Novel Immunomodulatory Flagellin-Like Protein FlaC in Campylobacter jejuni and Other Campylobacterales

Bacterial strains and culture conditions. Campylobacter jejuni strains NCTC 11168 (ATCC 700819) and 81-176 (77) were used. C. jejuni cultures were grown at 37°C or 42°C under microaerobic conditions (10% CO₂, 5% O₂, 85% N₂) in vented jars or under anaerobic conditions (10% CO₂, 10% H₂, 80% N₂) in a Scholzen incubator on blood agar plates (blood agar base II; Oxoid, Wesel, Germany), supplemented with 10% defibrinated horse blood (Oxoid) and standard antibiotics (10 mg/liter vancomycin, 3.2 mg/liter polymyxin B, 5 mg/liter trimethoprim, 4 mg/liter amphotericin B), or in brain heart infusion broth (Oxoid) with the addition of 2.5 g/liter yeast extract (Merck, Darmstadt, Germany). The C. jejuni mutant strains were grown on blood agar plates with standard antibiotic supplement and 20 mg/liter chloramphenicol for selection. For coincubation assays with eukaryotic cells and for bacterial lysate preparations (ultrasonicated), bacteria were grown on plates for approximately 24 h before being harvested. Escherichia coli strains DH5α and MC1061 were used for cloning. For the overexpression of proteins, the E. coli strain BL21(DE3) was used. E. coli was cultured in Luria-Bertani (LB) broth (Difco LB agar; Lennox, BD Biosciences, Heidelberg, Germany) or on LB plates containing 1.5% Bacto agar. When appropriate, ampicillin (500 mg/liter) or kanamycin (20 mg/liter) was added to the medium.

Cell types and culture conditions.Different cell lines were used for cell coincubation and transfection assays. Human embryonic kidney 293 cells (HEK293T) were used for transfection and expression of recombinant proteins and cultured in Dulbecco’s minimal essential medium (MEM) (Biochrom, Berlin, Germany) supplemented with 10% (vol/vol) fetal bovine serum (FBS). The HD-11 chicken macrophage-like cell line and the human monocytic cell line THP-1 were maintained in Iscove’s basal medium (IBM) (Biochrom) supplemented with 10% (vol/vol) FBS. The Lovo human colon epithelial cell line (78) was cultured in Dulbecco’s MEM and Ham’s F-12 (1:1 mixture) (Biochrom) supplemented with 10% (vol/vol) FBS. HD-11, THP-1, and Lovo cell lines stably transfected with the firefly luciferase gene under the control of an NF-κB promoter were continuously cultured in the presence of puromycin (5 g/liter). All cell lines were routinely kept at 37°C in a 5% CO₂ humidified atmosphere. Surface expression of TLR5 in THP-1 cells was verified by fluorescence-activated cell sorting (FACS) as previously published (79). Expression of tlr5 mRNA in chicken HD-11 cells has been previously shown (80), and we have confirmed the presence of the tlr5 transcript by semiquantitative reverse transcription-PCR (RT-PCR) in all cell lines used (not shown), except for Lovo cells. The functional activity of TLR5 was confirmed for all cell lines by ultrapure FliC stimulation and positive measurement of IL-8 secretion.

Generation of flaC mutants by allelic exchange and complementation of flaC. flaC mutants and flaC complementation strains in C. jejuni were generated by an allelic-exchange gene replacement strategy. Briefly, the flaC gene from strain 11168 was PCR amplified using primers CjflaC_F_BamHI and CjflaC_R_BamHI (Table 3) and cloned into pUC18. A unique internal BglII restriction site in the flaC gene was used to insert the chloramphenicol resistance cassette (CAT), derived from the plasmid pBHpC8 (81). The plasmid containing interrupted flaC was used for the natural transformation of two C. jejuni strains, 11168 and 81-176, and chloramphenicol-resistant clones were recovered after 3 days. The flaC mutant of the C. jejuni strain 81-176 was complemented by allelic gene replacement of flaC in the rdxA locus together with a gentamicin (Gm; aminoglycoside acetyltransferase, aac) resistance cassette from pUC1813apra (82). The rdxA gene from strain 81-176 was PCR amplified using primers CjrdxA_XbaI_F1 and CjrdxA_XbaI_R1 (Table 3) and cloned into pUC18 (Table 4). An internal BglII restriction site in the rdxA gene was used to insert flaC from strain 11168. This construct was amplified via inverse PCR using primers CjrdxA_SpeI_F3 and CjrdxA_ClaI_R3 (Table 3) and served for the insertion of the PCR-amplified Gm cassette (with primers Gm1_ClaI and Gm2_SpeI [Table 3]; template, the pUC1813apra plasmid) upstream of flaC using ClaI and SpeI restriction sites. The correct insertions after allelic exchange in CAT- or Gm-resistant clones recovered after natural transformation was confirmed by PCR using different primer combinations. The mutants and complementants showed no growth difference from the wild type in growth curve experiments (not shown). FlaC expression of the complementation strains was verified by Western blotting (not shown).

