologous (S3B) and heterologous (81-176) strains probed with the C. jejuni S3B-SPF sera. (A) OMPs were prepared from C. jejuni strains S3B and 81-176, separated by SDS-PAGE, and immunoblotted with the C. jejuni S3B-SPF sera. The numbers above the strips indicate the identification number of the serum samples. The two strips located on the right side show C. jejuni S3B and 81-176 OMP extracts probed with a goat anti-CadF antibody. The strip on the far right shows a C. jejuni S3B OMP extract processed as described for all other strips but without a primary antibody. The arrowheads indicate immunoreactive bands. The black arrow on the left indicates the position of the CadF protein. The positions of size markers are indicated on the left of the blots. (B) Table representing the presence or absence (blank) of bands that reacted with the maternal antibodies as determined from immunoblots.

Inspection of blots revealed that the S3B-SPF sera contained antibodies that reacted against strain-specific proteins and against proteins shared between the C. jejuni S3B and 81-176 strains (Fig. 2A). Proteins with apparent molecular masses of 90, 83, 65, 60, 56, 54, 42, 37, 26, and 20 kDa (bands 1 to 6, 9, 11, 14, and 16, respectively) were cross-reactive with both the C. jejuni S3B homologous and 81-176 heterologous strains. Immunoreactive proteins specific to C. jejuni S3B were observed at approximately 32, 28, and 16 kDa (bands 12, 13, and 17, respectively) (Fig. 2B). Immunoreactive proteins unique to C. jejuni 81-176 were observed at approximately 50, 45, 40, and 23 kDa (bands 7, 8, 10, and 15, respectively) (Fig. 2B). These results indicated that the chicks possessed both maternal antibodies that reacted against the particular C. jejuni strain with which the hens were colonized and maternal antibodies that reacted with proteins shared among C. jejuni strains.

Immunoblotting was performed to determine if the C. jejuni S3B-SPF sera contained antibodies reactive against CadF protein (Fig. 2A and B). This protein is a 37-kDa OMP that is essential for C. jejuni colonization of chickens (12, 14, 17, 25, 26, 51). A reactive band (band 11), corresponding to a protein with a molecular mass of 37 kDa, was observed in the OMP extracts from the C. jejuni S3B and 81-176 strains using each of the nine C. jejuni S3B-SPF sera (Fig. 2A and B). The bands observed at 37 kDa with the C. jejuni S3B-SPF sera had the same relative migration as the CadF protein detected using a goat anti-CadF-specific serum (Fig. 2A).

Identification of the bands recognized by the C. jejuni S3B-SPF sera.
Nano-LC/MS/MS was used to identify the C. jejuni membrane-associated antigens recognized by the C. jejuni S3B-SPF sera. The OMP extracts from the C. jejuni S3B and 81-176 strains were separated by SDS-PAGE and either stained with CBB-R250 or transferred to a PVDF membrane. The blot was incubated with a representative C. jejuni S3B-specific serum (no. 144 in Fig. 2) to identify the reactive proteins. Seventeen reactive bands were identified; 14 of the 17 bands were subjected to nano-LC/MS/MS.

Bands 1, 2, 4 to 10, and 12 to 16 were excised individually from the gel and subjected to tryptic digestion followed by nano-LC/MS/MS. Careful attention was paid to excise those protein bands that were in perfect alignment with the reactive bands in the corresponding immunoblot. The proteins identified are listed in Table 1. The predicted "best-fit" protein matches were OMPs with significant MASCOT scores and had molecular masses corresponding to the migrations of the proteins in an SDS-12.5% polyacrylamide gel. Confidence in protein matches was established using MudPIT scoring and an ion score cutoff of 0.05.

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TABLE 1. Predicted "best-fit" immunogenic membrane-associated C. jejuni proteins identified by nano-LC/MS/MS

Bands 3 and 11 were identified via immunoblot analysis using protein-specific sera, and band 17 was identified based on its apparent molecular mass. The 65-kDa protein (band 3) was identified as flagellin using an anti-C. jejuni flagellin serum, and the 37-kDa protein (band 11) was identified as CadF using an anti-C. jejuni CadF serum. We propose that the 16-kDa immunoreactive band (band 17), which was detected only in the OMP extracts prepared from the C. jejuni S3B strain, is lipooligosaccharide (LOS) (34).

