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Epitope Mapping of Porphyromonas gingivalis Heat-shock Protein and Human Heat-shock Protein in Human Atherosclerosis
J.-I. Choi1,*,
S.-W. Chung2,
H.-S. Kang3,
B.Y. Rhim4,
Y.-M. Park5,
U.-S. Kim1 and
S.-J. Kim1
1 Department of Periodontology and Research Institute for Oral Biotechology, School of Dentistry, 2 Department of Thoracic and Cardiovascular Surgery, School of Medicine, 3 Department of Molecular Biology, College of Natural Sciences, 4 Department of Pharmacology, and 5 Department of Microbiology, School of Medicine, Pusan National University, 1–10, Ami-Dong, Seo-Ku, Pusan 602–739, Korea;
Correspondence: * corresponding author, jrapa{at}pusan.ac.kr
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ABSTRACT
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To identify T- and/or cross-reactive B-cell epitopes of P. gingivalis and human heat-shock protein (HSP)60 in atherosclerosis patients, we synthesized 104 overlapping synthetic peptides spanning whole molecules of P. gingivalis HSP60 and human HSP60, respectively. T-cell epitopes of P. gingivalis HSP were identified with the use of previously established P. gingivalis HSP-reactive T-cell lines. B-cell epitopes of P. gingivalis HSP60 and human HSP60 were identified by the use of patients’ sera. Anti-P. gingivalis, anti-P. gingivalis HSP60, or anti-human HSP60 IgG antibody titers were higher in the atherosclerosis patients compared with the healthy subjects. Five immunodominant peptides of P. gingivalis HSP60, identified as T-cell epitopes, were also found to be B-cell epitopes. Moreover, 6 cross-reactive B-cell epitopes of human HSP60 were identified. It was concluded that P. gingivalis HSP60 might be involved in the immunoregulatory process of atherosclerosis, with common T- and/or B-cell epitope specificities and with cross-reactivity with human HSP60.
Key Words: Porphyromonas gingivalis • heat-shock protein • epitope • atherosclerosis
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INTRODUCTION
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Periodontal infection may be one of the risk factors for cardiovascular diseases (Chiu, 1999; Haraszthy et al., 2000; Okuda et al., 2001). This association is supported by the recent observation that Porphyromonas gingivalis (P. gingivalis), a primary periodontopathic pathogen, can invade endothelial cells (Deshpande et al., 1998).
Due to a considerably high degree of sequence homology between bacterial and human heat-shock proteins (HSP), this protein might be involved in autoimmune disease mechanisms operating in humans (Hansson, 2001; Wick et al., 2001). T-cell immune responses specific to bacterial or human HSP have been demonstrated in atherosclerosis (Kaufmann et al., 1990; Ross, 1993; Wick et al., 1995). The host immune system primed by HSP of a major periodontal pathogen, such as Porphyromonas gingivalis (P. gingivalis), can cross-react with its cognate mammalian counterpart in gingival connective tissue or arterial walls (Yamazaki et al., 2002). To provide evidence that P. gingivalis may be actively involved in the immunopathogenic process of atherosclerosis, we have recently reported T-cell responses specific to P. gingivalis or P. gingivalis HSP in atherosclerosis patients (Choi et al., 2001a, 2002).
In clarifying the immune mechanisms modulating the autoimmune diseases, it is critical that one identify the immunodominant epitope(s) of an infecting pathogen that is (are) exclusively recognized by T- and/or B-cells. In the present study, we have attempted to characterize P. gingivalis HSP-reactive T-cell lines, and to identify T- and/or B-cell epitopes of P. gingivalis HSP and human HSP.
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MATERIALS & METHODS
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Patient Selection Criteria
Patients who were subject to surgical intervention of atheromatous plaques were screened for destructive periodontal disease. Probing pocket depths and levels of attachment were measured for diagnosis of periodontal disease according to procedures described by the American Academy of Periodontology (1991). Periodontitis patients with elevated IgG antibody responses to only P. gingivalis were included in the study. Informed consent forms were obtained from patients, and the protocol was approved by the Institutional Review Board of Pusan National University Hospital. Clinical investigation was conducted according to Declaration of Helsinki principles. Clinically healthy subjects, without any noticeable history of systemic or destructive periodontal disease, were selected for the control group.
