APMIS
© 2014 APMIS. Published by John Wiley & Sons Ltd. DOI 10.1111/apm.12332
Interaction of Bordetella pertussis filamentous hemagglutinin with human TLR2: identification of the TLR2-binding domain HOSSEIN ASGARIAN-OMRAN,1,2 ALI AKBAR AMIRZARGAR,3 SACHA ZEERLEDER,4,5 MARZIEH MAHDAVI,6 GERARD VAN MIERLO,4 SHABNAM SOLATI,4 MAHMOOD JEDDI-TEHRANI,6 HODJATALLAH RABBANI,6 LEUCIEN AARDEN3 and FAZEL SHOKRI2,6 1
Department of Immunology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran; 2 Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran; 3 Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran; 4 Department of Immunopathology, Sanquin Research Institute, Amsterdam; 5Department of Hematology, Academic Medical Center, Amsterdam, The Netherlands; and 6Avicenna Research Institute, Monoclonal Antibody Research Center, ACECR, Tehran, Iran
Asgarian-Omran H, Amirzargar AA, Zeerleder S, Mahdavi M, van Mierlo G, Solati S, Jeddi-Tehrani M, Rabbani H, Aarden L, Shokri F. Interaction of Bordetella pertussis filamentous hemagglutinin with human TLR2: identification of the TLR2-binding domain. APMIS 2014. Filamentous hemagglutinin (FHA) is a major adhesion and virulence factor of Bordetella pertussis and also a main component of acellular pertussis vaccines. Interaction of FHA with different receptors on human epithelial and immune cells facilitates entrance and colonization of bacteria as well as immunomodulation of the host immune response. Three overlapping segments of the FHA gene were cloned in a prokaryotic expression vector and the recombinant proteins were purified. These recombinant fragments along with the native FHA protein were employed to assess their potential Toll-like receptor (TLR) stimulatory effects and to localize the TLR binding region. TLR stimulation was monitored by applying HEK293-Blue cell lines cotransfected with TLR2, 4, or 5 and a NF-jB reporter gene. Culture supernatants were checked for secretion of the reporter gene product and IL-8 as indicators of TLR stimulation. Native FHA was found to strongly stimulate TLR2, but not TLR4 or TLR5 transfected cells. Among recombinant FHA fragments only the fragment spanning amino acid residues 1544–1917 was able to exhibit the TLR2 stimulating property of FHA. Interaction of FHA with TLR2 suggests its involvement in induction of the innate immune system against Bordetella pertussis. The TLR2-binding domain of FHA may contribute to immunoprotection against pertussis infection. Key words: Bordetella pertussis; filamentous hemagglutinin; innate immunity; Toll-like receptor; TLR2. Fazel Shokri, Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. e-mail: [email protected]
The mammalian innate immune system senses microbial structures known as pathogen associated molecular patterns (PAMPs) through a variety of pattern recognition receptors (PRRs) present on the surface and in the cytoplasm of different cell types (1). One of the most important and wellstudied groups of PRRs is Toll-like receptors (TLRs) which are currently composed of 10 different members (TLR1-10) in human (2). TLR family members are homo- or hetero-dimeric molecules Received 1 January 2014. Accepted 6 May 2014
which can bind to various microbial products and initiate the immune responses to infectious diseases (2). TLR2 is a cell surface protein which is expressed both as a homo- and hetero-dimers with TLR1 or TLR6 (3). Human TLR2 trigger pro-inflammatory responses to pathogens by interacting with a broad range of PAMPs such as bacterial lipopeptides, peptidoglycan, lipoteichoic acid, fungal zymosan, glycosylphosphatidylinositol-anchored mucin-like glycoproteins from Trypanosoma cruzi and hemagglutinin from measles virus (3). 1
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Whooping cough, pertussis, is an acute respiratory disease caused by the gram-negative bacteria, Bordetella pertussis (Bp) (4). Vaccination against pertussis was initiated in the 1940s by inoculation with heat inactivated whole cell bacteria (4). In spite of dramatic reduction in pertussis cases after vaccination with whole cell bacteria, reactogenecity and side effects of this vaccine which were more remarkable in neonates and children, prompted researchers to shift to acellular pertussis subunit vaccines (Ac) consisting of a cocktail of 2–5 immunogenic antigens of Bp including pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin, and fimberiae 2–3 antigens (5). Filamentous hemagglutinin (FHA) is the major adhesion and immunomodulator molecule of Bp which is synthesized as a large 367 kDa precursor protein and modified to a mature 220 kDa form by proteolytic degradation at the N- and C-terminus (6). This protein has long been recognized as a highly immunoprotective antigen with crucial roles in pertussis pathogenesis (7). Different binding domains have been identified for FHA which mediate the attachment and entrance of the bacteria to the epithelial cells (8–10). Interaction of Bp with the target epithelial cells via these domains has been shown to induce pro-inflammatory and pro-apoptotic signals which contribute to the pathogenesis of pertussis (11). In this study, we have demonstrated for the first time that FHA binds to human TLR2 and induces secretion of the pro-inflammatory cytokine IL-8 following activation of the NF-kB signaling pathway. The TLR2 binding site was mapped to a region of FHA spanning the amino acids 1544–1917, using three overlapping recombinant fragments of FHA. MATERIALS AND METHODS Cell lines, bacterial strains, culture media, and reagents: Bordetella pertussis Tohama I strain (ATCC, Manassas, CA, USA) was employed for FHA gene amplification and production of recombinant FHA proteins. The bacteria were grown on Bordet-Gengou agar (Becton Dickinson, Franklin Lakes, NJ, USA) supplemented with 15% defibrinated sheep blood, 1% v/v glycerol, 1% v/w proteose peptone (DIFCO, Franklin Lakes, NJ, USA) and 40 lg/ mL cephalexin for 72 h. Both E. coli strains JM109 and BL21 (DE3) were purchased from Novagen (Merck KGaA, Darmstadt, Germany) and cultured in LB agar containing 1% w/v peptone (Merck KGaA), 0.5% w/v yeast extract (Merck KGaA), 0.6% w/v NaCl and 1.5% w/v agar (Merck KGaA). LB broth medium constituents were the same as LB agar without agar. HEK293-Blue-TLR2 (transfected with human TLR2, CD14 and NF-jB reporter gene), HEK293-Blue-TLR4
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(transfected with human TLR4, CD14, MD2 and NF-jB reporter gene) and HEK293-Blue-TLR5 (transfected with human TLR5 and NF-jB reporter gene) cell lines (Invivogen, San Diego, CA, USA) were used for TLR stimulation studies. Cells were cultured in IMDM medium supplemented with 5% fetal calf serum (FCS) (Gibco-Biocuh, Paisley, Scotland), 100 lg/mL streptomycin, 100 IU/mL penicillin, 100 lg/mL Normocin (Invivogen) and selective antibiotics according to the manufacture’s instruction. All cells were cultured at 37 °C in a humidified atmosphere with 5% CO2. Native FHA was obtained from Sigma-Aldrich (St. Louis, MO, USA) and was also purified in our lab using an antibody affinity-based column from Bp culture supernatant and characterized by SDS-PAGE and Western blotting (data not shown). Staphylococcus aureus Cowan I strain cells (SAC) (Pansorbin, Calbiochem, La Jolla, CA, USA), E. coli lipopolysacharaide (LPS) (Invivogen) and flagellin (Invivogen) were used as commercial TLR2, 4 and 5 agonists, respectively, to optimize the stimulation conditions of TLR-transfected HEK293-Blue cells.
