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Surface protease of Treponema denticola hydrolyzes C3 and influences function of polymorphonuclear leukocytes
Microbes and Infection 8 (2006) 1758e1763 www.elsevier.com/locate/micinf
Original article
Surface protease of Treponema denticola hydrolyzes C3 and influences function of polymorphonuclear leukocytes Tomoko Yamazaki a, Meguru Miyamoto b, Satoru Yamada a, Katsuji Okuda b, Kazuyuki Ishihara b,* a b
Department of Periodontics, Tokyo Dental College, 1-2-2 Masago, Mihama-ku, Chiba 261-8502, Japan Department of Microbiology, Tokyo Dental College, 1-2-2 Masago, Mihama-ku, Chiba 261-8502, Japan Received 9 September 2005; accepted 8 February 2006 Available online 24 April 2006
Abstract Treponema denticola is a dominant microorganism in human periodontal lesions. One of the major virulence factors of this microorganism is its chymotrypsin-like surface protease, dentilisin. The purpose of this study was to evaluate the effect of dentilisin on human polymorphonuclear leukocytes (PMNs). We used chemiluminescence to assess production of O 2 by PMNs against T. denticola ATCC 35405 and dentilisin-deficient mutant K1. T. denticola ATCC 35405 induced production of O 2 , whereas dentilisin-deficient K1 did not. We found that chymostatin, a protease inhibitor, strongly reduced the ability of T. denticola ATCC 35405 to induce production of, O 2 , whereas K1 was relatively unaffected. We also used Immunoblot and ELISA to evaluate the activation of complement by this microorganism in relation to PMNs. T. denticola ATCC 35405 hydrolyzed the a-chain of C3, producing iC3b. Furthermore, strain ATCC 35405 induced a larger release of MMP-9 from PMNs than strain K1. Dentilisin activated PMNs via complement pathways and may play a role in establishing periodontal lesions. Ó 2006 Elsevier SAS. All rights reserved. Keywords: Treponema denticola; Complement; Polymorphonuclear leukocyte; Protease; Dentilisin; Periodontitis
1. Introduction Spirochetes are helically shaped motile microorganisms. They have been implicated in chronic periodontitis and acute necrotizing ulcerative gingivitis [1e4]. In the presence of disease, the percentage of spirochetes has been reported to increase to as much as 50% of the subgingival bacteria [5,6]. One species of spirochete frequently isolated from periodontal lesions is Treponema denticola [7]. It has been reported that levels of T. denticola increased in correlation with the severity of periodontitis [8]. These findings suggest that this microorganism is a major pathogen in periodontitis. This microorganism possesses several pathogenic factors, including its major outer sheath protein and proteases [9,10]. Dentilisin is a prolyl-phenylalanine specific surface protease and a major
* Corresponding author. Tel.: þ81 43 270 3742; fax: þ81 43 270 3744. E-mail address: [email protected] (K. Ishihara). 1286-4579/$ - see front matter Ó 2006 Elsevier SAS. All rights reserved. doi:10.1016/j.micinf.2006.02.013
pathogen of T. denticola [11]. Dentilisin hydrolyzes host bioactive proteins [12] and is cytotoxic to periodontal ligament epithelial cells [13]. Polymorphonuclear leukocytes (PMNs) are predominant among the cells that interact with the microflora in the periodontal pocket [14,15]. We previously reported that T. denticola was located primarily on the apical surface of subgingival plaque in direct contact with the periodontal pocket epithelium [16]. Various functions of PMNs, such as phagocytosis, enzyme release, and chemotaxis in response to oral bacteria, have been implicated in the progression of periodontal disease. When PMNs are stimulated by bacteria, they undergo a respiration burst characterized by increased oxygen consumption [14,15]. In this case, superoxide free radicals (O 2 ), H2O2, and PMN enzymes can be released into the extracellular fluid. These PMN enzymes include several potentially destructive proteases, such as serine protease, elastase, cathepsin G, and also matrix metalloproteinases such as PMN collagenase (MMP-8) and PMN gelatinase B/type IV collagenase (MMP-9)
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[17,18]. MMP-9 has been found to be elevated in patients with periodontitis [19]. Sela et al. [20] found that T. denticola suppressed the production of superoxide. Ding et al. [17,18] showed that components of T. denticola such as Msp induced the production of matrix metalloproteinase by PMNs. Boehringer et al. [21] demonstrated that phagocytosed treponemes remained inside phagosomes. They found that the killing activity of spirochetes by PMNs was low, and that spirochetes affected the host defense mechanisms [21]. In this study, we evaluated the effect of dentilisin on PMN phagocytosis in order to clarify the interaction between T. denticola and PMNs. 2. Materials and methods 2.1. Growth conditions and preparation of sonic extract from T. denticola T. denticola ATCC 35405 and T. denticola dentilisindeficient mutant K1 [22] were maintained in TYGVS medium under anaerobic conditions as described previously [23]. T. denticola cells were cultured for 3 days in TYGVS and then harvested by centrifugation at 1000 g for 20 min. They were then washed twice with phosphate buffered saline (PBS, pH 7.2) and resuspended in PBS. The cells were sonicated for 10 min on ice with a Sonicator (Branson Sonic Power Co., Danbury, CT), and the soluble fraction was collected by centrifugation at 7000 g for 30 min. The fraction was stored at 20 C until measurement of protease activity. The protein concentration of the fraction was assayed with a DC protein assay kit (Bio-Rad, Hercules, CA). 2.2. Dentilisin activity assays Prolyl-phenylalanine specific protease activity was measured using a synthetic substrate, N-succinyl-L-alanyl-L-alanylL-prolyl-L-phenylalanine p-nitroanilide (SAAPNA, Sigma Chemical Company, St, Louis. MO), as described previously [11]. Briefly, each sonicate of T. denticola was added to 100 mM TriseHCl buffer (pH 8.0) containing 1.6 mM SAAPNA. The mixture was incubated at 37 C for 15 min, and then the reaction was stopped by adding 50 ml of 20% acetic acid. Release of p-nitroaniline was evaluated by measuring absorbance at 405 nm. 2.3. Chemiluminescence assay for O 2 The chemiluminescence assay was performed as described previously [24]. Briefly, a total of 50 ml venous blood was collected into tubes containing heparin by venipuncture from 3 healthy donors. Written informed consent was obtained from each donor. PMNs were separated by centrifugation at 500 g in a swing-out rotor at 20 C for 30 min using Polymorphrep (Axis-Shield PoC, Oslo, Norway). Isolated PMNs were washed with PBS and harvested following centrifugation at 400 g for 10 min at 20 C. The PMNs were then resuspended in 4 ml of RPMI-1640 medium (Nissui, Tokyo, Japan)