The C. jejuni S3B-SPF bands unique to C. jejuni 81-176 are flagellin.
A number of the bands were found to contain peptides that matched the FlaA or FlaB sequence as judged by nano-LC/MS/MS (Table 1). This finding raised the possibility that a particular band may have been immunoreactive because of the presence of flagellin protein subunits. To determine whether the reactivity of these bands was due to flagellin subunits or another protein distinct from flagellin, C. jejuni S3B and 81-176 OMP extracts were probed with the anti-C. jejuni flagellin serum (Fig. 3). As expected, the 65-kDa protein (band 3) reacted with the anti-C. jejuni flagellin serum. In addition, proteins of 50 kDa (band 7), 45 kDa (band 8), 40 kDa (band 10), and 23 kDa (band 15) were detected in the OMP extracts from C. jejuni strain 81-176 but not in C. jejuni S3B. The immunoreactive bands of 50 kDa, 45 kDa, 40 kDa, and 23 kDa were the only proteins unique to the C. jejuni 81-176 OMP extracts (i.e., not detected in the C. jejuni S3B OMP extracts). The bands of 50 kDa, 45 kDa, 40 kDa, and 23 kDa in the C. jejuni 81-176 OMP extracts were determined to be FlaA or FlaB by nano-LC/MS/MS.

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FIG. 3. C. jejuni 81-176 OMP extracts contain flagellin degradation products. Immunoblot analyses were performed with OMP extracts prepared from C. jejuni S3B and 81-176 and probed with either the C. jejuni S3B-SPF serum (no. 144) or anti-C. jejuni flagellin serum. Lanes: 1, profile of the C. jejuni 81-176 OMP extract probed with the C. jejuni S3B-SPF 144 serum sample; 2, profile of the C. jejuni 81-176 OMP extract probed with the anti-C. jejuni flagellin serum; 3, profile of the C. jejuni S3B OMP extract probed with the anti-C. jejuni flagellin serum. The arrowheads indicate the C. jejuni 81-176 flagellin bands recognized by C. jejuni S3B-SPF serum no. 144. The numbers listed adjacent to the arrowheads correspond to the band identification numbers in Fig. 2. The arrows show the C. jejuni 81-176 bands of less than 60 kDa that were recognized by the anti-C. jejuni flagellin serum determined to be FlaA or FlaB by nano-LC/MS/MS. The positions of size markers are indicated on the left.

C. jejuni S3B-specific antibodies inhibit the motility of the homologous strain but not that of the heterologous strain.
Motility assays were performed with the C. jejuni S3B-SPF sera and both the C. jejuni S3B (Fig. 4A) and 81-176 (Fig. 4B) strains. In contrast with the C. jejuni 81-176 strain, only the C. jejuni S3B strain showed a reduction in motility compared with the same strain with control sera harvested from birds not colonized with C. jejuni. This observation was true for both the C. jejuni S3B-SPF heat-inactivated sera and the C. jejuni S3B-SPF untreated sera, demonstrating that the reduction in motility is due to antibodies binding to the bacteria and not due to the action of complement.

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FIG. 4. C. jejuni S3B-SPF pooled sera contain antibodies that reduce the motility of C. jejuni S3B but not that of C. jejuni 81-176. Suspensions of bacteria were mixed with sera from chickens not inoculated with C. jejuni (control sera), pooled C. jejuni S3B-SPF sera, or heat-inactivated C. jejuni S3B-SPF pooled sera. The suspensions were spotted on semisolid MH medium with 0.4% agar as described in Materials and Methods. Motility plates and MH-blood plates were incubated for 48 h at 37°C under microaerobic conditions. All images shown were captured using the same scanning settings. (A) Motility assays performed with the C. jejuni S3B strain. (B) Motility assays performed with the C. jejuni 81-176 strain. The horizontal lines indicate the diameters of the spots (i.e., from the center to the edge of the bacterial zone).

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In the present study, we analyzed the reactivity of the C. jejuni S3B-SPF sera against C. jejuni WCLs by ELISA. Each of the C. jejuni S3B-SPF serum samples tested possessed antibodies that reacted against the C. jejuni WCLs at a level that was significantly greater (P < 0.005) than that of sera obtained from chickens that were not colonized with C. jejuni. In addition, the reactivity of the C. jejuni S3B-SPF sera against the WCLs from the C. jejuni S3B strain was significantly greater (P < 0.005) than that observed for the C. jejuni 81-176 strain. Thus, the increase in the reactivity of the C. jejuni S3B-SPF sera against the WCLs from the C. jejuni S3B strain versus the 81-176 strain may partially explain the delay in onset of colonization with the C. jejuni S3B-challenged chicks and the reduced rate of horizontal spread among the flock.

