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1 Department of Microbiology, School of Dentistry, Aichi-Gakuin University, Nagoya, Aichi 464-8650, Japan
2 Department of Oral and Maxillofacial Surgery II, School of Dentistry, Aichi-Gakuin University, Nagoya, Aichi 464-8650, Japan
3 Department of Endodontics, School of Dentistry, Aichi-Gakuin University, Nagoya, Aichi 464-8650, Japan
Correspondence
Yukitaka Murakami
yukimu{at}dpc.agu.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONADDENDUMREFERENCES |
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. Gene 371, 102–111] were determined. To verify the function of the S-layer proteins, three mutants with tfsA, tfsB, or both deleted were successfully constructed by a PCR-based overlapping method. S-layer proteins were completely lost in the double mutant. The single-deletion mutants appeared to lose one of the 230 and 270 kDa proteins. Thin-section microscopy clearly revealed that the 230 and 270 kDa proteins composed the S-layer. Although the S-layer proteins may be weakly related to haemagglutinating activity, these proteins were highly responsible for adherence to human gingival epithelial cells (Ca9-22) and KB cells. These results suggest that the S-layer proteins in T. forsythensis play an important role in the initiation stage of oral infection including periodontal disease.
Present address: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3202, USA.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONADDENDUMREFERENCES |
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; Sakamoto et al., 2002). T. forsythensis, Porphyromonas gingivalis and Treponema denticola form the so-called ‘red complex’, which is thought to be important in the pathogenesis and progression of destructive forms of periodontitis (Socransky et al., 1998; Holt & Ebersol, 2005). Mixed infections by P. gingivalis and T. forsythensis showed a synergic effect on enhanced abscess formation in a murine model (Takemoto et al., 1997; Yoneda et al., 2001). Recent studies have suggested an aetiological role of this organism in several forms of periodontal disease (Gersdorf et al., 1993; Takemoto et al., 1997; Haffajee et al., 1998; Yano-Higuchi et al., 2000), periradicular lesion (Gonçalves & Mouton, 1999; Siqueira & Rôças, 2003) and systemic disease (Noack et al., 2001; Hajishengallis et al., 2002).
Surface layers (S-layers) are regularly ordered proteins found as the outermost cell envelope components of many bacteria, representing approximately 10–15 % (w/w) of the total cellular protein of the bacterial cells (Boot & Pouwels, 1996). The majority of S-layers are composed of a single protein or glycoprotein with a molecular mass of 40–200 kDa (Sleytr & Messner, 1988; Sleytr et al., 1993; Sleytr & Beveridge, 1999; Sára & Sleytr, 2000). S-layers are considered to function as protective barriers, as molecular sieves, and in adhesion to cells, surface recognition, and maintenance of cell shape and envelope rigidity (Sleytr & Messner, 1988; Sleytr et al., 1993; Sleytr & Beveridge, 1999; Sára & Sleytr, 2000).
T. forsythensis has been reported to have an S-layer consisting of regularly arrayed subunits outside the outer membrane revealed by electron microscopy (Tanner et al., 1986; Kerosuo, 1988). Several studies reported that major high molecular mass proteins larger than 200 kDa were characteristic of T. forsythensis (Kerosuo, 1988; Gersdorf et al., 1993). We have shown by immunoelectron microscopy that the S-layer is composed of 230 and 270 kDa major proteins (Higuchi et al., 2000; Moriguchi et al., 2003). These S-layer proteins were shown to be specifically recognized by sera from patients with periodontitis (Yoneda et al., 2003). Recently, the S-layer protein was isolated and reported to be associated with haemagglutination, cell adhesion and invasion (Sabet et al., 2003).