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containing 0.5% gelatin at a concentration of 1.5 107 cells/ ml and held at room temperature. The T. denticola cells grown in TYGVS medium were harvested by centrifugation at 7000 g for 10 min and washed with PBS three times. They were then suspended at 1.8 108 cells/ml in veronal buffer containing 0.1% gelatin, 0.1% glucose, 4 mM MgCl2, and 0.6 mM CaCl2 (GGVB). In the inhibitor assay, T. denticola was treated with 30 mg/ml protease inhibitor chymostatin (Calblochem, San Diego, CA) at 37 C for 30 min, after which the cells were washed twice with PBS. The treated cells were then resuspended in GGVB at 1.8 108/ml. C5-deficient human serum preparation (Sigma Chemical Co.) or C3-deficient complement serum (Sigma) was used as the complement source. Six ml of 3.3 M luminol dissolved in dimethyl sulfoxide, 150 ml of PMNs (1.5 107 cells/ml), and 784 ml of GGVB were mixed and preincubated at 37 C for 30 min. The mixtures, 40 ml of bacteria, 10 ml of 100 mM CaCl2, and 10 ml of C5deficient complement or C3-deficient complement serum were mixed (total assay volume 1 ml) and transferred into chemiluminescence test tubes. Chemiluminescence was measured with an Auto-Lumicounter Model 1422EX (Microtech-Nichion, Chiba, Japan) at 37 C for 135 min. O2 production was expressed as relative luminescent units (rlu). 2.4. Evaluation of C3 fragmentation To clarify activation of C3 by dentilisin, we evaluated the level of proteolytic fragmentation of C3 with T. denticola ATCC 35405 and T. denticola K1. C3 at 0.1 mg/ml was incubated at 37 C for 17 h with sonicates of T. denticola at a protein concentration of 5 mg/ml in 100 mM TriseHCl buffer at pH 8.0. C3 degradation was evaluated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with 10e20% gradient gel (Daiichi Pure Chemicals, Tokyo). The separated proteins in the gel were blotted onto PVDF membranes and immunostained with goat anti-serum to human complement C3 IgG (MP Biomedicals, Irvine, CA). The C3 antibody immobilized on the membranes was then detected with peroxidaseconjugated rabbit anti-goat IgG (Bio-Rad, Hercules, CA) as described previously [22]. 2.5. Measurement of iC3b levels from C3 after treatment with T. denticola Bacterial cells (1.8 108 cells/ml), 100 mM CaCl2, and C5-deficient complement were incubated at 37 C for 30 and 60 min. The samples were cooled on ice for one minute and then centrifuged at 1000 g. The supernatant was stored at 80 C until used. Generation of iC3b was measured with an iC3b ELISA kit (QUIDEL Co., San Diego, USA) according to the manufacturer’s instructions. Briefly, 100 ml of diluted supernatant were added to the microassay well and incubated at room temperature for 30 min. After washing, 50 ml of iC3b antibody conjugated with perioxidase were added and incubated for 30 min. After washing, it was developed by adding 100 ml of substrate solution. The reaction was terminated
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with 50 ml of stop solution. Absorbance at 405 nm was evaluated with a Microplate reader (BioRad). 2.6. Induction of MMP-9 from PMN treated with T. denticola Furthermore, to evaluate the activation of PMNs by dentilisin, release of MMP-9 from PMNs was investigated with an ELISA system for MMP-9 (Amersham Bioscience, Uppsala, Sweden) according to the manufacturer’s instructions. Separated PMNs were adjusted at 1.5 107 cells/ml, and bacterial cells (1.8 108 cells/ml), 100 mM CaCl2, and C5-deficient complement were added as described above. The mixtures were incubated at 37 C for 30, 60, 90, and 120 min. After incubation, the culture supernatant was isolated. The samples obtained were cooled on ice for 1 min and stored at 80 C until used. One hundred ml of each sample were incubated at 37 C for 1 h in the flat-bottomed microwell of the MMP-9 ELISA kit. After washing, anti-MMP-9 antibody conjugated with horseradish peroxidase was added and incubated for 1 h. It was then washed and developed by adding substrate solution. Absorbance was measured at 450 nm with the Microplate reader (BioRad). Activation of MMP-9 by dentilisin was evaluated with the Matrix Metalloprotease-9 Biotrak, Activity Assay System (Amersham Bioscience) according to the manufacturer’s instructions. Briefly, PMNs were incubated with T. denticola as described above, and after 90 min, the culture supernatant was isolated. The samples obtained were cooled on ice for 1 min and stored at 80 C until used. One hundred ml of each sample were incubated at 37 C for 1 h in the provided flat-bottomed microwell. After washing, 50 ml of assay buffer were added and incubated at 37 C for 1.5 h. After incubation, 50 ml of detection reagent were added. The plate was incubated at 37 C for 1 h, and increase of absorbance at 450 nm from 0 to 1 h was evaluated.
ATCC 35405 and T. denticola K1 are shown in Fig. 1. Peaks of chemiluminescence occurred at 30 min. We found that T. denticola ATCC 35405 induced a significantly higher production of O 2 than did K1 (P < 0.01). The same difference in O 2 production between T. denticola ATCC 35405 and K1 was observed in the PMNs obtained from all three donors. To investigate the activation of complement by dentilisin, evaluation of superoxide production from PMNs by T. denticola was performed after treatment with protease inhibitors. Chemiluminescent activity with T. denticola ATCC 35405 decreased to 36.4% after treatment with chymostatin for 30 min. That with T. denticola K1 showed about 10% after treatment, as shown in Table 1. 3.3. Analysis of C3 proteolytic activation Hydrolysis of C3 by dentilisin was evaluated by incubation of C3 with T. denticola ATCC 35405 and K1. The results of SDS-PAGE and immunoblot after incubation are shown in Fig. 2. A 110-kDa C3 a-chain band was hydrolyzed into 37kDa and 35.5-kDa bands after incubation with T. denticola ATCC 35405; in contrast, no change was seen in this band in the C3 when incubated with mutant K1. To determine whether this activation was directly caused by the splitting of C3 into C3b, a chemiluminescence assay was performed using C3-deficient serum. As shown in Fig. 3, activation of PMNs by T. denticola ATCC 35405 was at almost the same level as that with T. denticola K1. To determine activation of C3 by dentilisin, we evaluated production of iC3b using an iC3b ELISA kit. The results are shown in Fig. 4. We found that generation of iC3b was significantly higher with T. denticola ATCC 35405 than with mutant K1 (P < 0.01). To confirm the involvement of dentilisin, the effect of a protease inhibitor was investigated. In the presence
2.7. Statistical analysis
3. Results 3.1. Enzymatic activity of protease Dentilisin activity in the sonicates from T. denticola ATCC 35405 and mutant K1 were assayed with SAAPNA. T. denticola ATCC 35405 showed SAAPNA-hydrolyzing activity at 0.768 OD405/mg protein h, while K1 only showed activity at 0.058 OD405/mg protein h. 3.2. Chemiluminescence assay (O 2)
Superoxide production by PMNs was evaluated with a chemiluminescence assay. Representative data for T. denticola
1.00E + 06
Relative luminescent units
Chemiluminescence activity was assessed and compared using the ManneWhitney U-test. The levels of the iC3b activity and MMP-9 were analyzed by repeated measurement oneway ANOVA followed by a Student-NewmaneKeuls test.