Construction and expression of recombinant proteins: As previously described, three overlapping regions from FHA coding sequence were selected and amplified from Bp genomic DNA for production of recombinant proteins designated rFHA1-3 (12).
Purification of the recombinant proteins: Recombinant protein purification was performed in a denaturing condition using the Acta Explorer Chromatography System (GE Healthcare Life Sciences, Uppsala, Sweden). Harvested bacterial pellets containing inclusion bodies were solubilized in 20 mL of lysis buffer containing 100 mM NaH2PO4, 100 mM NaCl, 30 mM Tris-HCL, pH:8 and incubated on ice for 1 h. This solution was continuously sonicated at 70% amplitude for 15 min to cell destruction and then centrifuged at 12 000 g for 10 min. Supernatants were discarded and pellets were resuspended in buffer A (100 mM NaH2PO4, 50 mM NaCl, 10 mM Tris-HCL, 30 mM imidazole, 8M urea, pH: 8) and incubated at room temperature for 1 h. After centrifugation at 12 000 g, pellets were discarded and supernatants containing His-tagged recombinant proteins were applied as starting materials on HisTrap HP (GE Healthcare Life Sciences) column, which has been equilibrated with buffer A. Following the binding of recombinant proteins to column, the protein refolding process was performed by applying a descending continuous gradient of urea concentration from 8 M to zero for 3 h. Target proteins were then eluted by applying ascending continuous gradient of imidazole concentrations from 30 mM–1 M. All recombinant proteins were eluted at a range of 230–250 mM of imidazole. The purity of selected fractions was checked by silver stained SDS-PAGE. To get rid of any contamination, the purified fractions collected from Ni-NTA agarose column were concentrated by Amicon Ultra-15 centrifugal filter units (Merck KGaA) and applied to Hiload 16/60 Superdex200 prep grade gel filtration column (GE Healthcare Life Sciences) equilibrated with 0.15 M PBS. The peaks corresponding to
© 2014 APMIS. Published by John Wiley & Sons Ltd
TLR2 TRIGGERING BY BORDETELLA PERTUSSIS FHA
molecular weight of the target proteins were selected and concentrated using Amicon Ultra-4 centrifugal filter units (Merck KGaA) followed by filtration with 0.2 lm membrane filters (Merck KGaA). Protein concentrations were determined by BCA colorimetric assay (Pierce, Rockford, IL, USA).
Western blot analysis of the recombinant FHA proteins:
measurement of secreted embryonic alkaline phosphatase (SEAP), the product of NF-jB reporter gene, and IL-8 in culture supernatants. Responsiveness and sensitivity of the HEK293-Blue-TLR2, 4 and 5 cell lines were initially assessed using their commercial ligands, including SAC, LPS, and flagellin, respectively (Fig. 1A). Each cell line
After assessment of the recombinant proteins by silverstained 10% SDS-PAGE, the reactivity of all recombinant proteins was confirmed by immunoblot analysis (12).
A
TLR stimulation assay: HEK293-Blue-TLR cells were seeded at 40 * 103/well on 96 well flat-bottom culture plates and stimulated with different concentrations of native and recombinant FHA as well as their commercial agonists. After 16 h incubation at 37 °C in a humidified atmosphere with 5% CO2, supernatants were harvested and the activity of secreted embryonic alkaline phosphatase (SEAP) was evaluated by incubation with QUANTI-Blue reagent (Invivogen) followed by OD measurements at 620 nm according to the manufacturer’s instruction. TLR stimulation is represented as fold increase at OD 620 nm of SEAP activity over that of untreated cells.
SEAP fold increase
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Quantification of secreted IL-8 by ELISA:
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Nave FHA-commercial Nave FHA-our purified Ag
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Statistical analysis: Data analysis was performed using SPSS 15.0 for Windows. Mann–Whitney U test, student’s t-test and One Way ANOVA test were appropriately used for statistical analysis. pvalues less than 0.05 were considered as statistically significant.
RESULTS Native FHA stimulates TLR2 but not TLR4 or TLR5 transformed cells:
To assess the interaction of the native and recombinant FHA proteins with different TLR molecules, the HEK293 cell lines double transfected with human TLR2, 4 or 5 and a NF-jB reporter gene (HEK293-Blue-TLR) were employed. Along with the TLR molecule and NF-jB reporter gene, both HEK293-TLR2 and -TLR4 cell lines were cotransfected with CD14 co-receptor molecule and HEK293-TLR4 cell line was cotransfected with MD2. TLR stimulation was monitored by © 2014 APMIS. Published by John Wiley & Sons Ltd
C IL8 concentration (pg/mL)
Level of IL-8 in culture supernatants of HEK293-BlueTLR2 cells were measured by Sanquin IL-8 ELISA kit (Sanquin, Amsterdam, The Netherlands) according to the manufacturer’s instructions. The kit sensitivity for IL-8 detection was 5 pg/mL.
