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1 Department of Oral Microbiology and Immunology, School of Dentistry, Seoul National University, Seoul 110-749, Republic of Korea
2 Dental Research Institute, School of Dentistry, Seoul National University, Seoul 110–749, Republic of Korea
Correspondence
Bong-Kyu Choi
bongchoi{at}snu.ac.kr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-), cyclooxygenase (COX)-2, and prostaglandin E2 (PGE2). The results indicate that MspTL may induce cell adhesion and inflammation via its N-terminal region.
, tumour necrosis factor- |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Dewhirst et al., 2000). Treponema lecithinolyticum is a small saccharolytic spirochaete with phospholipase A and C activities (Wyss et al., 1999), and belongs to the phylogenetic group IV oral Treponema that is frequently found in periodontitis (Choi et al., 1994; Moter et al., 2006; Wyss et al., 1999). Recently, these organisms were detected in infected root canals associated with either asymptomatic or symptomatic apical periodontitis (Siqueira & Rocas, 2003). With most oral treponemes, the proportion of subjects positive for certain species or phylotypes was higher in subjects with chronic and aggressive periodontitis than in the periodontitis resistant subjects (Moter et al., 2006). This difference was pronounced for treponemes of the phylogenetic groups II and IV, and for Treponema socranskii and T. lecithinolyticum. T. lecithinolyticum has been reported to activate matrix metalloproteinase-2 (MMP-2) in human gingival fibroblasts and periodontal ligament (PDL) cells (Choi et al., 2001a), and to induce osteoclast differentiation by a prostaglandin E2 (PGE2)-dependent mechanism (Choi et al., 2001b). The bacterium possesses the prcA-prtP gene, whose product exhibits chymotrypsin-like protease activity (Correia et al., 2003).
Bacterial surface proteins promote diverse activities, including adhesion, cytotoxicity, antigenicity and host cell stimulation. Recently we reported that the major surface protein (MspTL) of T. lecithinolyticum induced proinflammatory factors in host cells (Lee et al., 2005). MspTL (590 amino acids, including the signal peptide of 19 amino acids) is homologous to the Treponema maltophilum major surface protein, MspA, with 53 % identity and 59 % similarity. These homologous proteins stimulate the expression of interleukin (IL)-1, IL-6 and IL-8 and intercellular adhesion molecule (ICAM)-1 in THP-1 cells, a monocytic cell line, and primary cultured PDL cells. Identification of the functional regions of bacterial surface proteins will improve our understanding of bacteria–host interactions and may help diverse eradication strategies.
The purpose of this study was to examine the role of MspTL in cell adhesion/migration and to identify its proinflammatory regions using truncated recombinant MspTL polypeptides.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Purification of the recombinant proteins with Ni-NTA agarose and decontamination of endotoxin using polymyxin B agarose were also performed as described previously (Lee et al., 2005). The protein concentration was determined by bicinchoninic acid protein assay. The primers used for PCR to amplify MspTL and MspTL gene fragments were as follows: 5'-AACTGGATCCATGAACCTTCCGCCGAAGC-3' and 5'-CCCGGAGCTCTTACAGAGCGATTTTCGCCTT-3' for the MspTL gene; 5'-AACTGGATCCATGAACCTTCCGCCGAAGC-3' and 5'-CCCGGAGCTCTTAATCGTCGTCTATTTTGGT-3' for the gene of polypeptide 1 (PP1) (1–86 aa positions); 5'-AACTGGATCCATGAACGTTAATTTGCTTACC-3' and 5'-CCCGGAGCTCTTACAGAGCGATTTTCGCCTT-3' for the gene of PP2 (75–571 aa positions); 5'-AACTGGATCCATGCTGGGTTATGAACAAAAA-3' and 5'-CCCGGAGCTCTTACTTTACGAGTTCGCCGGAATA-3' for the gene of PP3 (167–421 aa positions); 5'-AACTGGATCCATGAAACTGCTTCCCGGTGTT-3' and 5'-CCCGGAGCTCTTACAGAGCGATTTTCGCCTT-3' for the gene of PP4 (385–571 aa positions). The underlined sequences are the introduced recognition sites for BamHI (GGATCC) and SacI (GAGCTC). Four nucleotides were added to 5' ends of these restriction sites. The mock extract was prepared from E. coli transformed with pQE-30 without the insert DNA, using the same chemicals that were used for recombinant MspTL polypeptides.