The antigens that the C. jejuni S3B-SPF maternal antibodies reacted against were identified by immunoblot analysis coupled with tandem MS of OMP extracts from C. jejuni 81-176. While a total of 60 proteins were identified in the OMP extracts from C. jejuni 81-176, far fewer proteins (i.e., 20) that reacted with the antibodies in the C. jejuni S3B-SPF sera were identified. These proteins were identified by excising bands from SDS-polyacrylamide gels that aligned with S3B-SPF immunoreactive bands, followed by nano-LC/MS/MS. A caveat with this method is that it is not possible to know with certainty the protein species that the antibodies bound when more than one protein is found within a band. In this regard, bands of less than 60 kDa that corresponded to flagellin were identified in the C. jejuni 81-176 OMP extracts. Nevertheless, it is unlikely that these reactive bands confounded the identification of the C. jejuni S3B-specific antigens for two reasons. First, the antiflagellin serum showed little reactivity against proteins of less than 50 kDa in the OMP extract from the C. jejuni S3B strain; this observation is in contrast with the multiple bands observed against proteins in the C. jejuni 81-176 OMP extract. Second, comparison of the banding pattern of the C. jejuni 81-176 OMP extracts with that of the C. jejuni S3B-SPF sera revealed distinct profiles compared to the anti-C. jejuni flagellin serum.

The C. jejuni S3B-SPF sera contained antibodies that reacted with the flagellar hook protein (FlgE2) and the FlaA and FlaB filament proteins in C. jejuni S3B and 81-176. FlgE2 has a molecular mass of 89.4 kDa and is required for motility, flagellar assembly, and protein secretion in C. jejuni (10, 15). The FlgE2 protein has been shown previously to be immunogenic (11, 22, 24, 49); anti-FlgE2 antibodies recognize surface-exposed epitopes on the assembled hook (35). The flagellum, or at least the flagellar filament protein FlaA, is known to be important for chicken colonization (29, 48). In addition, the flagellar filament proteins have consistently been identified as immunogenic (4). Although the predicted molecular mass of these proteins is around 59 kDa, glycosylation has been shown to alter the mass of the proteins by up to 10% depending on the level of modification (45). We observed a band of between 65 and 63 kDa as judged by immunoblot analysis with an anti-C. jejuni flagellin-specific serum. It is possible that this band represents glycosylated forms of the FlaA or FlaB protein, whereas the proteins with apparent molecular masses of less than 60 kDa represent degradation products.

The C. jejuni S3B-SPF sera contained antibodies that reacted against C. jejuni strain-specific proteins as well as proteins common among C. jejuni strains. For example, a 40-kDa immunoreactive protein, CmeA (band 9), was recognized in the OMP extracts of the C. jejuni S3B and 81-176 strains by all of the C. jejuni S3B-SPF sera, whereas a 54-kDa protein, presumably CmeC, was recognized primarily in the OMP extracts of the C. jejuni S3B strain. Together the CmeA, -B, and -C proteins comprise a resistance-nodulation-division (RND) efflux pump that is involved in resistance to a broad range of antimicrobials and bile salts (20, 21). CmeB is the inner membrane efflux transporter, whereas CmeA is localized in the periplasmic space and CmeC forms an outer membrane channel. CmeABC is widely distributed in C. jejuni isolates (20), and comparison of the deduced amino acid sequence of each protein from four C. jejuni strains (NCTC11169, RM1221, 81116, and 81-176) revealed that the sequence of each protein was well conserved (>98% similarity) among these strains. Previous work has demonstrated that the cmeABC operon is transcribed in chickens and that antibodies are generated against CmeC (19). Convalescent-phase serum from a C. jejuni-infected individual has also been found to contain antibodies reactive against CmeC (5).

Two outer membrane substrate-binding proteins involved in amino acid transport, CjaA (band 12) and CjaC (band 13), were identified. CjaA has been characterized as an extracytoplasmic solute receptor in a putative ATP-binding cassette-type cysteine transporter (27), while CjaC has been shown to be required for histidine transport (9). It is possible that amino acid transport system proteins may serve as good vaccine components, because C. jejuni is asaccharolytic and relies on exogenous sources of amino acids for energy production. In fact, both CjaA and CjaC have been proposed to be promising candidates for chicken vaccines in that they are highly immunogenic and expressed in multiple Campylobacter isolates recovered from both chickens and humans (31, 50). Wysznska et al. (50) showed that chickens orally immunized with avirluent Salmonella expressing C. jejuni CjaA developed serum IgG and mucosal IgA antibody responses against C. jejuni membrane proteins. Additionally, immunization with this same strain greatly reduced the ability of a heterologous C. jejuni isolate to colonize the cecum. We found that the C. jejuni S3B-SPF sera contained antibodies that reacted against the CjaA and CjaC proteins in the C. jejuni S3B strain, but not in the 81-176 strain. Pawelec et al. (32) demonstrated genetic diversity in both cjaA and cjaC among C. jejuni isolates, with as much as 16% variation noted at the nucleotide level. The relevant antigenic surfaces of CjaA and CjaC, although cross-reactive with each other, have not been identified. Thus, slight variations in the amino acid sequences may account for the reduced or absent antibody response to CjaA and CjaC in the C. jejuni 81-176 strain.