Concentration (µg/mL)
Fig. 1. Stimulatory response of TLR-transfected cell lines to their commercial ligands and native FHA. HEK293Blue-TLR2, 4, or 5 cells were treated with different concentrations of FHA, SAC, LPS or flagellin for 16 h. Secreted embryonic alkaline phosphatase (SEAP) or IL-8 (only for HEK293-Blue-TLR2 cells) levels were measured in culture supernatants. Mean values SEM of three or more independent experiments are indicated. For SEAP measurement, data are represented as optical density of SEAP measurement at 620 nm or fold increase in SEAP activity over untreated cells. (A) HEK293-Blue-TLR2, 4, and 5 stimulatory responses to different concentrations or dilutions of their corresponding commercial ligands, including SAC, LPS and flagellin, respectively. The results of TLR2 stimulation were obtained using different fourfold dilutions of SAC starting from 1/2000. (B and C) HEK293-Blue-TLR2 stimulation in response to different concentrations of both commercial and our purified native FHA. Stimulatory response was assessed by measurement of SEAP (B) or IL-8 (C) levels in culture supernatants.
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responded only to its specific ligand, but not to ligands of the other TLRs (data not presented). Native FHA, both commercial and our purified preparations, strongly triggered TLR2 signaling at a low concentration, down to 0.2 lg/mL, in a dose-dependent manner as indicated by the secretion of both SEAP and IL-8 (Figs. 1B and C). The levels of SEAP and IL-8 induced by both preparations of native FHA were significant at all concentrations of FHA (p < 0.001). Despite strong stimulation of TLR2, FHA did not stimulate TLR4 or TLR5-expressing cell lines (data not presented). Mapping of the TLR2 binding region by recombinant FHA segments:
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Fig. 2. Analysis of the purified recombinant FHA fragments by SDS-PAGE. All proteins were purified through His-tag by HisTrap HP column followed by gel filtration. SDS-PAGE analysis was performed using a 10% polyacrylamide gel and silver stain.
SEAP fold increase
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rFHA1
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To localize the TLR2 binding region within FHA molecule, three overlapping recombinant fragments from FHA were produced. The coding sequences for these overlapping regions were amplified from genomic DNA of Bp. These fragments include rFHA1 (from aa 1201 to aa 1584 – a total of 384aa), rFHA2 (from aa 1545 to aa 1917 – a total of 373aa) and rFHA3 (from aa 1878 to aa 2250 – a total of 373aa). Agarose gel electrophoresis of the amplified PCR products from rFHA1-3 confirmed their 1152, 1119, and 1119 bp sizes, respectively (12). All three sequences were separately inserted into pET22b(+) vector and expressed in E. coli BL21 (DE3) as inclusion bodies. The recombinant proteins were purified by HisTrap HP agarose column after solubilization with 8 M urea and gradual renaturation using continuous descending gradients of urea in sodium phosphate buffer. Recovered proteins were finally passed through a gel filtration column to remove contaminating proteins. Purity of all fragments was checked by SDS-PAGE (Fig. 2) and confirmed by immunoblotting using three different preparations of polyclonal anti-FHA and anti-His tag antibodies (12). To identify which part of the native FHA is responsible for TLR2 triggering activity, potential TLR2 binding activities of all three recombinant fragments were tested on HEK293-TLR cell lines. The results showed that only rFHA2 segment (amino acids 1544–1917) stimulates TLR2 pathway in a dose-dependent manner as indicated by the secretion of both SEAP (Fig. 3A) and IL-8 (Fig. 3B) in culture supernatants. No stimulation was observed with rFHA1 or rFHA3 fragments. SEAP and IL-8 levels induced by rFHA2 were significant at all concentrations of rFHA2 compared to untreated cells (p < 0.01). Neither TLR4 nor TLR5 transfected cells were stimulated in response to all rFHA1-3 fragments (data not presented).
B 6000
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Fig. 3. Stimulatory response of TLR2-transfected cell line to recombinant FHA fragments. HEK293-Blue-TLR2 cell line was treated with different concentrations of rFHA1–3 for 16 h. Secreted embryonic alkaline phosphatase (SEAP) (A) or IL-8 (B) levels were measured in culture supernatants. Mean values SEM of three or more independent experiments are indicated. For SEAP measurement, data is represented as fold increase in SEAP activity over untreated cells.