In the ‘red complex’ bacteria, complete genome sequences of P. gingivalis and Treponema denticola have been determined (Nelson et al., 2003; Seshadri et al., 2004). A genome sequencing project for Tannerella forsythensis is also in progress at The Institute for Genomic Research (TIGR). From information on N-terminal and internal amino acid sequences of the 230 and 270 kDa proteins, we attempted to identify S-layer protein genes (tfsA and tfsB, respectively, following the names in a recent paper by Lee et al., 2006) utilizing unfinished genome sequences, and then obtain deletion mutants to elucidate the function of the S-layer proteins. In this paper, we also describe the role of the S-layer proteins in haemagglutination and adherence to host cells, determined using S-layer gene deletion mutants.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONADDENDUMREFERENCES |
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. T. forsythensis ATCC 43037 was originally obtained from A. Tanner (The Forsyth Institute, Boston, MA, USA). All T. forsythensis were grown on Brucella HK agar (Kyokuto Pharmaceutical Industrial) supplemented with 5 % (v/v) laked rabbit blood, 2.5 µg haemin ml–1, 5 µg menadione ml–1, 0.1 mg DTT ml–1, 10 µg N-acetylmuramic acid ml–1 (Wyss, 1989; Braham & Moncla, 1992) (blood agar) and appropriate antibiotics when necessary at 37 °C for 7 days under anaerobic conditions [10 % (v/v) CO2, 10 % (v/v) H2 and 80 % (v/v) N2]. A Zero Blunt TOPO PCR cloning kit, which included the plasmid vector pCR-Blunt II-TOPO, was purchased from Invitrogen, and Escherichia coli TOP10 carrying pCR-Blunt II-TOPO was grown in Luria–Bertani medium supplemented with 25 µg kanamycin ml–1. E. coli DH5
carrying pKD260 was grown in Luria–Bertani medium supplemented with 25 µg chloramphenicol (Cp) ml–1 as described previously (Ueda & Yoshimura, 2003).
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) described for deletion mutants in P. gingivalis (Nagano et al., 2005). DNA fragments that allowed the replacement of tfsA, tfsB, or both genes with the cat gene in the T. forsythensis chromosome were constructed. The primers and their annealing sites are shown in Table 2 and Fig. 1(a), respectively. Briefly, the cat gene, encoding chloramphenicol acetyltransferase (CAT), was amplified from the ATG start codon to the TAA stop codon with primers AGU-01 and AGU-02 to generate a 660 bp product from pKD260. For construction of the tfsA-deletion cassette, the flanking sequence upstream of tfsA was amplified with primers AGU-71 and AGU-72, which have homology to the 5' end of the cat fragment. The flanking sequence downstream of tfsA was amplified with AGU-73 and AGU-74, which have homology to the 3' end of the cat fragment. The cat, tfsA-upstream and tfsA-downstream fragments were used as templates for overlap extension PCR to generate a deletion cassette in which tfsA was replaced by cat.
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, except that the organism was grown on the plates for 7 days, scraped, washed and suspended to obtain competent cells (1011 cells ml–1). The plasmid constructs (5 µg) were linearized by digestion with endonucleases KpnI and XbaI and introduced into electrocompetent cells of T. forsythensis (1010 cells). After 12 h anaerobic incubation in Trypticase soy broth (Becton Dickinson) supplemented with 2.5 mg yeast extract ml–1, 2.5 µg haemin ml–1, 5 µg menadione ml–1, 0.1 mg DTT ml–1, 10 µg N-acetylmuramic acid ml–1 and 5 % (v/v) Fildes extract (Oxoid), the pulsed cells were plated on blood agar supplemented with 12 µg Cp ml–1 and the plates were incubated anaerobically at 37 °C for 7 days. Possible recombinants were verified by PCR.
Membrane preparation.
Separation of whole envelopes from T. forsythensis strains was performed essentially as described previously (Murakami et al., 2002b, 2004). Briefly, bacterial cells were washed with 10 mM HEPES/NaOH (pH 7.4) containing 0.15 M NaCl and then resuspended with 10 mM HEPES/NaOH (pH 7.4) containing 0.1 mM N--p-tosyl-L-lysine chloromethyl ketone, 0.2 mM PMSF and 0.1 mM leupeptin (HEPES buffer). The cells were disrupted by sonication and the remaining undisrupted bacterial cells were removed by centrifugation at 1000 g for 10 min. The envelope was collected as a pellet by centrifugation at 100 000 g for 60 min at 4 °C. The pellet was washed once by resuspending it in HEPES buffer and recentrifuged. The protein content of the membrane preparation was estimated by the method of Bradford (1976).
SDS-PAGE and Western blotting.
SDS-PAGE was performed in a 1.0 mm thick 10 % (w/v) gel as described by Lugtenberg et al. (1975). The samples were usually solubilized in SDS buffer with 2-mercaptoethanol at 100 °C for 5 min (Hongo et al., 1999). The gels were stained with Coomassie brilliant blue R-250 (CBB). Western blotting was performed as described previously (Nishikawa & Yoshimura, 2001). Proteins in the SDS-polyacrylamide gel were electrophoretically transferred onto a nitrocellulose membrane. The membrane was blocked with 1 % (w/v) BSA (fraction V) in Tris-buffered saline [TBS, 20 mM Tris/HCl (pH 7.5) containing 0.5 M NaCl]. Then the membrane was reacted with the antiserum followed by incubation with peroxidase-conjugated anti-rabbit IgG (MP Biomedicals). After the membrane was washed, signals of the S-layer proteins were detected with 0.01 % (w/v) 4-chloro-1-naphthol in TBS supplemented with hydrogen peroxide.
