1 Department of Oral Microbiology, School of Dentistry, Health Sciences University of Hokkaido, Hokkaido 061-0293, Japan
2 Department of Food Science and Technology, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493, Japan
3 Divisions of Oral Molecular Pharmacology, Department of Developmental and Reconstructive Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8588, Japan
4 Divisions of Microbiology and Oral Infection, Department of Developmental and Reconstructive Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8588, Japan
Correspondence
Arihide Kamaguchi
kamaguti{at}hoku-iryo-u.ac.jp
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ABSTRACT |
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INTRODUCTION |
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The initial step of Por. gingivalis attachment to the oral tissue has been shown to be fimbriae-mediated (Hamada et al., 1994; Sojar et al., 1999
, 2002
). In addition, the attachment can be achieved by Por. gingivalis adherence to micro-organisms that have already colonized the periodontal regions (Slots & Gibbons, 1978
). Adhesive interactions among bacterial cells can be observed as co-aggregation in vitro (Kolenbrander, 1988
). We have previously reported that Por. gingivalis can co-aggregate with Prevotella (Pre.) intermedia (Kamaguchi et al., 2001
). Pre. intermedia is detected not only in infected periodontal regions but also in the normal gingival crevice, indicating that Pre. intermedia is one of the early colonizers in periodontal niches (Loesche et al., 1982
; Slots & Listgarten, 1988
; Kononen, 1993
; Raber-Durlacher et al., 1994
; Ashimoto et al., 1996
). Therefore, it is possible that Por. gingivalis participates in the periodontal biofilm by adhering to pre-existing Pre. intermedia.
Previously, we found that co-aggregation between Por. gingivalis and Pre. intermedia was inhibited by L-arginine and L-lysine, and by the potent Rgp/Kgp inhibitors leupeptin and N-p-tosyl-L-lysine chloromethyl ketone hydrochloride (Kamaguchi et al., 2001
). Also, analysis with Por. gingivalis mutant strains revealed that the Rgp-/Kgp-related genes might be responsible for co-aggregation. In this study, we investigated aggregation factors of Por. gingivalis causing co-aggregation between Por. gingivalis and Pre. intermedia. We cloned a DNA region encoding one of the putative aggregation factors and found that this region encoded one of the adhesin domains (HGP17) within rgpA and kgp. In addition, recombinant HGP17-conjugated Sepharose 4B beads bound to Pre. intermedia.
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METHODS |
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Purification of aggregation factors from Por. gingivalis vesicles and antiserum preparation.
Por. gingivalis vesicles were prepared from culture supernatants essentially according to Grenier & Mayrand (1987). Vesicle fractions were solubilized with 3 % (w/v) n-heptyl
-D-thioglucoside (HTG) and then applied to a gel-filtration column (TOYOPEARL HW 65F, 250x800 mm) equilibrated with 50 mM Tris/HCl buffer (pH 7·2) containing 1 % (w/v) HTG. The solubilized proteins were eluted with the same buffer. Aggregation-positive fractions were collected and applied to an arginineSepharose 4B column (Amersham Biosciences, 10x200 mm). The column was washed with a large volume of 50 mM Tris/HCl buffer (pH 7·2), followed by 1 M NaCl to remove non-specifically absorbed proteins. Specifically absorbed proteins were eluted with 0·5 M L-arginine. After the eluted protein fractions were dialysed against water, the aggregation activity of each fraction was determined. Fractions showing aggregation activity were pooled and subjected to SDS-PAGE; the major protein bands were extracted. The purity of the extracted proteins was then examined by SDS-PAGE. Antisera were raised in rabbits against the purified 18 kDa protein, 41 kDa protein and 44 kDa protein. Each protein (50 µg) was mixed with Freund's complete adjuvant, and the mixtures were injected into rabbits subcutaneously three times at 10-day intervals. Ten days after the third injection, each protein (50 µg) was injected intravenously into the rabbits. Serum was obtained from each rabbit 3 days after the last injection.
Aggregation assay.