DISCUSSION The mammalian innate immune system is the first defending line encountering microbial pathogens (2). TLR molecules are a broad range of innate immune receptors which respond quickly to the © 2014 APMIS. Published by John Wiley & Sons Ltd
TLR2 TRIGGERING BY BORDETELLA PERTUSSIS FHA
presence of microbial infections by induction of pro-inflammatory responses and bridging between innate and adaptive immune mechanisms (3). Thus identification of the microbial molecules which mediate the TLR-induced immune responses could be very helpful for understanding the microbial pathogenesis mechanisms and designing new targets for vaccination and therapy. In this study, we demonstrated for the first time that FHA, one of the immunodominant antigens of Bordetella pertussis, interacts with human TLR2, but not TLR4 or TLR5. By constructing three overlapping recombinant FHA fragments, we showed that a region of FHA spanning the amino acid residues 1544–1917 is responsible for this binding activity. The three recombinant FHA fragments were expressed in E. coli as inclusion bodies and subsequently purified using a combination of His-tag and gel filtration techniques. Assessment of the purified proteins by SDS-PAGE and immunoblotting suggests isolation of highly pure preparations. All three recombinant FHA1-3 fragments were purified using the same chromatography columns and methodology in different preparations. Both native FHA preparations employed in this study (commercial source and our purified antigen) which were purified from the culture supernatant of B. pertussis, as well as rFHA2 fragment strongly stimulated TLR2 pathway. Our purified native FHA was purified by affinity chromatography using a Sepharose coupled anti-FHA antibody column, which enables preparation of a highly pure FHA by removal of all contaminating molecules (data not shown). It has long been known that the pertussis component of the triple diphtheria-tetanus-pertussis (DTP) vaccines induces adjuvant activity and can potentiate the protective immune responses against other components of the vaccine (13). Similar activities have also been reported for the two immunoprotective antigens of Bp, pertussis toxin (PT), and FHA (14–17). However, the mechanisms responsible for this adjuvant activity had not been identified. Engaging of different TLR molecules by bacterial components like TLR2 by FHA could be an explanation for highly adjuvant activity of Bordetella pertussis. Several studies have reported involvement of TLR4 and TLR2 in the host protective immune response and vaccine-induced immunity against Bp using different approaches (18–25). Similar to LPS isolated from some gram-negative bacteria, Bp lipooligosaccharide (LOS) is able to bind and stimulate TLR4-expressing cells, but this effect is CD14-independent (26). In addition to LOS, PT has also been demonstrated to act as a potent
© 2014 APMIS. Published by John Wiley & Sons Ltd
triggering factor of TLR4 pathway (24, 25). PT and LOS could activate different T helper subtypes and induce protective immune responses through TLR4 stimulation (24, 26). There are some conflicting reports about involvement of TLR2 in Bp infection (20, 21, 24, 25, 27). While Wang et al., using TLRtransfected HEK293 cells have reported that PT has no effects on TLR2 (25), Nasso et al., using the same methodology showed that this toxin can weakly activate this pathway (24). It has also been reported that wild type whole cell Bp is a potent stimulator of TLR2 and this stimulatory activity is not associated to adenylate cyclase toxin of bacteria, but the bacterial component responsible for this activity was not identified (20). In agreement with this study, Schreibelt et al. have recently demonstrated that Infanrix, a commercial acellular pertussis vaccine containing PT, FHA and pertactin antigens, strongly activates TLR2 signaling pathway (27). Our findings regarding robust TLR2 stimulation by FHA extend and confirm the available published data on the triggering of TLR2 pathway by Bp. In the last two decades, many properties have been attributed to FHA as a major immunomodulating factor of Bp (7, 28–30). It was shown to induce apoptosis and pro- or anti-inflammatory responses in macrophages or macrophage-like cell lines (11, 31). Ishibashi et al. have reported that FHA up-regulates ICAM-1 expression on respiratory epithelial cells and binding of RGD motif to VLA5 is responsible for this phenomenon (32). They have also shown that NF-kB activation and signal transduction is necessary for FHA-dependent ICAM-1 over-expression on epithelial cells (33). The role of NF-kB signaling pathway in immunomodulatory mechanisms of FHA was later studied by Abramson and colleagues who showed that exposure of human monocytes or a monocyte-derived macrophage cell line (U-937) to FHA induces rapid activation of NF-kB pathway (29). The immunomodulatory properties of FHA have recently been investigated in more detail by analyzing the genome-wide transcript changes of human PBMC following stimulation with FHA (30). Since NF-kB signaling pathway is up-regulated by most TLR molecules including TLR2, so TLR2 activation by FHA which is shown in this study could explain the NF-kB stimulation and up-regulation by Bordetella pertussis FHA. Mature FHA is a large protein with 220 kDa molecular weight and almost 2370 amino acids length resulting from mainly C-terminal truncation of the 367 kDa precursor (6, 34). There are at least three identified binding domains or motifs in this molecule, including RGD domain (amino acids 1097–1099) which binds to integrin CR3 (very late antigen 5- VLA5) mainly expressed on macrophages
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(8), carbohydrate recognition domain (CRD) (amino acids 1141–1279) which is responsible for bacterial attachment to ciliated cells (9) and heparin binding domain (HBD) (amino acids 442–863) involved in interaction with sulfated carbohydrates on epithelial cells or extra-cellular matrix (10). In addition, epitope mapping studies have demonstrated that most immunodominant epitopes recognized by B cells and T cells are located in the Cterminal part of mature FHA (35–38). Considering these structural characteristics of FHA, we decided to construct three overlapping recombinant segments from the C-terminal region to localize the domain involved in TLR2 binding. Using these recombinant fragments, we were able to explore that the TLR2-binding region lies within amino acid residues 1544–1917 of the FHA molecule. Our findings need to be extended by narrowing down the TLR2 recognizing epitope(s) of FHA through production of recombinant FHA subfragments or by using a synthetic peptide library. In summary, our findings of TLR2 interaction and NF-kB activation by Bordetella pertussis FHA suggest involvement of this bacterial antigen in activation of the innate immune system. The immunostimulatory function was mapped to a region within amino acid residues 1544–1917 of FHA. Given the immunoprotective function of FHA against Bp infection, this region might serve as an important immunodominant domain involved in the control of Bp infection through induction of both the innate and adaptive immune responses.
DISCLOSURE The authors declare no financial/commercial conflicts of interest.
This study was supported in part by grants from the Ministry of Health and Medical Education of Iran and Avicenna Research Institute, ACECR. The authors are grateful to Jalal Khoshnoodi for his technical assistance.