1.20E + 06
8.00E + 05 ATCC 35405 Dentilisin-deficient K1 6.00E + 05
4.00E + 05
2.00E + 05
0.00E + 00
30
60
90
120
min Fig. 1. Chemiluminescence responses of human PMNs. PMNs (1.5 107 cells/ml) were exposed to 1.8 108 cells/ml of freshly harvested T. denticola ATCC 35405 and dentilisin-deficient K1 in presence of C5deficient serum.
T. Yamazaki et al. / Microbes and Infection 8 (2006) 1758e1763 Table 1 Effect of protease inhibitor on superoxide production induced by T. denticola % inhibitiona
T. denticola ATCC 35405þ chymostatin Mutant K1þ chymostatin
36.4 16.5 10.0 5.61
1.20E + 06
1.00E + 06
Relative luminescent units
Strain
Inhibition of superoxide production evaluated by chemiluminescence assay of T. denticola (1.8 108 cells/ml) treated with chymostatin (30 mg/ml). a Mean standard error of three experiments done in duplicate samples.
of the protease inhibitor, chymostatin, production of iC3b was reduced from 12.20 2.21 to 1.78 1.04 at 30 min and from 6.27 2.70 to 1.65 1.49 at 60 min. A t-test showed that these reductions were statistically significant (P < 0.01).
8.00E + 05
ATCC 35405 with C3 deficeint-serum
6.00E + 05
Dentilisin-deficient K1 with C3-deficient serum
4.00E + 05
2.00E + 05
0.00E + 00
3.4. Release of PMN-proteinases during phagocytosis To evaluate the activation of PMNs by T. denticola, we measured release of MMP-9. As shown Fig. 5 the culture supernatant of PMNs incubated with T. denticola contained significantly higher MMP-9 than that incubated with mutant K1 from 30 to 120 min (P < 0.01). The activity of MMP-9 after exposure to T. denticola was also investigated. As shown in Fig. 6, activation of MMP-9 by exposure to T. denticola was confirmed, and this activity was significantly reduced by chymostatin. 4. Discussion The present study demonstrated that T. denticola dentilisin is capable of activating complement. Significant differences were found in the production of superoxide and release of MMP-9 between T. denticola ATCC 35405 and K1. Treatment with protease inhibitor chymostatin induced a reduction in production of O 2 , iC3b production and active MMP 1
2
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30
60
90
120
min Fig. 3. Chemiluminescence responses of human PMNs using C3b-deficient serum. PMNs (1.5 107 cells/ml) were exposed to 1.8 108 cells/ml of freshly harvested T. denticola ATCC 35405 and dentilisin-deficient K1 in presence of C3-deficient serum.
production. These results implicate dentilisin in the activation of PMNs via activation of complement. They suggest that enhancement of chemiluminescence was caused by activation of complement. C3b is a key factor in opsonization [15]. Phagocytes bear receptors on their surfaces that are specifically reactive with C3b and contribute to enhanced opsonization of foreign particles [25]. Our immunoblot analysis demonstrated that T. denticola ATCC 35405 hydrolyzed the a-chain of C3, and that K1 did not. C3a and C3b are generated by degradation of C3 and by activation of complement [14]. We confirmed that iC3b was produced from C3 by wild-type T. denticola. iC3b has the ability to bind complement receptor type 3 (CR3) [26]. CR3 is expressed on the plasma membrane of mammalian PMNs, most
3
** **
175 KDa
18
** **
16
83 KDa
14
62 KDa
47.5 KDa
iC3b µg/ml
12
35405 Dentilisin-deficient K1 Control
10 8 6 4 2 0
32.5 KDa Fig. 2. Immunoblot analysis of C3. C3 (0.1 mg/ml) after incubation with T. denticola ATCC 35405 and dentilisin-deficient K1 using anti-human complement C3 serum. Lane 1: T. denticola ATCC 35405; lane 2: mutant K1; lane 3: control (complement alone). Arrowheads indicate high-molecularmass oligomeric protein and a-chain and b-chain of C3 band.
30 min
60 min
**P < 0.01 Fig. 4. Generation of iC3b from C3 by T. denticola. C5-deficient complement and 1.8108 cells/ml T. denticola after incubation at 37 C for 30 and 60 min. iC3b concentrations are shown as mean (pg) standard deviation. One-way ANOVA for repeated measurements was used for intergroup comparisons. A Student-NewmaneKeuls test was used for multiple comparisons. (*P < 0.01).
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*
ATCC 35405
500
Dentilisin-deficient K1
450
*
*
*
Control
400
MMP-9 ng/ml
350
* *
300 250 200
*
* *
*
150 100 50 0
30 min
60 min
90 min
120 min
* P < 0.05 Fig. 5. Induction of MMP-9 release by T. denticola. PMNs were incubated with 1.8 108 cells/ml of T. denticola in the presence of C5-deficient complement at 37 C for 30, 60, 90 and 120 min. MMP-9 concentrations of culture supernatants are shown as mean (pg) standard deviation. One-way ANOVA for repeated measurements was used for intergroup comparisons. A StudentNewmaneKeuls test was used for multiple comparisons. (*P < 0.01).
mononuclear phagocytes and natural killer cells, and contributes to opsonization. These results indicate that T. denticola dentilisin hydrolizes C3 and activates PMNs via a complement pathway, producing the C3-derived iC3b. In the present study, chemiluminescence and complement analysis were performed in the absence of any specific
** **
*
100 90
Active MMP-9 (ng/ml)
80 70 60 50 40 30 20 10 0
ATCC35405
ATCC35405 + Chymostatin
Control
* P < 0.05, **P<0.01 Fig. 6. Induction of active MMP-9 release by T. denticola. PMNs were incubated with 1.8 108 cells/ml of T. denticola in the presence of C5-deficient complement at 37 C for 90 min. Amounts of active MMP-9 concentrations of culture supernatants are shown as mean (pg) standard deviation. Oneway ANOVA for repeated measurements was used for intergroup comparisons. A Student-NewmaneKeuls test was used for multiple comparisons. (*P < 0.01).