We show for the first time that CadF, a well-characterized 37-kDa fibronectin-binding protein, is immunogenic in chickens (12-14, 17, 23, 25, 26, 51). This finding is consistent with previous observations that a reactive 37-kDa band was observed with maternally derived anti-Campylobacter antibodies (39). Noteworthy is that the CadF protein, which is necessary for C. jejuni colonization of chickens (51), was detected in both the C. jejuni S3B and 81-176 strains. Antibodies against the CadF protein have also been detected in convalescent-phase sera from C. jejuni-infected individuals (5, 13).

Several C. jejuni OMPs, which have been studied less intensely, were also found to be immunogenic. These proteins included PEB3, MapA, and CJJ81176_0586. The function of PEB3 is not known (33). MapA is an outer membrane lipoprotein that has been used as an identification tool to distinguish between C. jejuni and Campylobacter coli (43) and to detect and diagnose individuals with C. jejuni infection (3). CJJ81176_0586 has been identified as a hypothetical OMP based on its amino acid sequence.

It is well known that chicks are at least partially protected from colonization by C. jejuni through the actions of maternal antibodies. However, the mechanisms of maternal antibody protection have yet to be fully elucidated. Maternal antibodies may interfere with bacterial motility, promote clearance by agglutination, block ion/nutrient transport, decrease viability through complement-mediated killing, and/or block the interaction between bacterial adhesins and host cell intestinal receptors (42). Sahin et al. (37) demonstrated the bactericidal activity of the C. jejuni S3B-SPF sera against the S3B strain of C. jejuni in the presence of complement; however, the C. jejuni S3B-SPF sera had no effect on a heterologous strain of C. jejuni (21190). Similar to the previous observations with regard to the specificity of the maternal antibodies, the C. jejuni S3B-SPF pooled heat-inactivated sera reduced the motility of C. jejuni S3B but not that of the C. jejuni 81-176 strain. While it reasonable to hypothesize that the antibodies generated against a given strain could reduce the colonization of the same C. jejuni strain by inhibiting bacterial motility, it is noteworthy that this effect was observed only against the C. jejuni strain with which the hens were colonized. This protection, or inhibition in motility, is likely conferred by antibodies that react with the domains of flagellin that are accessible to antibody binding (i.e., surface-exposed or accessible domains). Thus, we hypothesize that a reduction in motility could be observed against other C. jejuni strains depending on the amino acid composition of the flagellin subunits. The limited amount of sera available prevented the performance of assays to test whether the C. jejuni S3B-SPF sera could cause bacterial agglutination or block the binding of C. jejuni to host intestinal cells, as well as other functional assays.

In summary, we have identified C. jejuni proteins that react with antibodies contained in 2-day SPF White Leghorn chicks that were hatched from hens inoculated with C. jejuni. We believe that these data provide a foundation for a variety of future studies, including those to determine the efficacy of various C. jejuni proteins as vaccine candidates. This study provides a list of proteins against which antibodies are generated in hens and transferred to chicks; these maternal antibodies provide chicks with partial protection against C. jejuni colonization. In addition to flagellin and CadF, the maternal antibodies reacted against other proteins that have been less intensely studied. Additional experiments are required to determine the subset of proteins that constitute the C. jejuni protective antigens. Noteworthy is that antibodies directed against the flagellins diminished the motility of the homologous C. jejuni strain as judged by in vitro assays. Additional studies are needed to dissect the functional attributes of the antibodies generated in poultry against C. jejuni.

 
We are grateful to Qijing Zhang from the Department of Veterinary Microbiology and Preventive Medicine at Iowa State University, Ames, for providing the maternal antibody sera. We also thank Gerhard Munske and Chongjie Zhu from Washington State University for assistance with mass spectrometric analysis. Finally, we thank Phil Mixter (School of Molecular Biosciences, Washington State University), Jason Neal-McKinney (School of Molecular Biosciences, Washington State University), and Orhan Sahin for critical reviews of the manuscript.

This work was supported from funds awarded to M.E.K. from Food Safety Coordinated Agricultural Project (FS-CAP) 2005-35212-15287 and funds provided by the USDA National Research Initiative's Food Safety 32.0 program (2006-35201-17305). The WSU Chemistry/Proteomics Core Laboratory is supported by funding from NIH-NCRR (contract 1S10RR022538-01).

 
* Corresponding author. Mailing address: School of Molecular Biosciences, Washington State University, Pullman, WA 99164-4234. Phone: (509) 335-5039. Fax: (509) 335-1907. E-mail: konkel{at}wsu.edu

Published ahead of print on 19 September 2008.

Supplemental material for this article may be found at http://aem.asm.org/.

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Applied and Environmental Microbiology, November 2008, p. 6867-6875, Vol. 74, No. 22
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