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specificity. Proc Natl Acad Sci USA 2005;102:18578– 83. Abramson T, Kedem H, Relman DA. Modulation of the NF-kappaB pathway by Bordetella pertussis filamentous hemagglutinin. PLoS ONE 2008;3:e3825. Dieterich C, Relman DA. Modulation of the host interferon response and ISGylation pathway by B. pertussis filamentous hemagglutinin. PLoS ONE 2011;6:e27535. McGuirk P, Mills KH. Direct anti-inflammatory effect of a bacterial virulence factor: IL-10-dependent suppression of IL-12 production by filamentous hemagglutinin from Bordetella pertussis. Eur J Immunol 2000;30:415–22. Ishibashi Y, Nishikawa A. Bordetella pertussis infection of human respiratory epithelial cells up-regulates intercellular adhesion molecule-1 expression: role of filamentous hemagglutinin and pertussis toxin. Microb Pathog 2002;33:115–25. Ishibashi Y, Nishikawa A. Role of nuclear factorkappa B in the regulation of intercellular adhesion molecule 1 after infection of human bronchial epithelial cells by Bordetella pertussis. Microb Pathog 2003;35:169–77. Relman DA, Domenighini M, Tuomanen E, Rappuoli R, Falkow S. Filamentous hemagglutinin of Bordetella pertussis: nucleotide sequence and crucial role in adherence. Proc Natl Acad Sci USA 1989;86:2637– 41. Di Tommaso A, Domenighini M, Bugnoli M, Tagliabue A, Rappuoli R, De Magistris MT. Identification of subregions of Bordetella pertussis filamentous hemagglutinin that stimulate human T-cell responses. Infect Immun 1991;59:3313–5. Leininger E, Bowen S, Renauld-Mongenie G, Rouse JH, Menozzi FD, Locht C, et al. Immunodominant domains present on the Bordetella pertussis vaccine component filamentous hemagglutinin. J Infect Dis 1997;175:1423–31. Wilson DR, Siebers A, Finlay BB. Antigenic analysis of Bordetella pertussis filamentous hemagglutinin with phage display libraries and rabbit anti-filamentous hemagglutinin polyclonal antibodies. Infect Immun 1998;66:4884–94. Piatti G. Identification of immunodominant epitopes in the filamentous hemagglutinin of Bordetella pertussis. FEMS Immunol Med Microbiol 1999;23:235–41.
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© 2014 APMIS. Published by John Wiley & Sons Ltd. DOI 10.1111/apm.12332
Interaction of Bordetella pertussis filamentous hemagglutinin with human TLR2: identification of the TLR2-binding domain HOSSEIN ASGARIAN-OMRAN,1,2 ALI AKBAR AMIRZARGAR,3 SACHA ZEERLEDER,4,5 MARZIEH MAHDAVI,6 GERARD VAN MIERLO,4 SHABNAM SOLATI,4 MAHMOOD JEDDI-TEHRANI,6 HODJATALLAH RABBANI,6 LEUCIEN AARDEN3 and FAZEL SHOKRI2,6 1
Department of Immunology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran; 2 Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran; 3 Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran; 4 Department of Immunopathology, Sanquin Research Institute, Amsterdam; 5Department of Hematology, Academic Medical Center, Amsterdam, The Netherlands; and 6Avicenna Research Institute, Monoclonal Antibody Research Center, ACECR, Tehran, Iran
Asgarian-Omran H, Amirzargar AA, Zeerleder S, Mahdavi M, van Mierlo G, Solati S, Jeddi-Tehrani M, Rabbani H, Aarden L, Shokri F. Interaction of Bordetella pertussis filamentous hemagglutinin with human TLR2: identification of the TLR2-binding domain. APMIS 2014. Filamentous hemagglutinin (FHA) is a major adhesion and virulence factor of Bordetella pertussis and also a main component of acellular pertussis vaccines. Interaction of FHA with different receptors on human epithelial and immune cells facilitates entrance and colonization of bacteria as well as immunomodulation of the host immune response. Three overlapping segments of the FHA gene were cloned in a prokaryotic expression vector and the recombinant proteins were purified. These recombinant fragments along with the native FHA protein were employed to assess their potential Toll-like receptor (TLR) stimulatory effects and to localize the TLR binding region. TLR stimulation was monitored by applying HEK293-Blue cell lines cotransfected with TLR2, 4, or 5 and a NF-jB reporter gene. Culture supernatants were checked for secretion of the reporter gene product and IL-8 as indicators of TLR stimulation. Native FHA was found to strongly stimulate TLR2, but not TLR4 or TLR5 transfected cells. Among recombinant FHA fragments only the fragment spanning amino acid residues 1544–1917 was able to exhibit the TLR2 stimulating property of FHA. Interaction of FHA with TLR2 suggests its involvement in induction of the innate immune system against Bordetella pertussis. The TLR2-binding domain of FHA may contribute to immunoprotection against pertussis infection. Key words: Bordetella pertussis; filamentous hemagglutinin; innate immunity; Toll-like receptor; TLR2. Fazel Shokri, Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. e-mail: [email protected]
The mammalian innate immune system senses microbial structures known as pathogen associated molecular patterns (PAMPs) through a variety of pattern recognition receptors (PRRs) present on the surface and in the cytoplasm of different cell types (1). One of the most important and wellstudied groups of PRRs is Toll-like receptors (TLRs) which are currently composed of 10 different members (TLR1-10) in human (2). TLR family members are homo- or hetero-dimeric molecules Received 1 January 2014. Accepted 6 May 2014
which can bind to various microbial products and initiate the immune responses to infectious diseases (2). TLR2 is a cell surface protein which is expressed both as a homo- and hetero-dimers with TLR1 or TLR6 (3). Human TLR2 trigger pro-inflammatory responses to pathogens by interacting with a broad range of PAMPs such as bacterial lipopeptides, peptidoglycan, lipoteichoic acid, fungal zymosan, glycosylphosphatidylinositol-anchored mucin-like glycoproteins from Trypanosoma cruzi and hemagglutinin from measles virus (3). 1
ASGARIAN-OMRAN et al.