antibody. Specific antibodies have a major impact on the susceptibility of bacteria to phagocytosis, and enhance opsonization. In our preliminary results, enhancement of opsonization was also low in mutant K1 compared with wild-type strain in the presence of antibody. Our results show that dentilisin generated iC3b, and that this generation was significantly reduced by chymostatin. Shenkein et al. [27] suggested C3 activation by accumulation of C3 component on the surface of T. denticola strains TD2 and FM. The results in the present study agree with that report. It has been reported that the lipoproteins of T. denticola enhanced chemiluminescence [28] and that PMNs were activated by the Msp of T. denticola [17,18,21]. Our results indicate that, in addition to other stimulants such as Msp, this microorganism activates PMNs with dentilisin via activation of complement. In the present study, PMNs were activated by dentilisin. It has been reported that T. denticola adheres to PMNs and is ingested into phagosomes [18,21]. Observations indicate that phagocytosed treponemes remain inside the phagosome, even after 60 min [21]. It has been suggested that the humoral immune response to T. denticola is not capable of resolving T. denticola infection [29], and it has also been shown that mouse antibody against T. denticola only slowed initial T. denticola replication and did not alter the maximum growth in vitro. In our preliminary results, growth of T. denticola ATCC 35405 over a two-day period after treatment with PMNs and complement was almost the same as growth when not treated with PMNs or complement (data not shown). It is possible that, while T. denticola activates PMNs, killing of the microorganism is attenuated. Dentilisin activated the production of reactive oxygen and released MMP-9 from PMNs. Released MMP-9 possessed activity, and this release was significantly reduced by chymostatin. Uncontrolled releases of reactive oxygen radicals and proteinases from neutrophils are known to cause cell injury [14,15,30,31]. Following stimulation by bacterial antigens, PMNs produce O 2 via the metabolic pathway of the ‘respiratory burst’ catalyzed by NADPH oxidase during phagocytosis. O 2 is released into the phagosomal space and also into the extracellular environment [30]. It has been suggested that reactive oxygens such as O 2 and H2O2 play a role in the activation of osteoclasts, in addition to their bactericidal function [32]. It has also been demonstrated that reactive oxygen molecules are capable of activating latent PMN collagenase in gingival crevices [33,34] and damaging connective tissues as part of the pathology of periodontal disease. It has also been reported that reactive oxygen degrades components of the extracellular matrix [30]. Furthermore, it has been demonstrated that MMP-2 and MMP-9 are capable of damaging connective tissues as part of the pathology of periodontal disease [35]. Our previous results have indicated that the protease mutant forms significantly smaller lesions in mice than does the wild type [24]. Further study is needed to elucidate the role of the activation of PMNs by dentilisin in colonization by T. denticola and inflammation of periodontal tissue. Taken together, these results indicate that the activation of production of reactive oxygen and MMP-9 by T. denticola
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dentilisin may be involved in the process of progressive breakdown of periodontal tissues. The present findings of this study support a new virulence profile for T. denticola. Acknowledgements This study was partially supported by grant 16591837 from the Ministry of Education, Science, Sport, Culture and Technology of Japan, grant 5A01 from the Oral Health Science Center of Tokyo Dental College and a grant from Tsuchiya Foundation for the promotion of culture. We thank Mr. Jeremy Williams for editing this manuscript. References [1] W.J. Loesche, S.A. Syed, B.E. Laughon, J. Stoll, The bacteriology of acute necrotizing ulcerative gingivitis, J. Periodontol. 53 (1982) 223e230. [2] R.W. Rowland, J. Mestecky, J.C. Gunsolley, R.B. Cogen, Serum IgG and IgM levels to bacterial antigens in necrotizing ulcerative gingivitis, J. Periodontol. 64 (1993) 195e201. [3] G.C. Armitage, W.R. Dickinson, R.S. Jenderseck, S.M. Levine, D.W. Chambers, Relationship between the percentage of subgingival spirochetes and the severity of periodontal disease, J. Periodontol. 53 (1982) 550e556. [4] M.A. Listgarten, S. Levin, Positive correlation between the proportions of subgingival spirochetes and motile bacteria and susceptibility of human subjects to periodontal deterioration, J. Clin. Periodontol. 8 (1981) 122e138. [5] W.J. Loesche, The Biology of Parasitic Spirochetes, in: R.C. Johnson (Ed.), Academic Press, NY, 1976, pp. 61e275. [6] M.A. Listgarten, Structure of the microbial flora associated with periodontal health and disease in man. A light and electron microscopic study, J. Periodontol. 47 (1976) 1e18. [7] K. Ishihara, K. Okuda, Molecular analysis for pathogenicity of oral treponemes, Microbiol. Immunol. 43 (1999) 495e503. [8] L.G. Simonson, C.H. Goodman, J.J. Bial, H.E. Morton, Quantitative relationship of Treponema denticola to severity of periodontal disease, Infect. Immun. 56 (1988) 726e728. [9] J.C. Fenno, K.H. Muller, B.C. McBride, Sequence analysis, expression, and binding activity of recombinant major outer sheath protein (Msp) of Treponema denticola, J. Bacteriol. 178 (1996) 2489e2497. [10] V.J. Uitto, D. Grenier, E.C. Chan, B.C. McBride, Isolation of a chymotrypsinlike enzyme from Treponema denticola, Infect. Immun. 56 (1988) 2717e2722. [11] K. Ishihara, T. Miura, H.K. Kuramitsu, K. Okuda, Characterization of the Treponema denticola prtP gene encoding a prolyl-phenylalanine-specific protease (dentilisin), Infect. Immun. 64 (1996) 5178e5186. [12] P.-L. Makinen, K.K. Makinen, S. Syed, Role of the chymotrypsin-like membrane associated proteinase from Treponema denticola ATCC 35405 in inactivation of bioactive peptides, Infect. Immun. 63 (1995) 3567e3575. [13] J.C. Fenno, P.M. Hannam, W.K. Leung, M. Tamura, V.J. Uitto, B.C. McBride, Cytopathic effects of the major surface protein and the chymotrypsinlike protease of Treponema denticola, Infect. Immun. 66 (1998) 1869e1877. [14] N.S. Taichman, J. Lindhe, Textbook of Clinical Periodontology, in: J. Lindhe (Ed.), Munkusgaard, Copenhagen, 1989, pp. 153e192. [15] T.E. Van Dyke, J. Vaikuntam, Neutriphil function and dysfunction in periodontal disease, Curr. Opinion Periodont. 1 (1994) 19e27. [16] T. Kigure, A. Saito, K. Seida, S. Yamada, K. Ishihara, K. Okuda, Distribution of Porphyromonas gingivalis and Treponema denticola in human subgingival plaque at different periodontal pocket depths examined by immunohistochemical methods, J. Periodont. Res. 30 (1995) 332e341.