SDS-PAGE.
Purified recombinant polypeptides were separated by a standard SDS-PAGE system (10 % gel) and visualized by staining with 0.1 % Coomassie blue R-250.
Cell-binding assay.
The effect of MspTL on cell binding was analysed using the human monocytic cell line THP-1 and human dermal microvascular endothelial cells (HMEC-1), which were purchased from the American Type Culture Collection. Binding was assessed as described by Al-Numani et al. (2003). THP-1 cells were cultured in RPMI 1640 medium (Gibco-BRL) with 10 % FBS (fetal bovine serum) overnight and stimulated with 10 µg MspTL ml–1 for 12 h in serum-free medium. In parallel, HMEC-1 cells were cultured in MCDB131 medium (Gibco-BRL) supplemented with 15 % FBS, 1 % antibiotics (50 U penicillin ml–1 and 50 µg streptomycin sulphate ml–1), 0.1 µg hydrocortisone ml–1 (Sigma-Aldrich) and 0.1 µg epidermal growth factor ml–1 (Invitrogen), seeded in 96-well microtitre plates at a density of 1x104 per well, and cultured to confluence. HMEC-1 cells were then stimulated with 10 µg MspTL ml–1 in serum-free medium for 12 h. MspTL-stimulated THP-1 cells (1x106 cells per well) were washed, resuspended in serum-free RPMI 1640 medium, and added to MspTL-stimulated HMEC-1 for 6, 12 and 24 h in a humidified atmosphere with 5 % CO2. Alternatively, non-stimulated THP-1 cells were added to MspTL-stimulated HMEC-1 cells or MspTL-stimulated THP-1 cells were added to non-stimulated HMEC-1 cells. After incubation, non-adherent THP-1 cells were carefully washed off with PBS. The remaining cells were fixed with absolute ethanol and left to dry. The cells were then stained with 0.1 % methylene blue in 100 mM borate buffer (pH 8.7) for 10 min and washed with 10 mM borate buffer three times. After adding 0.1 M HCl (100 µl per well) at 37 °C for 30 min to solubilize the dye bound by the cells, the A650 was measured. All assays were performed in triplicate and the binding of non-stimulated THP-1 cells to non-stimulated HMEC-1 cells was used for basal cell adherence. The number of bacteria equivalent to 10 µg of MspTL was approximately 5x108 cells (Lee et al., 2005).
Transendothelial migration assay.
The ability of MspTL to induce monocyte transmigration across an endothelial cell monolayer was assessed using the Transwell plate system (Corning). HMEC-1 cells (5x104 cells per well) were seeded in the upper chamber of Transwell culture plates (6.5 mm Transwell inserts with 8 µm pore, Corning Costar) and cultured to confluence. Confluence of the monolayer was confirmed by microscopy. HMEC-1 cells were stimulated with 10 µg MspTL ml–1 in serum-free MCDB131 medium for 24 h, and in parallel, THP-1 cells were separately stimulated with 10 µg MspTL ml–1 in serum-free RPMI 1640 medium for 24 h. After changing the medium in the upper chamber, MspTL-stimulated THP-1 cells (1x106 cells per well) were placed on top of the HMEC-1 cells in the upper chamber and allowed to migrate through the HMEC-1 monolayer into the lower chamber at 37 °C for 20 h in a humidified atmosphere with 5 % CO2. The migration assay was also performed using MspTL-stimulated THP-1 cells/non-stimulated HMEC-1 or non-stimulated THP-1 cells/MspTL-stimulated HMEC-1. The medium was taken from the lower chamber and migrated THP-1 cells were counted under a microscope using a cell-counting haemocytometer.
Treatment of host cells with MspTL polypeptides.