Plate motility assay.The motility of C. jejuni wild-type strains and the corresponding flaC mutants and complemented strains was tested in a six-well plate format with semisolid medium, containing 2.8% (wt/vol) brucella broth (BD Biosciences), 0.3% (wt/vol) Bacto Agar (BD Biosciences), 5% (vol/vol) heat-inactivated horse serum (Gibco, Darmstadt, Germany), and standard antibiotics (see above). Bacteria were grown overnight on blood agar plates, adjusted to a final optical density at 600 nm (OD₆₀₀) of 1.00, and inoculated with a pipette tip into the center of motility agar wells. The plates were then cultured for 2 days at 37°C under microaerobic or anaerobic conditions in a Scholzen incubator, and the swim diameter in the agar after the incubation was measured in comparison to a negative control (C. jejuniflgR mutant).

DNA and protein methods.DNA methods were performed according to standard protocols (83) using enzymes produced by New England Biolabs (NEB, Ipswich, NJ), Invitrogen (Carlsbad, California), or Roche (Basel, Switzerland). DNA from bacteria or tissue was prepared using the Qiagen Tissue Amp kit according to the manufacturers’ instructions, with slight modifications. PCRs were performed using Taq polymerase (Roche) or Phusion polymerase (NEB), if products with high amplification accuracy were required. Sanger sequencing technology was applied for sequencing of the cloned plasmids. Protein amounts were determined by bichinchoninic acid (BCA) protein assay (Thermo Scientific, Pierce, Rockford, IL), and protein analysis was performed by separation on denaturing 12% sodium dodecyl sulfate (SDS) polyacrylamide gels and Western immunoblot detection according to standard methods (84). Equal amounts of protein were loaded in each lane of the gels. Antibodies for labeling were used as indicated in the results. Visualization of immuno-reactive bands was obtained using the Super Signal West Pico chemiluminescent substrate (Pierce, Thermo Scientific, Bonn, Germany) and enhanced-chemiluminescence (ECL) hyperfilm (GE Healthcare, Piscataway, NJ). Analysis of phosphoproteins (P-p38, anti-P-p38 antibody, rabbit 92115; Cell Signalling Technology; used at a 1:1,000 dilution) of coincubated cells was performed on Western blots from cleared cell lysates prepared in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, protease inhibitor cocktail Complete [Roche], phosphatase inhibitor cocktail PhosStop [Roche]). Anti-p38 MAPK antibody (rabbit 92125; Cell Signalling Technology) was used on the same blots as a control for total p38. Quantification of chemiluminescent signals for the P-p38 blots was performed by densitometric measurements using the software ImageJ (http://rsb.info.nih.gov/ij/ ) and normalizing against signal intensities in each lane obtained using the p38 antibody. Additional normalization was performed against the corresponding loading controls visualized with the aid of anti-actin antibody (Chemicon MAB1501; mouse monoclonal antibody; used at a 1:2,000 dilution). In the case of sequential antibody application, membranes were stripped with Restore Western blot stripping buffer (Thermo Scientific, Pierce).