Aggregation of Pre. intermedia caused by Por. gingivalis vesicles or protein fractions isolated from the vesicles was examined as described previously (Kamaguchi & Baba, 1995). Briefly, the vesicles or protein fractions (1 ml) were mixed with 500 µl of the co-aggregation buffer and incubated at room temperature for 30 min. For determination of the inhibitory effect of antisera on aggregation, antisera 10xdiluted with the co-aggregation buffer were used in place of the co-aggregation buffer. One millilitre of a Pre. intermedia cell suspension (OD600 value of 1·8) was then added to the mixture and shaken at 150 r.p.m. at room temperature for 1 h. The cell suspension was centrifuged at 84 g for 1 min by the method of Kinder & Holt (1989)
; planktonic cells in the supernatant were counted by microscopy with a bacteria-counting chamber (Erma). As a control, Pre. intermedia cell suspensions were added to the aggregation buffer without vesicles or protein fractions, since Pre. intermedia shows auto-aggregation. Aggregation activity was expressed as a percentage and was calculated as follows. Aggregation (%)=[1-(A/B)]x100, where A is the number of planktonic cells in the supernatant of a cell suspension mixed with vesicles or proteins, and B is the number of planktonic cells in the supernatant of a cell suspension mixed with buffer. A value of 100 % represents no planktonic cells in the supernatant of a cell suspension mixed with vesicles.
Construction of the Por. gingivalis genomic library and screening with anti-18 kDa protein antiserum.
Genomic DNA from Por. gingivalis ATCC 33277T was isolated according to Smith et al. (1989). EcoRI-digested Por. gingivalis genomic DNA fragments were ligated into the unique EcoRI site of the phage
gt11. The phage DNA was packaged in vitro with the Ready-To-Go Lambda Packaging kit (Amersham Bioscience) and transformed into E. coli Y1090 to construct the genomic library. Clones were screened with anti-18 kDa protein antiserum according to Sambrook & Russell (2001)
.
DNA sequencing.
Inserted DNA from gt11 clones was amplified by PCR with
gt11 MCS primers. The DNA of the PCR product was sequenced directly using an automated DNA sequencer (ABI PRISM 310 Genetic Analyser; Applied Biosystems) with the ABI PRISM BigDye terminator cycle sequencing kit (Applied Biosystems).
Construction and purification of GST fusions.
DNA fragments encoding the rgpA HGP17 and HGP44 domains were generated by PCR using the following protocol. The rgpA HGP17 domain was PCR-amplified using primers 5'-TCCACGGGATCCAATGGCGCC-3' (forward; BamHI site is underlined) and 5'-TGCGAGGTCGACCTTGGCTTC-3' (reverse; SalI site is underlined); these primers amplified a region of 524 bp (target region 47385362). The rgpA HGP44 domain was PCR-amplified using primers 5'-AGGAATTCCCCCGAACTTCTT-3' (forward; EcoRI site is underlined) and 5'-AGACTCGAACGTCGACGTGAA- 3' (reverse; SalI site is underlined); these primers amplified a region of 1302 bp (target region 30824384). For both amplifications, PCRs were carried out in a total volume of 50 µl consisting of 4 µl of 2·5 mM dNTPs, 2·5 µl each primer pair (10 pmol µl-1), 0·2 µl Taq polymerase (5 U µl-1) and 35 µl H2O. PCR amplification parameters were as follows: initial denaturation at 94 °C for 5 min; 35 cycles at 92 °C for 1 min, 57 °C for 1 min and 72 °C for 1 min; final extension at 72 °C for 2 min.
The PCR-amplified DNA fragments for HGP17 and HGP44 were digested with BamHI/SalI and EcoRI/SalI, respectively, and ligated into pGEX-6P-3 digested with the same restriction enzyme combinations, resulting in pAK100 (GSTHGP17 fusion) and pAK101 (GSTHGP44 fusion). E. coli BL21 (DE3) cells containing the GST fusion plasmids were grown at 20 °C in LB broth containing 100 µg ampicillin ml-1 to an OD600 value of 0·4. After IPTG (0·1 mM) had been added, the cultures were incubated at 20 °C for 2 h. Cells were then harvested and disrupted by ultrasonication. GST fusion proteins were detected using Western blot analysis with a GST detection module (Amersham Biosciences) or were observed via the overproduced protein bands from SDS-PAGE analysis. The GST fusion proteins were purified from the cell extracts by glutathioneSepharose 4B column chromatography. Recombinant HGP17 and HGP44 proteins (60 µg in 100 µl-1) were obtained by cleavage of GST fusion proteins with 8 U PreScission protease (Amersham Bioscience). After cleavage of the GST fusion proteins binding to the glutathioneSepharose 4B beads with PreScission protease, the recombinant HGP17 and HGP44 proteins were eluted from the glutathioneSepharose 4B column with elution buffer. The eluates were then applied to the glutathioneSepharose 4B column to remove contaminated GST and PreScission protease, which was a GST fusion protein.
Absorption analysis of Pre. intermedia to GST fusion-protein-conjugated Sepharose 4B beads.