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[17] Y. Ding, V.J. Uitto, M. Haapasalo, K. Lounatmaa, Y.T. Konttinen, T. Salo, D. Grenier, T. Sorsa, Membrane components of Treponema denticola trigger proteinase release from human polymorphonuclear leukocytes, J. Dent. Res. 75 (1996) 1986e1993. [18] Y. Ding, M. Haapasalo, E. Kerosuo, K. Lounatmaa, A. Kotiranta, T. Sorsa, Release and activation of human neutrophil matrix metalloand serine proteinases during phagocytosis of Fusobacterium nucleatum, Porphyromonas gingivalis and Treponema denticola, J. Clin. Periodontol. 24 (1997) 237e248. [19] S. Seguier, B. Gogly, A. Bodineau, G. Godeau, N. Brousse, Is collagen breakdown during periodontitis linked to inflammatory cells and expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human gingival tissue? J. Periodontol. 72 (2001) 1398e1406. [20] M.N. Sela, A. Weinberg, R. Borinsky, S.C. Holt, Inhibition of superoxide production in human polymorphonuclear leukocytes by oral treponemal factors, Infect. Immun. 56 (1988) 589e594. [21] H. Boehringer, P.H. Berthold, N.S. Taichman, Studies on the interaction of human neutrophils with plaque spirochetes, J. Periodont. Res. 21 (1986) 195e209. [22] K. Ishihara, H.K. Kuramitsu, T. Miura, K. Okuda, Dentilisin activity affects the organization of the outer sheath of Treponema denticola, J. Bacteriol. 180 (1998) 3837e3844. [23] K. Ishihara, H.K. Kuramitsu, Cloning and expression of a neutral phosphatase gene from Treponema denticola, Infect. Immun. 63 (1995) 1147e1152. [24] T. Nakagawa, T. Sims, Q. Fan, J. Potempa, J. Travis, L. Houston, R.C. Page, Functional characteristics of antibodies induced by Arggingipain (HRgpA) and Lys-gingipain (Kgp) from Porphyromonas gingivalis, Oral Microbiol. Immunol. 16 (2001) 202e211. [25] R.B. Sim, V. Malhotra, A.J. Day, A. Erdei, Structure and specificity of complement receptors, Immunol. Lett. 14 (1987) 183e190. [26] P. Gasque, Complement: a unique innate immune sensor for danger signals, Mol. Immunol. 41 (2004) 1089e1098. [27] H.A. Schenkein, C.R. Berry, Activation of complement by Treponema denticola, J. Dent. Res. 70 (1991) 107e110. [28] M.N. Sela, A. Bolotin, R. Naor, A. Weinberg, G. Rosen, Lipoproteins of Treponema denticola: their effect on human polymorphonuclear neutrophils, J. Periodont. Res. 32 (1997) 455e466. [29] L. Kesavalu, S.C. Holt, J.L. Ebersole, Lack of humoral immune protection against Treponema denticola virulence in a murine model, Infect. Immun. 67 (1999) 5736e5746. [30] R.J. Waddington, R. Moseley, G. Embery, Reactive oxygen species: a potential role in the pathogenesis of periodontal diseases, Oral. Dis. 6 (2000) 138e151. [31] M. Battino, P. Bullon, M. Wilson, H. Newman, Oxidative injury and inflammatory periodontal diseases: the challenge of anti-oxidants to free radicals and reactive oxygen species, Crit. Rev. Oral. Biol. Med. 10 (1999) 458e476. [32] B.E. Bax, A.S. Alam, B. Banerji, C.M. Bax, P.J. Bevis, C.R. Stevens, B.S. Moonga, D.R. Blake, M. Zaidi, Stimulation of osteoclastic bone resorption by hydrogen peroxide, Biochem. Biophys. Res. Commun. 183 (1992) 1153e1158. [33] T. Sorsa, H. Saari, Y.T. Konttinen, K. Suomalainen, S. Lindy, V.J. Uitto, Non-proteolytic activation of latent human neutrophil collagenase and its role in matrix destruction in periodontal diseases, Int. J. Tissue React. 11 (1989) 153e159. [34] K. Suomalainen, T. Sorsa, O. Lindy, H. Saari, Y.T. Konttinen, V.J. Uitto, Hypochlorous acid induced activation of human neutrophil and gingival crevicular fluid collagenase can be inhibited by ascorbate, Scand. J. Dent. Res. 99 (1991) 397e405. [35] M. Makela, T. Salo, V.J. Uitto, H. Larjava, Matrix metalloproteinases (MMP-2 and MMP-9) of the oral cavity: cellular origin and relationship to periodontal status, J. Dent. Res. 73 (1994) 1397e1406.
Original article
Surface protease of Treponema denticola hydrolyzes C3 and influences function of polymorphonuclear leukocytes Tomoko Yamazaki a, Meguru Miyamoto b, Satoru Yamada a, Katsuji Okuda b, Kazuyuki Ishihara b,* a b
Department of Periodontics, Tokyo Dental College, 1-2-2 Masago, Mihama-ku, Chiba 261-8502, Japan Department of Microbiology, Tokyo Dental College, 1-2-2 Masago, Mihama-ku, Chiba 261-8502, Japan Received 9 September 2005; accepted 8 February 2006 Available online 24 April 2006
Abstract Treponema denticola is a dominant microorganism in human periodontal lesions. One of the major virulence factors of this microorganism is its chymotrypsin-like surface protease, dentilisin. The purpose of this study was to evaluate the effect of dentilisin on human polymorphonuclear leukocytes (PMNs). We used chemiluminescence to assess production of O 2 by PMNs against T. denticola ATCC 35405 and dentilisin-deficient mutant K1. T. denticola ATCC 35405 induced production of O 2 , whereas dentilisin-deficient K1 did not. We found that chymostatin, a protease inhibitor, strongly reduced the ability of T. denticola ATCC 35405 to induce production of, O 2 , whereas K1 was relatively unaffected. We also used Immunoblot and ELISA to evaluate the activation of complement by this microorganism in relation to PMNs. T. denticola ATCC 35405 hydrolyzed the a-chain of C3, producing iC3b. Furthermore, strain ATCC 35405 induced a larger release of MMP-9 from PMNs than strain K1. Dentilisin activated PMNs via complement pathways and may play a role in establishing periodontal lesions. Ó 2006 Elsevier SAS. All rights reserved. Keywords: Treponema denticola; Complement; Polymorphonuclear leukocyte; Protease; Dentilisin; Periodontitis
1. Introduction Spirochetes are helically shaped motile microorganisms. They have been implicated in chronic periodontitis and acute necrotizing ulcerative gingivitis [1e4]. In the presence of disease, the percentage of spirochetes has been reported to increase to as much as 50% of the subgingival bacteria [5,6]. One species of spirochete frequently isolated from periodontal lesions is Treponema denticola [7]. It has been reported that levels of T. denticola increased in correlation with the severity of periodontitis [8]. These findings suggest that this microorganism is a major pathogen in periodontitis. This microorganism possesses several pathogenic factors, including its major outer sheath protein and proteases [9,10]. Dentilisin is a prolyl-phenylalanine specific surface protease and a major