Lysis and fractionation of C. jejuni. C. jejuni lysates were generated by resuspending bacteria grown on blood agar plates in an appropriate volume of 0.9% NaCl and lysing by sonication (Branson Sonifier 450). To separate the soluble (cytoplasmic) and insoluble (membrane-associated) fractions of bacteria, C. jejuni lysates were centrifuged for 20 min at 9,000 × g and 4°C. The pellet, enriched for membrane-associated proteins, was resuspended in an appropriate volume of 0.9% NaCl; the supernatant represented the soluble bacterial fraction. For the preparation of secreted proteins, C. jejuni was grown in liquid culture to a defined OD₆₀₀ of 0.4 to 0.6. Bacteria were harvested by centrifugation for 20 min at 9,000 × g. Subsequently, the supernatant, which contains secreted proteins, was filtered through a 0.22-µm-pore-size sterile filter (polyethersulfone [PES] membrane, Millipore Express; Millex GP, Schleicher & Schuell). Precipitation of secreted proteins was conducted with trichloroacetic acid (TCA) overnight at room temperature, followed by centrifugation for 15 min at 14,000 × g and 4°C and a final washing step with ice-cold 100% acetone. Secreted proteins were resuspended in modified SDS-PAGE loading buffer (20% [vol/vol] 5× SDS-PAGE loading buffer, 25% [vol/vol] Tris-HCl [1 M, pH = 8.0] in distilled water). For enriching surface-associated proteins, which contain, among others, flagellar proteins, C. jejuni was grown on blood agar plates for 2 to 3 days. Bacteria were resuspended in 0.9% NaCl, and proteins were sheared off the bacterial surface by repeatedly (up to 30 times) pushing the suspension through a 23-gauge needle. Sheared-off material was separated from bacterial cells by differential centrifugation steps: 20 min at 9,000 × g and ultracentrifugation for 1 h at 40,000 × g (Beckman Optima100 ultracentrifuge) and 4°C. Surface-associated proteins were resuspended in Tris buffer (100 mM Tris-HCl, pH 7.5). All bacterial fractions were analyzed on Western blots using anti-FlaC antiserum and respective fractionation controls (not shown).

Recombinant expression and purification of C. jejuni FlaC and S. enterica serovar Typhimurium FliC.For recombinant expression of C. jejuni protein FlaC in E. coli, the FlaC expression plasmid pCJ1024 (Table 4) was constructed, using primers CjFlaC_F3 and CjFlaC_R2 (Table 3), and transformed into E. coli BL21(DE3). Expression of FlaC was induced at early exponential phase (OD₆₀₀ = 0.6) by adding 0.5 mM isopropyl-d-thiogalactopyranoside (IPTG). Bacteria were harvested after they reached the stationary phase (4 to 5 h), resuspended in appropriate volumes of lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM MgCl₂), and lysed by sonication. To separate the soluble from the insoluble fraction, the whole-cell lysate was centrifuged for 20 min at 9,000 × g and 4°C. The recombinantly expressed FlaC contained 6×His tags on its C and N termini and was purified from the soluble bacterial fraction by affinity chromatography using Ni²⁺-nitrilotriacetic acid (NTA)-Sepharose beads (Qiagen, Hilden, Germany) under denaturing conditions. The purification procedure comprised several wash steps with buffer B (8 M urea, 100 mM NaH₂PO₄, 100 mM Tris-HCl, pH 8.0) and buffer C (6 M urea, 100 mM NaH₂PO₄, 20 mM Tris-HCl, pH 6.3), with decreasing pH. Elution of the protein was achieved by using buffers with further decreased pH: buffer D (6 M urea, 100 mM NaH₂PO₄, 20 mM Tris-HCl, pH 5.8) and buffer E (6 M urea, 100 mM NaH₂PO₄, 20 mM Tris-HCl, pH 4.5). The single fractions were analyzed by SDS-PAGE with respect to their FlaC content; selected elution fractions were pooled and dialyzed against at least three changes of PBS using a dialysis chamber (Slide-A-Lyzer, molecular weight cutoff [MWCO], 3,500; Thermo Scientific). For coincubation experiments of eukaryotic cells with purified FlaC, an additional purification step by elution of the protein from an SDS gel in an Electro Eluter (model 422; Bio-Rad, Munich, Germany) was performed. Eluted protein was again dialyzed several times against cell culture-grade PBS, using pyrogen-free microconcentrator tubes (MWCO, 10,000; Amicon filter devices; Millipore) for centrifugation at 8,000 × g and 4°C. Expression and purification of Salmonella flagellin FliC was performed accordingly. The purity and amounts of ultrapure recombinant flagellins were finally checked on silver- or Coomassie blue-stained SDS gels (Fig. S7). Limulus assays (Limulus amebocyte lysis [LAL] chromogenic endpoint assay; Cambrex) of recombinant flagellins prepared by this method did not detect LPS above the detection limit of 1 endotoxin unit (EU) per µg of protein (13), and the preparations can thus be considered virtually free of contaminations by other TLR ligands. Before coincubation with cells, flagellar preparations were diluted in 1× PBS (cell culture grade) and monomerized by ultrasonication for 1 min. In cell activation experiments, the protein nature of the activating principle (purified FlaC) was again verified by heat-treating the protein preparations as a control, which led to a complete loss of activity (not shown).