BSA (100 µl of 1 mg ml-1) was added to 2 ml of a Pre. intermedia ATCC 25611T cell suspension (108 cells ml-1), and incubated at room temperature for 10 min. One-hundred microlitres of the BSA-treated Pre. intermedia cell suspension and 100 µl of co-aggregation buffer were mixed with 50 µl of GSTHGP17, GSTHGP44 or GST-conjugated Sepharose 4B beads. GSTHGP17- and GSTHGP44-conjugated Sepharose 4B beads were prepared as follows. Cells of E. coli BL21 (DE3)(pAK100) overproducing GSTHGP17 or E. coli BL21 (DE3)(pAK101) overproducing GSTHGP44 were sonicated and centrifuged. The resulting supernatants (5 ml) were mixed with 100 µl of glutathioneSepharose 4B beads. After incubation for 30 min at room temperature, the mixture was washed three times with PBS and then suspended in 1 ml PBS. The suspension was incubated at room temperature for 1 h, washed three times with co-aggregation buffer and resuspended in 1 ml co-aggregation buffer.
Gel electrophoresis and immunoblot analysis.
SDS-PAGE (12·5 %) was performed according to the method of Laemmli (1970). Gels were stained with Coomassie brilliant blue R-250. For immunoblotting, proteins were electrophoretically transferred to a PVDF membrane using a semi-dry blotting system (ATTO). The blotted membranes were immunostained with anti-18 kDa protein antiserum or anti-44 kDa protein antiserum, and signals were detected using an ECL detection system (Amersham Biosciences).
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RESULTS |
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Molecular cloning of a gene encoding the Por. gingivalis 18 kDa protein
Molecular cloning of a gene encoding the Por. gingivalis 18 kDa protein was performed using gt11 and anti-18 kDa protein antiserum. About 2000 plaques were screened, with one positive plaque obtained. Phage DNA was purified from the positive plaque. DNA sequencing revealed that the phage contained a 726 bp insert (data not shown). Interestingly, the insert DNA lacked EcoRI sites at both ends, although EcoRI digests of Por. gingivalis genomic DNA had been ligated into the
gt11 EcoRI site, suggesting that illegitimate ligation might have taken place, resulting in a positive clone. From similarity searches against the GenBank database (BLAST), the cloned DNA sequence was found to correspond to an intragenic region of the Por. gingivalis H66 rgpA gene encoding Rgp (GenBank accession no. U15282). The rgpA gene encodes a proteolytic domain and four adhesin domains (HGP44, HGP15, HGP17 and HGP27). As shown in Fig. 2
, the cloned DNA encoded the HGP15 C-terminal region, the entire HGP17 region and the HGP27 N-terminal region. This DNA also had homology to kgp (GenBank accession no. U54691). In addition, the 5' end (nucleotides 1348) of the cloned DNA sequence had homology to hagA (GenBank accession no. U41807). These results indicated that the 18 kDa protein is closely related to the HGP17 domain protein encoded by rgpA and kgp.
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DISCUSSION |
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We obtained aggregation-active protein fractions from Por. gingivalis vesicles by gel filtration and arginineSepharose 4B column chromatography, purified three major proteins with molecular masses of 44, 41 and 18 kDa from the protein fractions, and generated antisera against these proteins. The antisera against the 18 and 44 kDa proteins showed inhibition of Por. gingivalis vesicle-mediated aggregation of Pre. intermedia, while the antiserum against the 41 kDa protein failed to inhibit this aggregation. Molecular cloning of the gene encoding the Por. gingivalis 18 kDa protein by using the anti-18 kDa antiserum revealed that the 18 kDa protein was encoded by rgpA and kgp as a domain protein (HGP17). The rgpA and kgp genes encode polyproteins: proteolytic and adhesin domain proteins (Kadowaki et al., 1994; Okamoto et al., 1996
). The adhesin domains consist of HGP44, HGP15, HGP17 and HGP27. HGP44 and HGP17 are believed to be involved in haemagglutination, while HGP15 has the ability to bind haemoglobin (Curtis et al., 1996
; Booth & Lehner, 1997
; Kelly et al., 1997
; Nakayama et al., 1998
; Shi et al., 1999
; Shibata et al., 1999
).