Whooping cough, pertussis, is an acute respiratory disease caused by the gram-negative bacteria, Bordetella pertussis (Bp) (4). Vaccination against pertussis was initiated in the 1940s by inoculation with heat inactivated whole cell bacteria (4). In spite of dramatic reduction in pertussis cases after vaccination with whole cell bacteria, reactogenecity and side effects of this vaccine which were more remarkable in neonates and children, prompted researchers to shift to acellular pertussis subunit vaccines (Ac) consisting of a cocktail of 2–5 immunogenic antigens of Bp including pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin, and fimberiae 2–3 antigens (5). Filamentous hemagglutinin (FHA) is the major adhesion and immunomodulator molecule of Bp which is synthesized as a large 367 kDa precursor protein and modified to a mature 220 kDa form by proteolytic degradation at the N- and C-terminus (6). This protein has long been recognized as a highly immunoprotective antigen with crucial roles in pertussis pathogenesis (7). Different binding domains have been identified for FHA which mediate the attachment and entrance of the bacteria to the epithelial cells (8–10). Interaction of Bp with the target epithelial cells via these domains has been shown to induce pro-inflammatory and pro-apoptotic signals which contribute to the pathogenesis of pertussis (11). In this study, we have demonstrated for the first time that FHA binds to human TLR2 and induces secretion of the pro-inflammatory cytokine IL-8 following activation of the NF-kB signaling pathway. The TLR2 binding site was mapped to a region of FHA spanning the amino acids 1544–1917, using three overlapping recombinant fragments of FHA. MATERIALS AND METHODS Cell lines, bacterial strains, culture media, and reagents: Bordetella pertussis Tohama I strain (ATCC, Manassas, CA, USA) was employed for FHA gene amplification and production of recombinant FHA proteins. The bacteria were grown on Bordet-Gengou agar (Becton Dickinson, Franklin Lakes, NJ, USA) supplemented with 15% defibrinated sheep blood, 1% v/v glycerol, 1% v/w proteose peptone (DIFCO, Franklin Lakes, NJ, USA) and 40 lg/ mL cephalexin for 72 h. Both E. coli strains JM109 and BL21 (DE3) were purchased from Novagen (Merck KGaA, Darmstadt, Germany) and cultured in LB agar containing 1% w/v peptone (Merck KGaA), 0.5% w/v yeast extract (Merck KGaA), 0.6% w/v NaCl and 1.5% w/v agar (Merck KGaA). LB broth medium constituents were the same as LB agar without agar. HEK293-Blue-TLR2 (transfected with human TLR2, CD14 and NF-jB reporter gene), HEK293-Blue-TLR4
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(transfected with human TLR4, CD14, MD2 and NF-jB reporter gene) and HEK293-Blue-TLR5 (transfected with human TLR5 and NF-jB reporter gene) cell lines (Invivogen, San Diego, CA, USA) were used for TLR stimulation studies. Cells were cultured in IMDM medium supplemented with 5% fetal calf serum (FCS) (Gibco-Biocuh, Paisley, Scotland), 100 lg/mL streptomycin, 100 IU/mL penicillin, 100 lg/mL Normocin (Invivogen) and selective antibiotics according to the manufacture’s instruction. All cells were cultured at 37 °C in a humidified atmosphere with 5% CO2. Native FHA was obtained from Sigma-Aldrich (St. Louis, MO, USA) and was also purified in our lab using an antibody affinity-based column from Bp culture supernatant and characterized by SDS-PAGE and Western blotting (data not shown). Staphylococcus aureus Cowan I strain cells (SAC) (Pansorbin, Calbiochem, La Jolla, CA, USA), E. coli lipopolysacharaide (LPS) (Invivogen) and flagellin (Invivogen) were used as commercial TLR2, 4 and 5 agonists, respectively, to optimize the stimulation conditions of TLR-transfected HEK293-Blue cells.
Construction and expression of recombinant proteins: As previously described, three overlapping regions from FHA coding sequence were selected and amplified from Bp genomic DNA for production of recombinant proteins designated rFHA1-3 (12).
Purification of the recombinant proteins: Recombinant protein purification was performed in a denaturing condition using the Acta Explorer Chromatography System (GE Healthcare Life Sciences, Uppsala, Sweden). Harvested bacterial pellets containing inclusion bodies were solubilized in 20 mL of lysis buffer containing 100 mM NaH2PO4, 100 mM NaCl, 30 mM Tris-HCL, pH:8 and incubated on ice for 1 h. This solution was continuously sonicated at 70% amplitude for 15 min to cell destruction and then centrifuged at 12 000 g for 10 min. Supernatants were discarded and pellets were resuspended in buffer A (100 mM NaH2PO4, 50 mM NaCl, 10 mM Tris-HCL, 30 mM imidazole, 8M urea, pH: 8) and incubated at room temperature for 1 h. After centrifugation at 12 000 g, pellets were discarded and supernatants containing His-tagged recombinant proteins were applied as starting materials on HisTrap HP (GE Healthcare Life Sciences) column, which has been equilibrated with buffer A. Following the binding of recombinant proteins to column, the protein refolding process was performed by applying a descending continuous gradient of urea concentration from 8 M to zero for 3 h. Target proteins were then eluted by applying ascending continuous gradient of imidazole concentrations from 30 mM–1 M. All recombinant proteins were eluted at a range of 230–250 mM of imidazole. The purity of selected fractions was checked by silver stained SDS-PAGE. To get rid of any contamination, the purified fractions collected from Ni-NTA agarose column were concentrated by Amicon Ultra-15 centrifugal filter units (Merck KGaA) and applied to Hiload 16/60 Superdex200 prep grade gel filtration column (GE Healthcare Life Sciences) equilibrated with 0.15 M PBS. The peaks corresponding to
© 2014 APMIS. Published by John Wiley & Sons Ltd
TLR2 TRIGGERING BY BORDETELLA PERTUSSIS FHA
molecular weight of the target proteins were selected and concentrated using Amicon Ultra-4 centrifugal filter units (Merck KGaA) followed by filtration with 0.2 lm membrane filters (Merck KGaA). Protein concentrations were determined by BCA colorimetric assay (Pierce, Rockford, IL, USA).
Western blot analysis of the recombinant FHA proteins:
measurement of secreted embryonic alkaline phosphatase (SEAP), the product of NF-jB reporter gene, and IL-8 in culture supernatants. Responsiveness and sensitivity of the HEK293-Blue-TLR2, 4 and 5 cell lines were initially assessed using their commercial ligands, including SAC, LPS, and flagellin, respectively (Fig. 1A). Each cell line
After assessment of the recombinant proteins by silverstained 10% SDS-PAGE, the reactivity of all recombinant proteins was confirmed by immunoblot analysis (12).
A
TLR stimulation assay: HEK293-Blue-TLR cells were seeded at 40 * 103/well on 96 well flat-bottom culture plates and stimulated with different concentrations of native and recombinant FHA as well as their commercial agonists. After 16 h incubation at 37 °C in a humidified atmosphere with 5% CO2, supernatants were harvested and the activity of secreted embryonic alkaline phosphatase (SEAP) was evaluated by incubation with QUANTI-Blue reagent (Invivogen) followed by OD measurements at 620 nm according to the manufacturer’s instruction. TLR stimulation is represented as fold increase at OD 620 nm of SEAP activity over that of untreated cells.