We used THP-1 cells to assess the biological activities of MspTL polypeptides. THP-1 cells were cultured in RPMI 1640 with 10 % FBS. The cells were seeded in 35 mm cell culture dishes at a density of 1x106 cells ml–1 and cultured overnight. After replacing the medium with serum-free medium, the cells were stimulated with 0.16 µM of recombinant polypeptides for 12–24 h. This concentration of polypeptides was equivalent to 10 µg ml–1 of MspTL, which we used in our previous study (Lee et al., 2005). The cells were harvested for RNA isolation and flow cytometry analysis, and the conditioned culture supernatants were stored at –70 °C for ELISA. HMEC-1 cells (1x105 cells ml–1) were cultured in 35 mm cell culture dishes to confluence. After replacing the medium with the serum-free medium, the cells were stimulated with 10 µg MspTL ml–1 for 12 h and analysed for ICAM-1 and IL-8 mRNA expression by RT-PCR as described below. Non-stimulated cells and cells stimulated with mock extracts were used as negative controls.
Expression of ICAM-1 by flow cytometry.
THP-1 cells were stimulated with recombinant MspTL polypeptides for 24 h and the expression of ICAM-1 was evaluated by flow cytometry after reacting cells with anti-human ICAM-1 antibody and subsequently with FITC-labelled IgG antibody as described previously (Lee et al., 2005).
RT-PCR and real-time RT-PCR.
Total RNA was extracted from the cells stimulated with MspTL or polypeptides of MspTL using TRIzol reagent (Invitrogen). cDNA was synthesized by mixing RNA (1 µg) and Maxime RT premix (iNtRON) in a 20 µl reaction volume and incubating the mixture at 42 °C for 1 h. RT-PCR was performed to analyse the expression of ICAM-1 and IL-8 in HMEC-1 by MspTL as described previously (Lee et al., 2005). For real-time RT-PCR, the cDNA (1 µl) was mixed with 10 µl SYBR Premix Ex Taq (Takara Bio) and primer pairs (0.2 pmol each) in a 20 µl reaction volume, followed by PCR for 40 cycles with 95 °C denaturation for 15 s, 60 °C annealing for 15 s and 72 °C extension for 33 s in an ABI PRISM 7500 Fast Real-Time PCR System (Applied Biosystems). The PCR products were subjected to a melting curve analysis to verify a single amplification product. PCR without RT was performed as a negative control. The housekeeping gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference in order to normalize expression levels and quantify changes in gene expression between non-stimulated controls and MspTL polypeptide-stimulated cells. The expression fold change of each gene was determined from the difference in cycle numbers to reach a threshold value between control and MspTL-stimulated cells and was expressed in a log2 ratio. The genes analysed by real-time RT-PCR were ICAM-1, IL-1β, IL-6, IL-8, tumour necrosis factor-alpha (TNF-) and cyclooxygenase (COX)-2. For the inhibition assay, the cells were pretreated with NS-398 (Sigma-Aldrich), a specific COX-2 inhibitor, for 30 min before treatment with MspTL polypeptides. The sequences of the primers for real-time RT-PCR were as follows: 5'-CATATGCCATGCAGCTACAC-3' and 5'-AGTTGTATGTCCTCATGGTG-3' for ICAM-1; 5'-AGCTGTACCCAGAGAGTCC-3' and 5'-ACCAAATGTGGCCGTGGTTT-3' for IL-1β; 5'-AACCTGTCCACTGGGCACA-3' and 5'-TCTGGCTCTGAAACAAAGGAT-3' for IL-6; 5'-GTGAAGGTGCAGTTTTGCCA-3' and 5'-TCTCCACAACCCTCTGCAC-3' for IL-8; 5'-CAGGGACCTCTCTCTAATCA-3' and 5'-AGCTGGTTATCTCTCAGCTC-3' for TNF-; 5'-AAGCTGGGAAGCCTTCTCTA-3' and 5'-GTGCTGGGCAAAGAATGCAA-3' for COX-2; and 5'-GTGGTGGACCTGACCTGC-3' and 5'-TGAGCTTGACAAAGTGGTCG-3' for GAPDH.