N-terminal or internal amino acid sequence analysis.
The S-layer proteins (230 and 270 kDa) from T. forsythensis ATCC 43037 were excised from CBB-stained SDS-PAGE gels. N-terminal amino acid sequences or internal amino acid sequences after digestion with Staphylococcus aureus V8 protease were analysed as described previously (Murakami et al., 2002b; Imai et al., 2005).
Preparation of specific antisera.
Synthetic peptides derived from less-related amino acid sequences selected in the 230 and 270 kDa proteins (CR510LDPKNKKKDYSPVMGR526 and R10VKPYDNAKPTLKFK24C-NH4, respectively, numbered from the N-terminal amino acid estimated in ORFs of contig 5077) were purchased from the Peptide Institute. After peptide conjugation to thyroglobulin using m-maleimidobenzoyl-N-hydroxysuccinimide ester (Pierce Biotechnology), as described in the company's instructions, each peptide was emulsified with complete Freund's adjuvant and injected into rabbits subcutaneously four times at 2-week intervals. Specific antisera that reacted to both the 230 and 270 kDa proteins of T. forsythensis were prepared as described previously (Higuchi et al., 2000).
Haemagglutination assay.
Bacterial cells were washed once with 10 mM phosphate buffer (pH 7.0) containing 0.15 M NaCl (PBS) and resuspended in the same buffer (OD600 1.0). The haemagglutinating activity was determined at room temperature (20–22 °C) in a round-bottomed microtitre plate (Murakami et al., 2002a). The bacterial sample (50 µl) was diluted in twofold series in PBS, and 50 µl of a 2 % (v/v) suspension of chicken or human erythrocytes was added. The mixtures were left standing for 90 min. The titre was expressed as the reciprocal of the highest dilution showing positive agglutination.
For the screening of haemagglutination inhibition, 25 µl of the cell suspension and 50 µl of the erythrocyte suspension were sequentially added to 25 µl of the solution containing the inhibitor to be tested (Murakami et al., 2002a). The concentration of the cell suspension was adjusted to twice the minimal concentration at which distinct haemagglutination occurred. Haemagglutination was recorded after 90 min. The reaction mixture that did not contain the substances was used as a positive control of haemagglutination.
Cell cultures.
Cell lines Ca9-22 (JCRB0625, epithelial cell-like gingival carcinoma cell line) and KB (JCRB9027) were obtained from Health Science Research Resources Bank, Osaka, Japan. KB cells are now known to be HeLa cells (Masters, 2002). The cells were grown at 37 °C for 2–3 days in 5 % (v/v) CO2 in Dulbecco's Modified Eagle Medium (DMEM; Invitrogen) supplemented with 10 % (v/v) fetal calf serum (Invitrogen).
Bacterial adherence assay by confocal laser scanning microscopy.
Ca9-22 and KB cells were subcultured into Lab-Tek II-chamber slides (Nalge Nunc International) and grown for 24 h before adherence study. Ca9-22 or KB cells grown to near confluence of 105 cells per well were washed with PBS to remove unattached cells from the chamber slides. Then T. forsythensis cells, which were washed with PBS and resuspended in DMEM at a concentration of 107 cells ml–1, were added at a m.o.i. of 50. After incubation under 5 % CO2 at 37 °C for 3 h, chamber slides were washed three times with PBS, each followed by gentle shaking for 15 min at room temperature, and fixed with 4 % (v/v) paraformaldehyde at 4 °C overnight. After the fixed cells had been washed three times with TBS, they were permeabilized with TBS containing 0.05 % (v/v) Triton X-100 at 37 °C for 30 min. They were washed again and then blocked with TBS containing 1 % (w/v) BSA at 37 °C for 30 min. Bacterial cells on chamber slides were labelled with anti-T. forsythensis whole-cell antserum (1 : 1000 dilution) for 60 min at 37 °C and washed five times with TBS containing 0.05 % Tween 20. Then bacterial cells were incubated with the FITC-conjugated goat IgG fraction to the rabbit IgG F(ab')2 secondary antibody (1 : 1000 dilution, ICN Pharmaceuticals). Actin filaments in Ca9-22 or KB cells were simultaneously stained with tetramethylrhodamine isothiocyanate (TRITC)-labelled phalloidin (1 µg ml–1, Sigma-Aldrich) for 60 min at 37 °C in the dark. After five washes with TBS containing 0.05 % (v/v) Tween 20, chamber slides were mounted onto a slide containing 10 µl SlowFade Light Antifade (Invitrogen). Immunostained slides were kept in the dark until analysed. Adherent bacteria on the cell surfaces were examined by the laser scanning system Radience 2100 (Bio-Rad) mounted on an Axiovert 200 inverted microscope (Carl Zeiss) equipped with a x63 objective.