Generation of antiserum against C. jejuni FlaC.An antiserum raised against the recombinantly expressed and purified FlaC from C. jejuni was generated in rabbits by repeated intradermal injection in combination with Freund’s adjuvant (BioGenes, Berlin, Germany).

Anti-His tag pulldown assay.To investigate protein-protein interactions, pulldown analysis was performed. Two milligrams of purified recombinant 6×His-FlaC–6×His per sample was incubated for 30 min at 4°C with a 20-µl slurry of Cobalt (Co²⁺) beads (Talon beads; BD Biosciences) under continuous mixing in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, protease inhibitor cocktail Complete [Roche], phosphatase inhibitors). An incubation step with cleared lysates (soluble fraction) of TLR5-V5 plasmid-transfected HEK293T cells (200 µg of lysate per sample) was performed, followed by coincubation for 1 h at 4°C under continuous mixing. After unbound protein was removed by stringent wash steps with imidazole-containing buffer (60 to 80 mM imidazole in PBS), the beads were resuspended in appropriate volumes of 2.5× SDS-PAGE loading buffer, and protein interactions were analyzed on Western blots. Precipitated TLR5 was detected using an antibody against the C-terminal V5 epitope tag (mouse monoclonal R960-25; Invitrogen Life Technologies).

Cell transfection with plasmid DNA and protein expression analysis.HEK293T cells, which were seeded in 24-well plates, were used for transfection experiments at 80% confluence. One hour before transfection, the medium was changed to 0.5 ml OptiMEM medium (Gibco) containing 5% fetal calf serum (FCS). The plasmid of interest (0.4 mg), which was prepared with an endo-free plasmid midi-kit (Qiagen, Hilden, Germany), was transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. To allow expression of transfected constructs, cells were further incubated for 24 h to 48 h before being harvested. Protein expression was then analyzed by Western immunoblotting.

Coincubation of eukaryotic cells with proteins or live C. jejuni strains.For coincubation experiments with live C. jejuni strains or purified proteins, cells were seeded into 96 (4 × 10⁴ cells in 100 µl per well)-, 24 (2 × 10⁵ cells in 1 ml per well)-, or 6 (2 × 10⁶ cells in 3 ml per well)-well plates 24 h prior to the infection. Media were replaced 30 min before the coincubations. For bacterial coincubations, cells were infected with freshly resuspended bacteria at an MOI of 10, 20, 25, or 50 and synchronized by centrifugation (300 × g, room temperature, 10 min). Equal bacterial amounts at the endpoint of cell coincubation with live C. jejuni bacteria were verified by Western immunoblotting using an anti-C. jejuni antiserum produced in rabbits.

In order to test the activation potential of protein preparations, cells were coincubated with different amounts of sonicated proteins (ultrapure flagellins) for 3 to 4 h, and a 96-well plate format was used for determining NF-κB activation of stable luciferase reporter cells. NF-κB-mediated luciferase activation was measured using SteadyGlow firefly luciferase substrate (Promega Inc., Madison, WI) in a Victor3 Wallac 1420 microtiter plate luminometer (GE Healthcare). The 24-well plate format was chosen for transfection experiments or for the further quantitative analysis of cytokine secretion (IL-8) or P-p38 analysis after coincubation with ultrapure proteins, live bacteria, or bacterial lysates. Equal MOIs were used to compare live bacteria between different strains; for cell coincubation with bacterial lysates, equal protein amounts of each lysate (BCA assay) were used for the different strains.

For assaying TLR4 specificity, polymyxin B (Sigma Aldrich) was added to the cells 1 h prior to starting the further incubation steps. For the 2-day experiments assessing tolerization against cell activation by E. coli LPS (TLR4 ligand ultrapure grade, from E. coli O111:B4; List Biological Laboratories Inc., Campbell, CA), THP-1 or HD-11 cells (2 × 10⁴ per well) were incubated in 96-well plates. One day after being seeded, the cells were activated by adding proteins or activation controls, respectively. Three hours and 19 h after the primary activation, NF-kB-dependent luciferase activity was determined using Promega SteadyGlo luciferase buffer in selected wells. At this time point, ultrapure LPS was added as a secondary stimulus in the remaining experiment wells and incubated for a further 3-h period, followed by the last luciferase measurement. For all experiments and conditions involving LPS, the ultrapure LPS preparation was used. IL-8 in supernatants of cell activation experiments was routinely determined using a commercially available ELISA (IL-8 OptEIA ELISA system; Becton, Dickinson, Inc., BD Biosciences), according to the manufacturer’s instructions. Coincubation experiments using a 6-well plate format were performed for RNA preparations and real-time PCR analysis.