Measurement of Anti-P. gingivalis, Anti-P. gingivalis HSP60, and Anti-human HSP60 Serum IgG Antibody Titers
Recombinant P. gingivalis HSP60 was produced and purified from the P. gingivalis GroEL gene (Maeda et al., 1994), and the purity was verified as previously described (Choi et al., 2002). Microtiter plates coated with either formalinized P. gingivalis cells, P. gingivalis HSP60, or human HSP60 (StressGen, Victoria, BC, Canada), diluted in 10 mM phosphate buffer (Choi et al., 2000, 2001a,Choi et al., b), were incubated with an aliquot of serum samples. After samples were washed, peroxidase-conjugated mouse anti-human IgG (H+L) (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was added. The plates were washed, and an aliquot of tetramethylbenzidine (Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA) was added for incubation, followed by the addition of 0.18 M H2SO4 to stop the reaction. Optical densities were plotted as a function of the serum dilution factor for determination of the titer. Antibody titer was considered to be elevated if it was higher than the mean control titer + 3x the standard deviation.
Western Immunoblot
Recombinant P. gingivalis HSP60 or human HSP60 was subject to SDS-PAGE and electro-transferred to nitrocellulose membrane. After the membrane was blocked, human sera were added for incubation. The membrane was washed, followed by the addition of horseradish-peroxidase-conjugated mouse anti-human IgG (H+L) (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). After the membrane was washed, tetramethylbenzidine was added for color development. For identification of cross-reactivity of bacterial HSP among periodontopathogenic bacteria, bacterial cell lysates of heat-shocked P. gingivalis, Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Prevotella intermedia, Treponema denticola, and Fusobacterium nucleatum were subject to immunoblot with mouse anti-P. gingivalis HSP antisera.
Synthetic Peptide
We synthesized a total of 108 decapeptides, spanning the entire amino acid sequence of P. gingivalis GroEL and human HSP60, respectively, using an Epitope-Scanning Kit (Chiron Mimotopes, Clayton, Victoria, Australia). Peptides were designed to overlap by 5 amino acid residues.
T-cell Epitope Mapping
T-cells (1 x 105 cells/well) from previously established P. gingivalis HSP-reactive T-cell lines (Choi et al., 2002) were inclubated with synthetic peptides (5 µg/mL) of P. gingivalis HSP60 and antigen-presenting cells (5 x 106 cells/well). After 48 hrs of incubation, the cells were labeled with 3H-thymidine for an additional 6 hrs of incubation and counted in a liquid scintillation counter. Proliferation was presented as the stimulation index (SI): the ratio of the mean counts per minute (cpm) with antigen to the cpm without antigen. SI values of 3 or greater were considered to be positive.
Preparation of Conjugated Plate
Conjugation of the synthetic peptide of P. gingivalis HSP60 or human HSP60 to the microtiter plate (CovaLink plate, NUNC, Roskilde, Denmark) was done with a water-soluble 1-ethyl-(3-dimethyl-aminopropyl) (EDC) carbodiimide in the presence of N-hydroxy-succinamide (NHS). Each peptide was dissolved in dimethyl sulfoxide, diluted with 0.1 M carbonate-bicarbonate buffer, and used for coating plates.
B-cell Epitope Mapping
The conjugated plate was washed, and aliquots of serum samples were added and incubated. After the plates were washed, peroxidase-conjugated mouse anti-human IgG (H+L) (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was added. The plates were incubated and washed, and an aliquot of tetramethylbenzidine (Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA) was added for incubation, followed by the addition of 0.18 M H2SO4. Optical density means and the standard deviations of the ELISA signals to the peptides were calculated for each sample. The mean ± 3 standard deviation was assigned as the baseline, and each signal to the peptide was assigned a positive or negative response.
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RESULTS
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Clinical and Antibody Profiles of Patients
Six atherosclerosis patients, diagnosed as having arteriosclerosis obliterans in right or left superficial femoral arteries, or both, were screened and subjected to surgical intervention of atherosclerotic plaques. All of them were males, aged between 57 and 73 yrs, and they smoked. They demonstrated the characteristic features of severe periodontitis. They also had elevated IgG antibody titers to both P. gingivalis HSP60 and human HSP60 when compared with the control subjects (Table 1 ).