* Corresponding author. Tel.: þ81 43 270 3742; fax: þ81 43 270 3744. E-mail address: [email protected] (K. Ishihara). 1286-4579/$ - see front matter Ó 2006 Elsevier SAS. All rights reserved. doi:10.1016/j.micinf.2006.02.013
pathogen of T. denticola [11]. Dentilisin hydrolyzes host bioactive proteins [12] and is cytotoxic to periodontal ligament epithelial cells [13]. Polymorphonuclear leukocytes (PMNs) are predominant among the cells that interact with the microflora in the periodontal pocket [14,15]. We previously reported that T. denticola was located primarily on the apical surface of subgingival plaque in direct contact with the periodontal pocket epithelium [16]. Various functions of PMNs, such as phagocytosis, enzyme release, and chemotaxis in response to oral bacteria, have been implicated in the progression of periodontal disease. When PMNs are stimulated by bacteria, they undergo a respiration burst characterized by increased oxygen consumption [14,15]. In this case, superoxide free radicals (O 2 ), H2O2, and PMN enzymes can be released into the extracellular fluid. These PMN enzymes include several potentially destructive proteases, such as serine protease, elastase, cathepsin G, and also matrix metalloproteinases such as PMN collagenase (MMP-8) and PMN gelatinase B/type IV collagenase (MMP-9)
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[17,18]. MMP-9 has been found to be elevated in patients with periodontitis [19]. Sela et al. [20] found that T. denticola suppressed the production of superoxide. Ding et al. [17,18] showed that components of T. denticola such as Msp induced the production of matrix metalloproteinase by PMNs. Boehringer et al. [21] demonstrated that phagocytosed treponemes remained inside phagosomes. They found that the killing activity of spirochetes by PMNs was low, and that spirochetes affected the host defense mechanisms [21]. In this study, we evaluated the effect of dentilisin on PMN phagocytosis in order to clarify the interaction between T. denticola and PMNs. 2. Materials and methods 2.1. Growth conditions and preparation of sonic extract from T. denticola T. denticola ATCC 35405 and T. denticola dentilisindeficient mutant K1 [22] were maintained in TYGVS medium under anaerobic conditions as described previously [23]. T. denticola cells were cultured for 3 days in TYGVS and then harvested by centrifugation at 1000 g for 20 min. They were then washed twice with phosphate buffered saline (PBS, pH 7.2) and resuspended in PBS. The cells were sonicated for 10 min on ice with a Sonicator (Branson Sonic Power Co., Danbury, CT), and the soluble fraction was collected by centrifugation at 7000 g for 30 min. The fraction was stored at 20 C until measurement of protease activity. The protein concentration of the fraction was assayed with a DC protein assay kit (Bio-Rad, Hercules, CA). 2.2. Dentilisin activity assays Prolyl-phenylalanine specific protease activity was measured using a synthetic substrate, N-succinyl-L-alanyl-L-alanylL-prolyl-L-phenylalanine p-nitroanilide (SAAPNA, Sigma Chemical Company, St, Louis. MO), as described previously [11]. Briefly, each sonicate of T. denticola was added to 100 mM TriseHCl buffer (pH 8.0) containing 1.6 mM SAAPNA. The mixture was incubated at 37 C for 15 min, and then the reaction was stopped by adding 50 ml of 20% acetic acid. Release of p-nitroaniline was evaluated by measuring absorbance at 405 nm. 2.3. Chemiluminescence assay for O 2 The chemiluminescence assay was performed as described previously [24]. Briefly, a total of 50 ml venous blood was collected into tubes containing heparin by venipuncture from 3 healthy donors. Written informed consent was obtained from each donor. PMNs were separated by centrifugation at 500 g in a swing-out rotor at 20 C for 30 min using Polymorphrep (Axis-Shield PoC, Oslo, Norway). Isolated PMNs were washed with PBS and harvested following centrifugation at 400 g for 10 min at 20 C. The PMNs were then resuspended in 4 ml of RPMI-1640 medium (Nissui, Tokyo, Japan)
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containing 0.5% gelatin at a concentration of 1.5 107 cells/ ml and held at room temperature. The T. denticola cells grown in TYGVS medium were harvested by centrifugation at 7000 g for 10 min and washed with PBS three times. They were then suspended at 1.8 108 cells/ml in veronal buffer containing 0.1% gelatin, 0.1% glucose, 4 mM MgCl2, and 0.6 mM CaCl2 (GGVB). In the inhibitor assay, T. denticola was treated with 30 mg/ml protease inhibitor chymostatin (Calblochem, San Diego, CA) at 37 C for 30 min, after which the cells were washed twice with PBS. The treated cells were then resuspended in GGVB at 1.8 108/ml. C5-deficient human serum preparation (Sigma Chemical Co.) or C3-deficient complement serum (Sigma) was used as the complement source. Six ml of 3.3 M luminol dissolved in dimethyl sulfoxide, 150 ml of PMNs (1.5 107 cells/ml), and 784 ml of GGVB were mixed and preincubated at 37 C for 30 min. The mixtures, 40 ml of bacteria, 10 ml of 100 mM CaCl2, and 10 ml of C5deficient complement or C3-deficient complement serum were mixed (total assay volume 1 ml) and transferred into chemiluminescence test tubes. Chemiluminescence was measured with an Auto-Lumicounter Model 1422EX (Microtech-Nichion, Chiba, Japan) at 37 C for 135 min. O2 production was expressed as relative luminescent units (rlu). 2.4. Evaluation of C3 fragmentation To clarify activation of C3 by dentilisin, we evaluated the level of proteolytic fragmentation of C3 with T. denticola ATCC 35405 and T. denticola K1. C3 at 0.1 mg/ml was incubated at 37 C for 17 h with sonicates of T. denticola at a protein concentration of 5 mg/ml in 100 mM TriseHCl buffer at pH 8.0. C3 degradation was evaluated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with 10e20% gradient gel (Daiichi Pure Chemicals, Tokyo). The separated proteins in the gel were blotted onto PVDF membranes and immunostained with goat anti-serum to human complement C3 IgG (MP Biomedicals, Irvine, CA). The C3 antibody immobilized on the membranes was then detected with peroxidaseconjugated rabbit anti-goat IgG (Bio-Rad, Hercules, CA) as described previously [22]. 2.5. Measurement of iC3b levels from C3 after treatment with T. denticola Bacterial cells (1.8 108 cells/ml), 100 mM CaCl2, and C5-deficient complement were incubated at 37 C for 30 and 60 min. The samples were cooled on ice for one minute and then centrifuged at 1000 g. The supernatant was stored at 80 C until used. Generation of iC3b was measured with an iC3b ELISA kit (QUIDEL Co., San Diego, USA) according to the manufacturer’s instructions. Briefly, 100 ml of diluted supernatant were added to the microassay well and incubated at room temperature for 30 min. After washing, 50 ml of iC3b antibody conjugated with perioxidase were added and incubated for 30 min. After washing, it was developed by adding 100 ml of substrate solution. The reaction was terminated