RNA preparation and real-time PCR.For RNA preparation, cell pellets were resuspended in Qiagen RNeasy lysis buffer containing β-mercaptoethanol and transferred to lysing matrix D tubes containing ceramic beads (MPbio, Germany). The cells were lysed in a bead beater machine (FastPrep; Bio101 Thermo Scientific, Bonn, Germany) for 45 s. The RNA was further purified using the Qiagen RNeasy minikit according to the manufacturer’s instructions. Total RNA was quantitated in a NanoDrop 1000 device (Thermo Scientific, Bonn, Germany), and equal amounts of each sample were subjected to DNase I treatment (Roche). The residual content of DNA was determined by PCR using chicken glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers for HD-11 cells and human GAPDH primers for THP-1 and Lovo cells. If necessary, the residual DNA was removed by a second DNase I treatment and subsequent RNeasy column cleanup. cDNA was prepared from equivalent amounts (1 µg) of total RNA using Superscript III reverse transcriptase (Invitrogen) and oligo(dT) primers (Invitrogen) according to the manufacturer’s instructions.

Quantitative RT-PCR was performed using SYBR green mix (Qiagen) on equal amounts of cDNA (1 to 2 µl) and normalized to the results of a corresponding GAPDH or 16S rRNA gene control reaction mixture prepared from the same cDNAs (primers used for RT-PCR are listed in Table S2 in the supplemental material). Reactions were carried out in a Bio-Rad thermocycler (Bio-Rad C1000/CFX96 combined system [Bio-Rad, Hercules, CA]). Cycling conditions were as follows: denaturation for 10 min at 95°C and amplification for 40 cycles at 95°C for 15 to 45 s, at 59°C (or optimized according to the calculated annealing temperature of the primer pairs) for 15 s, and at 72°C for 30 s. For quantification of DNA amounts, a serial standard was prepared from a purified PCR product of the gene of interest. Each reaction was performed in triplicate.

Experimental administration of purified FlaC into SPF chicken and microbiota analyses.SPF eggs from white SPF Leghorn layer chickens were purchased from Valo BioMedia GmbH (Bremen, Germany). After hatching, birds were kept jointly in metal cages without bedding and supplied with conventional, autoclaved feed and water ad libitum. Two weeks after hatching, the birds were separated into two groups consisting of 8 animals each and further kept in two separate closed cages in the same room. At this time point, one group of birds received 25 µg of purified recombinant FlaC in 200 µl sterile cell culture-grade PBS via intracloacal administration. The mock-treated group was given 200 µl of only sterile PBS by the same route. The application of purified FlaC (25 µg) or PBS (control group) via the same route was repeated after 7 and 10 days. Fourteen days after the initial administration, birds were sacrificed and their organs (liver, cecal tonsils, and right-hand cecum after gentle removal of contents) collected for CFU determination of culturable bacteria. The complete left-side ceca and three different gut sections of each bird were cleared of contents and fixed in 4% paraformaldehyde (PFA) for histopathological analysis. Two tissue samples of the tips of each right-hand cecum were sampled and stored in RNA-Later (Qiagen) at −20°C for further RNA preparation and at −18°C for DNA isolation and subsequent microbiota analyses, respectively. Animal experiments were conducted according to the German animal protection laws and approved by the federal authorities (LAVES, Lower Saxony, Germany) under the administrative identification number 33.14-42502-04-13/1282. Microbiota analysis by 16S rRNA gene amplicon sequencing from total chicken cecal tip tissue DNA was performed using PCR products of primer pairs covering the bacterial 16S rRNA gene variable regions V1 to V3 on a Roche 454 GS FLX+ pyrosequencing platform (85). Microbiota sequence reads were analyzed as previously described, with modifications (85; detailed in Text S1 in the supplemental material).

Nucleotide sequence accession number.The microbiota data (16S rRNA gene sequences) are available under accession number PRJEB11550 (European Nucleotide Archives ENA at EMBL-EBI).