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Table 1. Clinical Profiles and IgG Antibody Titers against P. gingivalis, P. gingivalis HSP, and Human HSP in Atherosclerosis Patients and Healthy Control Subjects
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Western Immunoblot
Sera from atherosclerosis patients who had elevated IgG antibody to P. gingivalis HSP60 demonstrated strong cross-reactive patterns with P. gingivalis HSP60 and human HSP60, as evidenced by Western immunoblot (Fig. 1A). Sera from healthy controls reacted neither with P. gingivalis HSP60 nor with human HSP60. In addition, mouse anti-P. gingivalis HSP60 antisera recognized P. gingivalis HSP and cross-reacted with all the HSPs induced by heat-shock treatment of putative periodontopathogenic bacteria tested (Fig. 1B).
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Figure 1. (A) Western immunoblot pattern demonstrating the cross-recognition of P. gingivalis HSP60 and human HSP60 by sera obtained from six atherosclerosis patients (A1-A6). This was not observed in healthy control subjects (N1-N6). Pairs of two lanes represent serum reactivity of one subject, left panels being against P. gingivalis HSP 60 (P), right panels being against human HSP 60 (H), respectively. (B) In addition, mouse anti-P. gingivalis HSP60 antisera recognized P. gingivalis HSP and cross-reacted with all the HSPs induced by heat-shock treatment of putative periodontopathogenic bacteria tested. Pg, P. gingivalis; Aa, A. actinomycetemcomitans; Bf, B. forsythus; Fn, F. nucleatum; Pi, P. intermedia; Td, T. denticola.
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T-cell Epitope Mapping
T-cell lines established from each patient did not demonstrate any proliferative responses to other bacteria or bacterial HSP, as demonstrated by our previous observation (Choi et al., 2002). These T-cells showed multiple reactivities to peptides among the 108 overlapping peptides spanning the whole sequence of P. gingivalis HSP60. Of these, 5 peptides were designated as the major T-cell epitopes of which the SI values were 3 or greater in all six patients. The numbers and the respective amino acid sequences of the 5 peptides are listed in Table 2.
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Table 2. Common T- and B-cell Epitopes of P. gingivalis HSP60, and B-cell Epitopes Commonly Identified in P. gingivalis HSP60 and Human HSP60
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B-cell Epitope Mapping
Seropositivities of six patients’ sera against P. gingivalis HSP60 and human HSP60 are depicted as optical densities in Fig. 2. Fifteen antigenic peptides that showed positive signals in more than four out of six patients were designated as major B-cell epitopes of P. gingivalis HSP60 (peptides no. 3, 6, 15, 29, 33, 39, 53, 56, 69, 74, 75, 85, 93, 99, and 102). Of these, 5 were identical to T-cell epitopes (Table 2 ). In the same manner, major B-cell epitopes of human HSP60 were identified, being peptides no. 15, 29, 53, 56, 69, and 74 (Table 2).
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Figure 2. Bar diagram representing optical densities as a measure of positive reactivity of patient sera to 108 synthetic peptides spanning whole molecules of P. gingivalis HSP60 and human HSP60, respectively. The number indicates each patient. Fifteen antigenic peptides that showed positive signals in more than four out of six patients were designated as B-cell epitopes of P. gingivalis HSP60. In the same manner, B-cell epitopes of human HSP60 were identified.
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DISCUSSION
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HSPs are highly conserved, and, in the case of HSP60, approximately 60% sequence homology between the mycobacterial and human cognates has been observed (Jindal et al., 1989). Hence, T-cells and antibodies with specificity for conserved sequences are potentially auto-reactive, contributing to the pathogenesis of infectious diseases, including periodontal disease (Schoenfeld and Isenberg, 1988; Yamazaki et al., 2002). Previously, we have demonstrated P. gingivalis HSP-reactive T-cell lines from mononuclear cells isolated from atheromatous plaque (Choi et al., 2002). The T-cells were a mixture of CD4+ and CD8+ cells, producing the cytokines characteristic of both Th1 and Th2 subsets, suggesting that P. gingivalis HSP-specific T-cells "home" to the atheroma lesion, where P. gingivalis have infiltrated (Deshpande et al., 1998).