SEAP fold increase
B
Quantification of secreted IL-8 by ELISA:
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Statistical analysis: Data analysis was performed using SPSS 15.0 for Windows. Mann–Whitney U test, student’s t-test and One Way ANOVA test were appropriately used for statistical analysis. pvalues less than 0.05 were considered as statistically significant.
RESULTS Native FHA stimulates TLR2 but not TLR4 or TLR5 transformed cells:
To assess the interaction of the native and recombinant FHA proteins with different TLR molecules, the HEK293 cell lines double transfected with human TLR2, 4 or 5 and a NF-jB reporter gene (HEK293-Blue-TLR) were employed. Along with the TLR molecule and NF-jB reporter gene, both HEK293-TLR2 and -TLR4 cell lines were cotransfected with CD14 co-receptor molecule and HEK293-TLR4 cell line was cotransfected with MD2. TLR stimulation was monitored by © 2014 APMIS. Published by John Wiley & Sons Ltd
C IL8 concentration (pg/mL)
Level of IL-8 in culture supernatants of HEK293-BlueTLR2 cells were measured by Sanquin IL-8 ELISA kit (Sanquin, Amsterdam, The Netherlands) according to the manufacturer’s instructions. The kit sensitivity for IL-8 detection was 5 pg/mL.
Concentration (µg/mL)
Fig. 1. Stimulatory response of TLR-transfected cell lines to their commercial ligands and native FHA. HEK293Blue-TLR2, 4, or 5 cells were treated with different concentrations of FHA, SAC, LPS or flagellin for 16 h. Secreted embryonic alkaline phosphatase (SEAP) or IL-8 (only for HEK293-Blue-TLR2 cells) levels were measured in culture supernatants. Mean values SEM of three or more independent experiments are indicated. For SEAP measurement, data are represented as optical density of SEAP measurement at 620 nm or fold increase in SEAP activity over untreated cells. (A) HEK293-Blue-TLR2, 4, and 5 stimulatory responses to different concentrations or dilutions of their corresponding commercial ligands, including SAC, LPS and flagellin, respectively. The results of TLR2 stimulation were obtained using different fourfold dilutions of SAC starting from 1/2000. (B and C) HEK293-Blue-TLR2 stimulation in response to different concentrations of both commercial and our purified native FHA. Stimulatory response was assessed by measurement of SEAP (B) or IL-8 (C) levels in culture supernatants.
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responded only to its specific ligand, but not to ligands of the other TLRs (data not presented). Native FHA, both commercial and our purified preparations, strongly triggered TLR2 signaling at a low concentration, down to 0.2 lg/mL, in a dose-dependent manner as indicated by the secretion of both SEAP and IL-8 (Figs. 1B and C). The levels of SEAP and IL-8 induced by both preparations of native FHA were significant at all concentrations of FHA (p < 0.001). Despite strong stimulation of TLR2, FHA did not stimulate TLR4 or TLR5-expressing cell lines (data not presented). Mapping of the TLR2 binding region by recombinant FHA segments:
4
Fig. 2. Analysis of the purified recombinant FHA fragments by SDS-PAGE. All proteins were purified through His-tag by HisTrap HP column followed by gel filtration. SDS-PAGE analysis was performed using a 10% polyacrylamide gel and silver stain.
SEAP fold increase
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To localize the TLR2 binding region within FHA molecule, three overlapping recombinant fragments from FHA were produced. The coding sequences for these overlapping regions were amplified from genomic DNA of Bp. These fragments include rFHA1 (from aa 1201 to aa 1584 – a total of 384aa), rFHA2 (from aa 1545 to aa 1917 – a total of 373aa) and rFHA3 (from aa 1878 to aa 2250 – a total of 373aa). Agarose gel electrophoresis of the amplified PCR products from rFHA1-3 confirmed their 1152, 1119, and 1119 bp sizes, respectively (12). All three sequences were separately inserted into pET22b(+) vector and expressed in E. coli BL21 (DE3) as inclusion bodies. The recombinant proteins were purified by HisTrap HP agarose column after solubilization with 8 M urea and gradual renaturation using continuous descending gradients of urea in sodium phosphate buffer. Recovered proteins were finally passed through a gel filtration column to remove contaminating proteins. Purity of all fragments was checked by SDS-PAGE (Fig. 2) and confirmed by immunoblotting using three different preparations of polyclonal anti-FHA and anti-His tag antibodies (12). To identify which part of the native FHA is responsible for TLR2 triggering activity, potential TLR2 binding activities of all three recombinant fragments were tested on HEK293-TLR cell lines. The results showed that only rFHA2 segment (amino acids 1544–1917) stimulates TLR2 pathway in a dose-dependent manner as indicated by the secretion of both SEAP (Fig. 3A) and IL-8 (Fig. 3B) in culture supernatants. No stimulation was observed with rFHA1 or rFHA3 fragments. SEAP and IL-8 levels induced by rFHA2 were significant at all concentrations of rFHA2 compared to untreated cells (p < 0.01). Neither TLR4 nor TLR5 transfected cells were stimulated in response to all rFHA1-3 fragments (data not presented).
B 6000
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rFHA3
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Fig. 3. Stimulatory response of TLR2-transfected cell line to recombinant FHA fragments. HEK293-Blue-TLR2 cell line was treated with different concentrations of rFHA1–3 for 16 h. Secreted embryonic alkaline phosphatase (SEAP) (A) or IL-8 (B) levels were measured in culture supernatants. Mean values SEM of three or more independent experiments are indicated. For SEAP measurement, data is represented as fold increase in SEAP activity over untreated cells.