Several lines of evidence show that HGP17 is responsible for co-aggregation between Por. gingivalis and Pre. intermedia as a Por. gingivalis aggregation factor. First, partially purified protein fractions from Por. gingivalis vesicles that cause Pre. intermedia aggregation contain a protein with a molecular mass of 18 kDa. Second, antiserum against the 18 kDa protein markedly inhibited Por. gingivalis vesicle-mediated aggregation of Pre. intermedia. Third, one recombinant clone from the Por. gingivalis genomic library that reacted to antiserum against the 18 kDa protein contained a DNA region encoding HGP17. Fourth, the GSTHGP17-conjugated beads had the ability to bind Pre. intermedia. Finally, we found in a previous study that Por. gingivalis rgpA rgpB, rgpA kgp, rgpA rgpB kgp and rgpA kgp hagA mutants, which were producing reduced or negligible amounts of HGP17, failed to co-aggregate with Pre. intermedia (Kamaguchi et al., 2001).
We also found that the aggregation-active protein fractions contained the 44 kDa protein and that antiserum against the 44 kDa protein inhibited Por. gingivalis vesicle-mediated aggregation of Pre. intermedia. In addition, Pre. intermedia could bind to the GSTHGP44-conjugated beads. Since HGP17 and HGP44 have a common amino acid sequence region, the common region may contribute to the aggregation activity of these two proteins. The cross-reactivity of anti-18 kDa protein antiserum and anti-44 kDa protein antiserum to HGP17 and HGP44 may support this hypothesis (Fig. 3).
Although Pre. intermedia cells markedly adhered to GSTHGP17-conjugated beads, the recombinant GSTHGP17 proteins failed to aggregate Pre. intermedia cells when the proteins and the bacterial cells were mixed. In addition, the recombinant GSTHGP17 proteins could not suppress the binding of Pre. intermedia cells to GSTHGP17-conjugated beads. Moreover, Pre. intermedia cells that were pre-treated with the recombinant GSTHGP17 proteins failed to adhere to glutathioneSepharose 4B beads, suggesting that HGP17 proteins fixed on the solid surfaces may have the ability to bind Pre. intermedia cells, whereas free HGP17 proteins might lose the ability to bind. A similar phenomenon has been observed in the attachment of Actinomyces viscosus cells to apatitic surfaces fixed with salivary acidic proline-rich proteins (PRPs) (Gibbons & Hay, 1988). Gibbons & Hay (1988)
found that although PRP molecules adsorbed on apatitic surfaces interact strongly with A. viscosus cells, the same proteins in solution do not appear to bind to cells of the organism, nor do they affect its attachment to pellicles. Their explanation of this unexpected behaviour was that hidden molecular segments of PRPs became exposed as a result of conformational changes in the protein when it adsorbed to apatitic surfaces, which could react with the adhesins of A. viscosus cells. Further work is needed to clarify this issue.
L-arginine and L-lysine, and leupeptin and N-p-tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK), which are potent inhibitors of Rgp and Kgp, respectively, were found to suppress co-aggregation between Por. gingivalis and Pre. intermedia, suggesting that Rgp and Kgp activities may be involved in co-aggregation. However, Pre. intermedia adherence to the GSTHGP17-conjugated beads was not inhibited by the addition of L-arginine, L-lysine, leupeptin or TLCK (data not shown). HGP17, as well as other adhesin domain proteins, seems to be associated with the catalytic domain proteins on the cell surface. The conformation or location of HGP17 on the cell surface might be affected by a conformational change of the catalytic domain proteins caused by the inhibitory chemicals, resulting in loss of co-aggregation activity.
Proteinaseadhesin complexes, encoded by rgpA and kgp, appear to bind several human proteins such as fibrinogen, fibronectin and laminin (Pike et al., 1996). Since these complexes are on the cell surface (Bhogal et al., 1997
; DeCarlo & Harber, 1997
), they may play important roles in the attachment of Por. gingivalis to host-cell surfaces. HRgp, which consists of an Rgp domain and HGP44 (Pike et al., 1994
; Curtis et al., 1999
), actually adheres to erythrocytes and platelets, resulting in haemagglutination and platelet aggregation, respectively (Pike et al., 1994
; Shibata et al., 1999
; Lourbakos et al., 2001
). In this study, we demonstrated that HGP17 and HGP44 of the proteinaseadhesin complexes play an important role in Por. gingivalis vesicle-mediated aggregation of Pre. intermedia. This finding provides a novel function of the adhesin domains with regard to Por. gingivalis adherence. Interactions of various micro-organisms in the periodontal region result in a complex bacterial network in the gingival biofilm, which is believed to cause periodontal diseases. The adhesin domain proteins, such as HGP17 and HGP44, may make a significant contribution to the formation of the complex bacterial network as well as to the adhesion of Por. gingivalis to the cells of several different species of bacteria.
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Received 16 September 2002;
revised 2 December 2002;
accepted 3 February 2003.
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