Mean anti-P. gingivalis, anti-P. gingivalis HSP60, or human HSP60 IgG antibody titers in six atherosclerosis patients were higher when compared with the control subjects. Sera from atherosclerosis patients who had elevated IgG antibody to P. gingivalis HSP60 demonstrated cross-reactivity with human HSP60, as evidenced by Western blot results, suggesting that the autoimmune pathogenic mechanisms were regulated by P. gingivalis HSP in human atherosclerosis (Hansson, 2001; Wick et al., 2001; Choi et al., 2002; Yamazaki et al., 2002). Moreover, mouse anti-P. gingivalis HSP60 antisera recognized and reacted with P. gingivalis HSP as well as with all other HSPs induced by heat treatment of putative periodontopathogenic bacteria tested. Several authors have claimed the critical role of bacterial stress proteins or human HSP in recruiting immune cells which target antigens, consequently leading to the development of plaque and atheroma lesions (Kaufmann et al., 1990; Ross, 1993; Wick et al., 1995).
We performed the present study to scan anti-HSP60 serum antibody for specific linear B-cell epitopes on the P. gingivalis HSP60 proteins and compared them with T-cell epitopes. Interestingly, 5 of these epitopes were identified as common T-cell and B-cell epitopes in atherosclerosis patients (Table 2). When B-cell epitopes for corresponding sequences of P. gingivalis HSP60 or human HSP60 were compared, peptides no. 15, 29, 53, 56, 69, and 74 of P. gingivalis HSP60 and human HSP60 were identified as cross-reactive B-cell epitopes. Of these, peptide no. 15 of P. gingivalis HSP60 (VKEVASKTND) has also been identified as a B-cell epitope in periodontitis (Maeda et al., 2000), and the corresponding peptide no. 15 of human HSP60 (VQDVANNTNE) has been identified as a B-cell epitope in atherosclerosis (Metzler et al., 1997) and a T-cell epitope in atherosclerosis (unpublished observation). Of additional interest, peptide no. 56 of P. gingivalis HSP60 (PGFGDRRKAM) and human HSP60 (PGFGDNRKNQ), respectively, was consistently identified as a cross-reactive B-cell epitope in all the patients. This peptide has also been identified as a B-cell epitope in periodontitis (Maeda et al., 2000). These immunodominant cross-reactive epitopes might be critical in the immunopathogenic process in human atherosclerosis and could thus be candidate peptides for prospective vaccine development against atherosclerosis. We have established the epitope-specific T-cell lines from human atheromatous plaque to transfer adoptively into severe combined immunodeficiency (SCID) mice. This would eventually allow one to evaluate human immune responses for vaccine efficacy testing in experimental atherogenesis.
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ACKNOWLEDGMENTS
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We are thankful to Professor Yoji Murayama for providing P. gingivalis GroEL gene, Dr. K. Ishihara for providing bacterial culture, and S.W. Lee for his expertise in flow cytometry. This work was supported by Grant #1999-2-20500-004-3 from KOSEF.
Received for publication May 10, 2003.
Revision received August 28, 2004.
Accepted for publication September 14, 2004.
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REFERENCES
|
|---|
- American Academy of Periodontology (1991). Current procedural terminology. 6th ed. Chicago, IL.
- Chiu B (1999). Multiple infections in carotid atherosclerotic plaques. Am Heart J 138(Pt 2):S534–S536.[CrossRef][Medline]
[Order article via Infotrieve]
- Choi JI, Borrello MA, Smith E, Zauderer M (2000). Polarization of Porphyromonas gingivalis-specific helper T cell subsets by prior immunization with Fusobacterium nucleatum. Oral Microbiol Immunol 15:181–187.[Medline]
[Order article via Infotrieve]
- Choi J, Chung SW, Kim SJ, Kim SJ (2001a). Establishment of Porphyromonas gingivalis-specific T cell lines from atherosclerosis patients. Oral Microbiol Immunol 16:316–318.[Medline]
[Order article via Infotrieve]
- Choi JI, Borrello M, Smith E, Cutler CW, Sojar H, Zauderer M (2001b). Prior exposure of mice to Fusobacterium nucleatum modulates host response to Porphyromonas gingivalis. Oral Microbiol Immunol 16:338–344.[Medline]
[Order article via Infotrieve]
- Choi JI, Chung SW, Kang HS, Rhim BY, Kim SJ, Kim SJ (2002). Establishment of Porphyromonas gingivalis heat-shock protein-specific T cell lines from atherosclerosis patients. J Dent Res 81:344–348.