DISCUSSION The mammalian innate immune system is the first defending line encountering microbial pathogens (2). TLR molecules are a broad range of innate immune receptors which respond quickly to the © 2014 APMIS. Published by John Wiley & Sons Ltd
TLR2 TRIGGERING BY BORDETELLA PERTUSSIS FHA
presence of microbial infections by induction of pro-inflammatory responses and bridging between innate and adaptive immune mechanisms (3). Thus identification of the microbial molecules which mediate the TLR-induced immune responses could be very helpful for understanding the microbial pathogenesis mechanisms and designing new targets for vaccination and therapy. In this study, we demonstrated for the first time that FHA, one of the immunodominant antigens of Bordetella pertussis, interacts with human TLR2, but not TLR4 or TLR5. By constructing three overlapping recombinant FHA fragments, we showed that a region of FHA spanning the amino acid residues 1544–1917 is responsible for this binding activity. The three recombinant FHA fragments were expressed in E. coli as inclusion bodies and subsequently purified using a combination of His-tag and gel filtration techniques. Assessment of the purified proteins by SDS-PAGE and immunoblotting suggests isolation of highly pure preparations. All three recombinant FHA1-3 fragments were purified using the same chromatography columns and methodology in different preparations. Both native FHA preparations employed in this study (commercial source and our purified antigen) which were purified from the culture supernatant of B. pertussis, as well as rFHA2 fragment strongly stimulated TLR2 pathway. Our purified native FHA was purified by affinity chromatography using a Sepharose coupled anti-FHA antibody column, which enables preparation of a highly pure FHA by removal of all contaminating molecules (data not shown). It has long been known that the pertussis component of the triple diphtheria-tetanus-pertussis (DTP) vaccines induces adjuvant activity and can potentiate the protective immune responses against other components of the vaccine (13). Similar activities have also been reported for the two immunoprotective antigens of Bp, pertussis toxin (PT), and FHA (14–17). However, the mechanisms responsible for this adjuvant activity had not been identified. Engaging of different TLR molecules by bacterial components like TLR2 by FHA could be an explanation for highly adjuvant activity of Bordetella pertussis. Several studies have reported involvement of TLR4 and TLR2 in the host protective immune response and vaccine-induced immunity against Bp using different approaches (18–25). Similar to LPS isolated from some gram-negative bacteria, Bp lipooligosaccharide (LOS) is able to bind and stimulate TLR4-expressing cells, but this effect is CD14-independent (26). In addition to LOS, PT has also been demonstrated to act as a potent
© 2014 APMIS. Published by John Wiley & Sons Ltd
triggering factor of TLR4 pathway (24, 25). PT and LOS could activate different T helper subtypes and induce protective immune responses through TLR4 stimulation (24, 26). There are some conflicting reports about involvement of TLR2 in Bp infection (20, 21, 24, 25, 27). While Wang et al., using TLRtransfected HEK293 cells have reported that PT has no effects on TLR2 (25), Nasso et al., using the same methodology showed that this toxin can weakly activate this pathway (24). It has also been reported that wild type whole cell Bp is a potent stimulator of TLR2 and this stimulatory activity is not associated to adenylate cyclase toxin of bacteria, but the bacterial component responsible for this activity was not identified (20). In agreement with this study, Schreibelt et al. have recently demonstrated that Infanrix, a commercial acellular pertussis vaccine containing PT, FHA and pertactin antigens, strongly activates TLR2 signaling pathway (27). Our findings regarding robust TLR2 stimulation by FHA extend and confirm the available published data on the triggering of TLR2 pathway by Bp. In the last two decades, many properties have been attributed to FHA as a major immunomodulating factor of Bp (7, 28–30). It was shown to induce apoptosis and pro- or anti-inflammatory responses in macrophages or macrophage-like cell lines (11, 31). Ishibashi et al. have reported that FHA up-regulates ICAM-1 expression on respiratory epithelial cells and binding of RGD motif to VLA5 is responsible for this phenomenon (32). They have also shown that NF-kB activation and signal transduction is necessary for FHA-dependent ICAM-1 over-expression on epithelial cells (33). The role of NF-kB signaling pathway in immunomodulatory mechanisms of FHA was later studied by Abramson and colleagues who showed that exposure of human monocytes or a monocyte-derived macrophage cell line (U-937) to FHA induces rapid activation of NF-kB pathway (29). The immunomodulatory properties of FHA have recently been investigated in more detail by analyzing the genome-wide transcript changes of human PBMC following stimulation with FHA (30). Since NF-kB signaling pathway is up-regulated by most TLR molecules including TLR2, so TLR2 activation by FHA which is shown in this study could explain the NF-kB stimulation and up-regulation by Bordetella pertussis FHA. Mature FHA is a large protein with 220 kDa molecular weight and almost 2370 amino acids length resulting from mainly C-terminal truncation of the 367 kDa precursor (6, 34). There are at least three identified binding domains or motifs in this molecule, including RGD domain (amino acids 1097–1099) which binds to integrin CR3 (very late antigen 5- VLA5) mainly expressed on macrophages
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(8), carbohydrate recognition domain (CRD) (amino acids 1141–1279) which is responsible for bacterial attachment to ciliated cells (9) and heparin binding domain (HBD) (amino acids 442–863) involved in interaction with sulfated carbohydrates on epithelial cells or extra-cellular matrix (10). In addition, epitope mapping studies have demonstrated that most immunodominant epitopes recognized by B cells and T cells are located in the Cterminal part of mature FHA (35–38). Considering these structural characteristics of FHA, we decided to construct three overlapping recombinant segments from the C-terminal region to localize the domain involved in TLR2 binding. Using these recombinant fragments, we were able to explore that the TLR2-binding region lies within amino acid residues 1544–1917 of the FHA molecule. Our findings need to be extended by narrowing down the TLR2 recognizing epitope(s) of FHA through production of recombinant FHA subfragments or by using a synthetic peptide library. In summary, our findings of TLR2 interaction and NF-kB activation by Bordetella pertussis FHA suggest involvement of this bacterial antigen in activation of the innate immune system. The immunostimulatory function was mapped to a region within amino acid residues 1544–1917 of FHA. Given the immunoprotective function of FHA against Bp infection, this region might serve as an important immunodominant domain involved in the control of Bp infection through induction of both the innate and adaptive immune responses.
DISCLOSURE The authors declare no financial/commercial conflicts of interest.
This study was supported in part by grants from the Ministry of Health and Medical Education of Iran and Avicenna Research Institute, ACECR. The authors are grateful to Jalal Khoshnoodi for his technical assistance.
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