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with 50 ml of stop solution. Absorbance at 405 nm was evaluated with a Microplate reader (BioRad). 2.6. Induction of MMP-9 from PMN treated with T. denticola Furthermore, to evaluate the activation of PMNs by dentilisin, release of MMP-9 from PMNs was investigated with an ELISA system for MMP-9 (Amersham Bioscience, Uppsala, Sweden) according to the manufacturer’s instructions. Separated PMNs were adjusted at 1.5 107 cells/ml, and bacterial cells (1.8 108 cells/ml), 100 mM CaCl2, and C5-deficient complement were added as described above. The mixtures were incubated at 37 C for 30, 60, 90, and 120 min. After incubation, the culture supernatant was isolated. The samples obtained were cooled on ice for 1 min and stored at 80 C until used. One hundred ml of each sample were incubated at 37 C for 1 h in the flat-bottomed microwell of the MMP-9 ELISA kit. After washing, anti-MMP-9 antibody conjugated with horseradish peroxidase was added and incubated for 1 h. It was then washed and developed by adding substrate solution. Absorbance was measured at 450 nm with the Microplate reader (BioRad). Activation of MMP-9 by dentilisin was evaluated with the Matrix Metalloprotease-9 Biotrak, Activity Assay System (Amersham Bioscience) according to the manufacturer’s instructions. Briefly, PMNs were incubated with T. denticola as described above, and after 90 min, the culture supernatant was isolated. The samples obtained were cooled on ice for 1 min and stored at 80 C until used. One hundred ml of each sample were incubated at 37 C for 1 h in the provided flat-bottomed microwell. After washing, 50 ml of assay buffer were added and incubated at 37 C for 1.5 h. After incubation, 50 ml of detection reagent were added. The plate was incubated at 37 C for 1 h, and increase of absorbance at 450 nm from 0 to 1 h was evaluated.
ATCC 35405 and T. denticola K1 are shown in Fig. 1. Peaks of chemiluminescence occurred at 30 min. We found that T. denticola ATCC 35405 induced a significantly higher production of O 2 than did K1 (P < 0.01). The same difference in O 2 production between T. denticola ATCC 35405 and K1 was observed in the PMNs obtained from all three donors. To investigate the activation of complement by dentilisin, evaluation of superoxide production from PMNs by T. denticola was performed after treatment with protease inhibitors. Chemiluminescent activity with T. denticola ATCC 35405 decreased to 36.4% after treatment with chymostatin for 30 min. That with T. denticola K1 showed about 10% after treatment, as shown in Table 1. 3.3. Analysis of C3 proteolytic activation Hydrolysis of C3 by dentilisin was evaluated by incubation of C3 with T. denticola ATCC 35405 and K1. The results of SDS-PAGE and immunoblot after incubation are shown in Fig. 2. A 110-kDa C3 a-chain band was hydrolyzed into 37kDa and 35.5-kDa bands after incubation with T. denticola ATCC 35405; in contrast, no change was seen in this band in the C3 when incubated with mutant K1. To determine whether this activation was directly caused by the splitting of C3 into C3b, a chemiluminescence assay was performed using C3-deficient serum. As shown in Fig. 3, activation of PMNs by T. denticola ATCC 35405 was at almost the same level as that with T. denticola K1. To determine activation of C3 by dentilisin, we evaluated production of iC3b using an iC3b ELISA kit. The results are shown in Fig. 4. We found that generation of iC3b was significantly higher with T. denticola ATCC 35405 than with mutant K1 (P < 0.01). To confirm the involvement of dentilisin, the effect of a protease inhibitor was investigated. In the presence
2.7. Statistical analysis
3. Results 3.1. Enzymatic activity of protease Dentilisin activity in the sonicates from T. denticola ATCC 35405 and mutant K1 were assayed with SAAPNA. T. denticola ATCC 35405 showed SAAPNA-hydrolyzing activity at 0.768 OD405/mg protein h, while K1 only showed activity at 0.058 OD405/mg protein h. 3.2. Chemiluminescence assay (O 2)
Superoxide production by PMNs was evaluated with a chemiluminescence assay. Representative data for T. denticola
1.00E + 06
Relative luminescent units
Chemiluminescence activity was assessed and compared using the ManneWhitney U-test. The levels of the iC3b activity and MMP-9 were analyzed by repeated measurement oneway ANOVA followed by a Student-NewmaneKeuls test.
1.20E + 06
8.00E + 05 ATCC 35405 Dentilisin-deficient K1 6.00E + 05
4.00E + 05
2.00E + 05
0.00E + 00
30
60
90
120
min Fig. 1. Chemiluminescence responses of human PMNs. PMNs (1.5 107 cells/ml) were exposed to 1.8 108 cells/ml of freshly harvested T. denticola ATCC 35405 and dentilisin-deficient K1 in presence of C5deficient serum.
T. Yamazaki et al. / Microbes and Infection 8 (2006) 1758e1763 Table 1 Effect of protease inhibitor on superoxide production induced by T. denticola % inhibitiona
T. denticola ATCC 35405þ chymostatin Mutant K1þ chymostatin
36.4 16.5 10.0 5.61
1.20E + 06
1.00E + 06
Relative luminescent units
Strain
Inhibition of superoxide production evaluated by chemiluminescence assay of T. denticola (1.8 108 cells/ml) treated with chymostatin (30 mg/ml). a Mean standard error of three experiments done in duplicate samples.
of the protease inhibitor, chymostatin, production of iC3b was reduced from 12.20 2.21 to 1.78 1.04 at 30 min and from 6.27 2.70 to 1.65 1.49 at 60 min. A t-test showed that these reductions were statistically significant (P < 0.01).