- Deshpande RG, Khan MB, Genco CA (1998). Invasion of aortic and heart endothelial cells by Porphyromonas gingivalis. Infect Immun 66:5337–5343.[Abstract/Free Full Text]
- Hansson GK (2001). Immune mechanism in atherosclerosis. Arterioscler Thromb Vasc Biol 21:1876–1890.[Abstract/Free Full Text]
- Haraszthy VI, Zambon JJ, Trevisan M, Zeid M, Genco RJ (2000). Identification of periodontal pathogens in atheromatous plaques. J Periodontol 71:1554–1560.[CrossRef][Medline]
[Order article via Infotrieve]
- Jindal S, Dudani AK, Singh B, Harley CB, Gupta RS (1989). Primary structure of a human mitochondrial protein homologous to the bacterial and plant chaperonins and to the 65-kiloDalton mycobacterial antigen. Mol Cell Biol 9:2279–2283.[Abstract/Free Full Text]
- Kaufmann SHE, Schoel A, Wand-Wurttenberger A, Steinhoff U, Munk ME, Koga T (1990). T-cells, stress proteins, and pathogenesis of mycobacterial infections. Curr Top Microbiol Immunol 155:125–141.[Medline]
[Order article via Infotrieve]
- Maeda H, Miyamoto M, Hongyo H, Nagai A, Kurihara H, Murayama Y (1994). Heat shock protein 60 (GroEL) from Porphyromonas gingivalis: molecular cloning and sequence analysis of its gene and purification of the recombinant protein. FEMS Microbiol Lett 119:129–136.[CrossRef][Medline]
[Order article via Infotrieve]
- Maeda H, Miyamoto M, Kokeguchi S, Kono T, Nishimura F, Takashiba S, et al. (2000). Epitope mapping of heat shock protein 60 (GroEL) from Porphyromonas gingivalis. FEMS Immunol Med Microbiol 28:219–224.[CrossRef][Medline]
[Order article via Infotrieve]
- Metzler B, Schett G, Kleindienst R, van der Zee R, Ottenhoff T, Hajeer A, et al. (1997). Epitope specificity of anti-heat shock protein 65/60 serum antibodies in atherosclerosis. Arterioscler Thromb Vasc Biol 17:536–541.[Abstract/Free Full Text]
- Okuda K, Ishihara K, Nakagawa T, Hirayama A, Inayama Y, Okuda K (2001). Detection of Treponema denticola in atherosclerotic lesions. J Clin Microbiol 39:1114–1117.[Abstract/Free Full Text]
- Ross R (1993). The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 362:801–809.[CrossRef][Medline]
[Order article via Infotrieve]
- Schoenfeld Y, Isenberg DA (1988). Mycobacteria and autoimmunity. Immunol Today 9:178–182.[CrossRef][Medline]
[Order article via Infotrieve]
- Wick G, Kleindienst R, Schett G, Amberger A, Xu Q (1995). Role of heat shock protein 65/60 in the pathogenesis of atherosclerosis. Int Arch Allergy Immunol 107:130–131.[Medline]
[Order article via Infotrieve]
- Wick G, Perschinka H, Millonig G (2001). Atherosclerosis as an autoimmune disease: an update. Trends Immunol 22:665–669.[CrossRef][Medline]
[Order article via Infotrieve]
- Yamazaki K, Ohsawa Y, Tabeta K, Ito H, Ueki K, Oda T, et al. (2002). Accumulation of human heat shock protein 60-reactive T cells in the gingival tissues of periodontitis patients. Infect Immun 70:2492–2501.[Abstract/Free Full Text]
Journal of Dental Research, Vol. 83, No. 12,
936-940 (2004)
DOI: 10.1177/154405910408301209
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ABSTRACT
INTRODUCTION