ELISA.
The culture supernatants of THP-1 cells stimulated with MspTL polypeptides were assayed to determine IL-8 and PGE2 levels using ELISA kits from R&D Systems.
Statistical analysis.
The statistical significance of the differences between non-stimulated and MspTL polypeptide-stimulated cells or between MspTL polypeptide and MspTL polypeptide/inhibitor-treated cells was evaluated using Student's t-test. A P value of less than 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). ICAM-1 is a representative adhesion molecule for endothelial cells and IL-8 is a chemoattractant for various types of cells. In addition, both molecules contribute to cell adhesion and migration, and so we first analysed whether MspTL induced ICAM-1 and IL-8 in HMEC-1. As shown in Fig. 1, MspTL induced ICAM-1 and IL-8 mRNA expression. Next, we analysed the binding of monocytes to and transmigration through endothelial cell layers, using THP-1 and HMEC-1 cells. As shown in Fig. 2, MspTL significantly increased the binding of THP-1 cells to endothelial cells. The binding capacity was higher in experiments using both cell types stimulated with MspTL than in those where only one type of cell was stimulated with MspTL and the other was not stimulated.
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Expression of partial MspTL polypeptides
Truncated recombinant MspTL polypeptides were produced to determine the functional regions of MspTL with regard to host cell stimulation. MspTL and its partial polypeptides were successfully expressed in E. coli and the recombinant proteins were detected at the position of the predicted molecular masses when analysed by SDS-PAGE (Fig. 3).
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, PP1, 2 and 3 significantly induced mRNA expression of these factors in THP-1 cells as demonstrated by real-time RT-PCR. Among the polypeptides, PP1, which encompasses the first 86 amino acids of MspTL, induced ICAM-1 and IL-8 expression most significantly. PP4 exhibited minimal stimulating activity. To determine the induction at the protein level, we evaluated ICAM-1 expression by flow cytometry and IL-8 level by ELISA. As shown in Fig. 4(b), PP1 induced ICAM-1 and IL-8 expression most significantly and the level was equivalent to or higher than that of MspTL-induced cells. The dose-dependent induction of ICAM-1 expression by PP1 is shown in Fig. 4(c).
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, PP1 significantly upregulated mRNA of IL-1β, TNF-, IL-6 and COX-2 in THP-1 cells.
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, MspTL and PP1 significantly induced PGE2 in THP-1 cells. PGE2 levels were reduced to control levels when the cells were pretreated with NS-398, a specific COX-2 inhibitor, before treatment with MspTL or PP1.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In additional experiments, we observed that MspTL-induced ICAM-1 expression accounted for more than 80 % of that induced by the equivalent amounts of T. lecithinolyticum whole cells or lysates. Therefore, we assume that MspTL is a main player of host cell stimulation with respect to ICAM-1 induction. In this study, we further characterized MspTL with regard to its role in monocyte adhesion and transepithelial migration, and determined the proinflammatory regions using truncated recombinant MspTL polypeptides.
The interaction between monocytes and the endothelium is a key process in many inflammatory diseases, including periodontitis. Since MspTL induced IL-8, a classical chemokine, and ICAM-1, an adhesion molecule, in both monocytes and endothelial cells, we examined monocyte–endothelial cells interaction. MspTL induced monocyte adhesion to endothelial cells and transendothelial migration. These results suggest that MspTL may play an important role in the initial infiltration of monocytes into the periodontal tissues in periodontitis. Fimbriae of Porphyromonas gingivalis, a periodontal pathogen with strong proteolytic activities, have been reported to induce human monocyte adhesion to endothelial cells through the interaction between CD11b/CD18 and ICAM-1 (Harokopakis et al., 2006), depending on Toll-like receptor 2, Rac1 and phosphatidylinositol 3-kinase.