8.00E + 05
ATCC 35405 with C3 deficeint-serum
6.00E + 05
Dentilisin-deficient K1 with C3-deficient serum
4.00E + 05
2.00E + 05
0.00E + 00
3.4. Release of PMN-proteinases during phagocytosis To evaluate the activation of PMNs by T. denticola, we measured release of MMP-9. As shown Fig. 5 the culture supernatant of PMNs incubated with T. denticola contained significantly higher MMP-9 than that incubated with mutant K1 from 30 to 120 min (P < 0.01). The activity of MMP-9 after exposure to T. denticola was also investigated. As shown in Fig. 6, activation of MMP-9 by exposure to T. denticola was confirmed, and this activity was significantly reduced by chymostatin. 4. Discussion The present study demonstrated that T. denticola dentilisin is capable of activating complement. Significant differences were found in the production of superoxide and release of MMP-9 between T. denticola ATCC 35405 and K1. Treatment with protease inhibitor chymostatin induced a reduction in production of O 2 , iC3b production and active MMP 1
2
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30
60
90
120
min Fig. 3. Chemiluminescence responses of human PMNs using C3b-deficient serum. PMNs (1.5 107 cells/ml) were exposed to 1.8 108 cells/ml of freshly harvested T. denticola ATCC 35405 and dentilisin-deficient K1 in presence of C3-deficient serum.
production. These results implicate dentilisin in the activation of PMNs via activation of complement. They suggest that enhancement of chemiluminescence was caused by activation of complement. C3b is a key factor in opsonization [15]. Phagocytes bear receptors on their surfaces that are specifically reactive with C3b and contribute to enhanced opsonization of foreign particles [25]. Our immunoblot analysis demonstrated that T. denticola ATCC 35405 hydrolyzed the a-chain of C3, and that K1 did not. C3a and C3b are generated by degradation of C3 and by activation of complement [14]. We confirmed that iC3b was produced from C3 by wild-type T. denticola. iC3b has the ability to bind complement receptor type 3 (CR3) [26]. CR3 is expressed on the plasma membrane of mammalian PMNs, most
3
** **
175 KDa
18
** **
16
83 KDa
14
62 KDa
47.5 KDa
iC3b µg/ml
12
35405 Dentilisin-deficient K1 Control
10 8 6 4 2 0
32.5 KDa Fig. 2. Immunoblot analysis of C3. C3 (0.1 mg/ml) after incubation with T. denticola ATCC 35405 and dentilisin-deficient K1 using anti-human complement C3 serum. Lane 1: T. denticola ATCC 35405; lane 2: mutant K1; lane 3: control (complement alone). Arrowheads indicate high-molecularmass oligomeric protein and a-chain and b-chain of C3 band.
30 min
60 min
**P < 0.01 Fig. 4. Generation of iC3b from C3 by T. denticola. C5-deficient complement and 1.8108 cells/ml T. denticola after incubation at 37 C for 30 and 60 min. iC3b concentrations are shown as mean (pg) standard deviation. One-way ANOVA for repeated measurements was used for intergroup comparisons. A Student-NewmaneKeuls test was used for multiple comparisons. (*P < 0.01).
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*
ATCC 35405
500
Dentilisin-deficient K1
450
*
*
*
Control
400
MMP-9 ng/ml
350
* *
300 250 200
*
* *
*
150 100 50 0
30 min
60 min
90 min
120 min
* P < 0.05 Fig. 5. Induction of MMP-9 release by T. denticola. PMNs were incubated with 1.8 108 cells/ml of T. denticola in the presence of C5-deficient complement at 37 C for 30, 60, 90 and 120 min. MMP-9 concentrations of culture supernatants are shown as mean (pg) standard deviation. One-way ANOVA for repeated measurements was used for intergroup comparisons. A StudentNewmaneKeuls test was used for multiple comparisons. (*P < 0.01).
mononuclear phagocytes and natural killer cells, and contributes to opsonization. These results indicate that T. denticola dentilisin hydrolizes C3 and activates PMNs via a complement pathway, producing the C3-derived iC3b. In the present study, chemiluminescence and complement analysis were performed in the absence of any specific
** **
*
100 90
Active MMP-9 (ng/ml)
80 70 60 50 40 30 20 10 0
ATCC35405
ATCC35405 + Chymostatin
Control
* P < 0.05, **P<0.01 Fig. 6. Induction of active MMP-9 release by T. denticola. PMNs were incubated with 1.8 108 cells/ml of T. denticola in the presence of C5-deficient complement at 37 C for 90 min. Amounts of active MMP-9 concentrations of culture supernatants are shown as mean (pg) standard deviation. Oneway ANOVA for repeated measurements was used for intergroup comparisons. A Student-NewmaneKeuls test was used for multiple comparisons. (*P < 0.01).
antibody. Specific antibodies have a major impact on the susceptibility of bacteria to phagocytosis, and enhance opsonization. In our preliminary results, enhancement of opsonization was also low in mutant K1 compared with wild-type strain in the presence of antibody. Our results show that dentilisin generated iC3b, and that this generation was significantly reduced by chymostatin. Shenkein et al. [27] suggested C3 activation by accumulation of C3 component on the surface of T. denticola strains TD2 and FM. The results in the present study agree with that report. It has been reported that the lipoproteins of T. denticola enhanced chemiluminescence [28] and that PMNs were activated by the Msp of T. denticola [17,18,21]. Our results indicate that, in addition to other stimulants such as Msp, this microorganism activates PMNs with dentilisin via activation of complement. In the present study, PMNs were activated by dentilisin. It has been reported that T. denticola adheres to PMNs and is ingested into phagosomes [18,21]. Observations indicate that phagocytosed treponemes remain inside the phagosome, even after 60 min [21]. It has been suggested that the humoral immune response to T. denticola is not capable of resolving T. denticola infection [29], and it has also been shown that mouse antibody against T. denticola only slowed initial T. denticola replication and did not alter the maximum growth in vitro. In our preliminary results, growth of T. denticola ATCC 35405 over a two-day period after treatment with PMNs and complement was almost the same as growth when not treated with PMNs or complement (data not shown). It is possible that, while T. denticola activates PMNs, killing of the microorganism is attenuated. Dentilisin activated the production of reactive oxygen and released MMP-9 from PMNs. Released MMP-9 possessed activity, and this release was significantly reduced by chymostatin. Uncontrolled releases of reactive oxygen radicals and proteinases from neutrophils are known to cause cell injury [14,15,30,31]. Following stimulation by bacterial antigens, PMNs produce O 2 via the metabolic pathway of the ‘respiratory burst’ catalyzed by NADPH oxidase during phagocytosis. O 2 is released into the phagosomal space and also into the extracellular environment [30]. It has been suggested that reactive oxygens such as O 2 and H2O2 play a role in the activation of osteoclasts, in addition to their bactericidal function [32]. It has also been demonstrated that reactive oxygen molecules are capable of activating latent PMN collagenase in gingival crevices [33,34] and damaging connective tissues as part of the pathology of periodontal disease. It has also been reported that reactive oxygen degrades components of the extracellular matrix [30]. Furthermore, it has been demonstrated that MMP-2 and MMP-9 are capable of damaging connective tissues as part of the pathology of periodontal disease [35]. Our previous results have indicated that the protease mutant forms significantly smaller lesions in mice than does the wild type [24]. Further study is needed to elucidate the role of the activation of PMNs by dentilisin in colonization by T. denticola and inflammation of periodontal tissue. Taken together, these results indicate that the activation of production of reactive oxygen and MMP-9 by T. denticola
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