Quantitative bacterial adherence assay.
For quantitative bacterial adherence assay, Ca9-22 and KB cells were subcultured into 24-well culture plates (Corning) and grown to near confluence of 5x105 cells per well for 24 h. The cells were washed with PBS to remove unattached cells from the wells. Then T. forsythensis cell suspension at a concentration of 107 cells ml–1 was added at a m.o.i. of 100. After incubation under 5 % CO2 at 37 °C for 3 h, non-adherent bacteria were removed by washing three times with PBS. After the cells had been disrupted by the addition of sterile distilled water (100 µl) and incubated at 37 °C for 10 min, serial dilutions of the disrupted mixture were plated on blood agar plates and incubated anaerobically at 37 °C for 7 days. The numbers of viable adherent bacteria were determined by plating followed by counting the colonies that appeared. All assays were performed in triplicate.
Electron microscopy.
T. forsythensis whole cells were fixed at 4 °C for 2 h with 4 % (w/v) paraformaldehyde and 5 % (v/v) glutaraldehyde in 0.1 M cacodylate buffer. Samples were washed and post-fixed with 4 % (w/v) osmium tetraoxide for 90 min and then 0.5 % (w/v) uranyl acetate for 20 min. The fixed cells were dehydrated in ethanol and embedded in L-R white resin (London Resin). Ultrathin sections were stained with uranyl acetate and lead citrate. Cells were also negatively stained on carbon-coated grids with 2 % (w/v) uranyl acetate. Stained samples were observed using a transmission electron microscope (JEM-1210, JEOL).
Antimicrobial susceptibility.
All antibiotics (chloramphenicol, ampicillin, tetracycline, cephalothin, erythromycin, ofloxacin, bacitracin and vancomycin) were purchased from Sigma-Aldrich. Assays were performed essentially as described previously (Nagano et al., 2005). MIC was evaluated by agar dilution assay, as recommended by the National Committee for Clinical Laboratory Standards (1997). Briefly, serial dilutions of the corresponding antibiotics were added to blood agars. After precultured bacteria were grown on blood agar, and washed bacterial suspension (2 µl) was spotted on the antibiotic-containing agar. After 7 days of anaerobic incubation, the susceptibility breakpoints were determined.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONADDENDUMREFERENCES |
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). The sequences were compared with the unfinished genome of T. forsythensis obtained from the TIGR website (http://www.tigr.org) using the BLAST program (http://www.ncbi.nlm.nih.gov/blast/), and two ORFs encoding the S-layer proteins were found. Two genes, tfsA encoding a 230 kDa protein and tfsB encoding a 270 kDa protein, were tandemly located from 2833655 bp to 2837191 bp and 2837292 bp to 2841383 bp in contig 5077, respectively. In the case of tsfA, the stop codon (TAG) was found at 2835326 bp to 2835328 bp in the unfinished genome of T. forsythensis. Since the internal amino acid sequences (230K fragments 1, 2 and 3 in Table 3), determined using partial digests of pure 230 kDa protein, were distributed before and after the stop codon at the corresponding DNA region in contig 5077, we noticed that the stop codon was incorrectly assigned due to the incomplete sequence. The calculated molecular masses of TsfA and TsfB based on the ORFs are about 120 kDa and 140 kDa, respectively, much smaller than the apparent molecular masses of 230 kDa and 270 kDa, respectively. Therefore, we speculate that the S-layer proteins might be post-translationally modified.
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. Transformants were detected at a frequency of 10–7 to 10–6 per pulsed cell, and the number of transformants obtained was 200–2000 per µg plasmid DNA. To confirm the replacement of ORFs for 270 (tfsA) and/or 230 kDa proteins (tfsB) by cat, the cloning sites were amplified by PCR with the use of chromosomes from each mutant as a template with several primers as shown in Fig. 1(b). All the amplified products showed the predicted fragments (Fig. 1c). All the constructed mutants showed no difference from the parent strain in colony morphology or growth on blood agar plates.