To identify regions critical to MspTL function we designed partial polypeptides of MspTL and evaluated their ability to induce inflammatory host factors. The N-terminal region of MspTL (86 amino acids) exerted a biological activity equivalent to the whole protein in terms of inducing proinflammatory mediators like IL-1β, TNF-, IL-6, IL-8 and PGE2. Prediction of protein subcellular localization analysed using the program PSORTb (Gardy et al., 2005) resulted in the localization of MspTL in the outer membrane with a likelihood of 94.9 %. Predicted secondary structure of MspTL using the programs PSIPRED (Jones, 1999) and DSSP (Kabsch & Sander, 1983) revealed that most of the protein is composed of β-strands and loop regions. The N-terminal 86 amino acids of MspTL form the structure of three β-sheet membrane-spanning segments with two surface-exposed loops and two periplasmic short segments, when analysed with the program PRED-TMBB (Bagos et al., 2005) predicting the topology of β-barrel outer-membrane proteins. Therefore, the N terminus could be thought to come into contact with the host cells via the surface-exposed loops.
The major surface protein, 53 kDa Msp, of Treponema denticola, the most intensively studied oral treponeme, is well characterized and regarded as an important virulence factor. It is an adhesin that binds to host cells and various tissue proteins such as fibronectin, keratin, laminin, collagen, fibrinogen, hyaluronic acid and heparin (Edwards et al., 2005). Msp causes cytopathic effects, forming ion channels within lipid bilayers of epithelial cells and disrupting calcium signalling in fibroblasts (Fenno et al., 1998; Wang et al., 2001). It also triggers release of MMP-8, MMP-9, cathepsin G and elastase from neutrophils (Ding et al., 1996), suggesting its role in periodontal tissue destruction. Recently, using recombinant partial polypeptides of Msp and region-specific antibodies, Edwards et al. (2005) analysed epitopes involved in mediating adhesion and found that the N-terminal half of Msp, particularly the central variable sequence region, carries this function. Although MspTL of T. lecithinolyticum and Msp of T. denticola are not homologous proteins, they are among the most abundant proteins in these bacteria. Therefore, accumulating data on the functional motifs of such proteins in oral spirochaetes will provide ideas about what controls the pathogenesis of the bacteria.
Various inflammatory mediators are synthesized in response to periodontal pathogens and their products and contribute to the initiation and progression of periodontitis, ultimately leading to periodontal tissue destruction, including alveolar bone resorption. High levels of inflammatory mediators are detected in gingival crevicular fluid (GCF) and periodontal tissues of periodontitis patients compared to periodontally healthy individuals (Gorska et al., 2003; Sakai et al., 2006; Wang et al., 2003). The presence of IL-1β, IL-6, TNF- and PGE2 in GCF is correlated with clinical signs of periodontitis such as a significantly higher plaque index and gingival index, deeper pocket probing depth and greater loss of attachment (Champagne et al., 2003). These factors are also potent bone-resorbing mediators that induce alveolar bone resorption, leading to tooth loss. Previously, we observed that T. lecithinolyticum lysates induced osteoclastogenesis in a co-culture system composed of mouse bone marrow cells and calvaria-derived osteoblastic cells, and that this process was dependent on PGE2 (Choi et al., 2001b). MspTL may be the main player behind the increase of PGE2 by T. lecithinolyticum.
In summary, we have demonstrated the ability of MspTL to induce the expression of proinflammatory mediators involved in cell adhesion, inflammation and bone resorption. The N-terminal polypeptide of 86 aa was critical to the stimulation of host cells and the pathological effect of MspTL. We are further pursuing a fine map of the N-terminal region that will contribute to the identification of potential targets to prevent pathogenesis of the major surface protein of T. lecithinolyticum.
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ACKNOWLEDGEMENTS |
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Edited by: M. A. Curtis
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Gardy, J. L., Laird, M. R., Chen, F., Rey, S., Walsh, C. J., Ester, M. & Brinkman, F. S. L. (2005). PSORTb v.2.0: expanded prediction of bacterial protein subcellular localization and insights gained from comparative proteome analysis. Bioinformatics 21, 617–623.
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Received 1 February 2007;
revised 3 April 2007;
accepted 1 May 2007.
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