SDS-PAGE and Western blotting
Bacterial envelopes were subjected to SDS-PAGE. The parent strain showed major 230 and 270 kDa protein bands, whereas the single mutants had lost one of these bands and the double mutant both bands (Fig. 2). Specific antisera to the 230 and 270 kDa proteins, prepared using conjugated synthetic peptides as antigens, successfully discriminated the two proteins by Western blot analysis. Anti-230 kDa protein antiserum detected the 230 kDa protein in the parent and 
270, and anti-270 kDa protein antiserum exclusively detected the 270 kDa protein in the parent and 230, as predicted. No immunoreactive bands appeared in the double mutant, confirming that the 230 and 270 kDa proteins were products of tfsA and tfsB, respectively. The single-deletion mutants appeared to have lost the corresponding 230 or 270 kDa protein. Immunoreactive bands at around 100 kDa in Fig. 2(c) were derived from the 270 kDa protein because none of these bands was detected in 270 and 230-270.
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). These results clearly indicated that the S-layer was composed of the 230 and 270 kDa proteins. Even after deletion of the S-layer genes, the fusiform shape was not changed. Negative staining microscopy also showed that the S-layer-deficient mutants maintained almost the same cell shape as the wild-type (data not shown).
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230 and 270 showed two- to fourfold lower haemagglutination, whereas the double mutant retained almost the same activity (Table 4). Similar results were obtained when erythrocytes from different volunteers or chicken were used, and experiments were independently repeated. Haemagglutination inhibition assay using specific antibodies to the 270 and/or 230 kDa proteins revealed that the contribution of the S-layer proteins to haemagglutination was much less, because inhibition was almost the same as with pre-immune control serum (data not shown).
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). Since there was no distinctive difference among observed fields, a set of representative images of the parent and the mutants is shown. Similarly, the parent, but not the mutants, had the ability to adhere to KB cells (Fig. 4b); however, careful staining procedures were necessary to avoid detaching KB cells from the chamber slide surface.
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). Thus, the observations by confocal laser microscopy were quantitatively confirmed by viable-cell counting data.
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). Strong CAT activity was detected only in the mutants where cat had been introduced (data not shown). The mutants showed fourfold lower MICs to vancomycin (0.78 µg ml–1) than the wild-type (3.13 µg ml–1). However, MICs of other antibiotics (chloramphenicol, ampicillin, tetracycline, cephalothin, erythromycin, ofloxacin and bacitracin) did not differ significantly between the parent and the three mutants. Most MIC tests were repeated three times.
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DISCUSSION |
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; Sharma et al., 1998, 2005; Hughes et al., 2003; Ishikura et al., 2003; Inagaki et al., 2005). Honma et al. (2001) reported the generation of specific gene knockout mutants of T. forsythensis for the first time using a suicide plasmid. We followed their procedure to obtain deletion mutants; however, no mutants were obtained. Therefore, we employed PCR-based overlapping methods, which were successful in producing deletion mutants in P. gingivalis (Nagano et al., 2005). The gene to be deleted by this method should at least have a strong promoter in front of it to express the cat gene adequately. Since the S-layer proteins were expressed in a large amount in T. forsythensis, the cat gene would be strongly transcribed by the promoter of tfsA and tfsB. Consequently, Cp-resistant mutants could be selected. To the best of our knowledge, this is the first report of gene deletion being systematically performed in T. forsythensis studies. This method would be useful to determine the functions of other putative virulence factors in T. forsythensis.
Very recently, Lee et al. (2006) reported S-layer genes from T. forsythensis. Sequences of the genes reported (accession no. AY23857) completely matched those we analysed. Although we initially speculated that the S-layer proteins (230 and 270 kDa) might be single gene products due to cross-immunoreactivity in a previous paper (Higuchi et al., 2000), these proteins turned out to be encoded by two genes, tfsA and tfsB, when the unfinished genome sequence became available soon after. tfsA and tfsB form an operon structure according to Northern blot and RT-PCR analyses (Lee et al., 2006). Both gene products, which are reported to exhibit 24 % similarity to each other (Lee et al., 2006), seemed to be expressed in almost the same amount, judged from intensities of the protein bands in CBB-stained gels (Higuchi et al., 2000). It was hypothesized that S-layer genes of T. forsythensis might belong to a novel class, since two large proteins were encoded by two separate genes (Lee et al., 2006). Generally, S-layers are composed of a single protein or glycopotein subunit (Sára & Sleytr, 2000). Although several lactobacilli possess multiple S-layer protein genes, these genes can be differentially or simultaneously expressed depending on growth conditions (Åvall-Jääskeläinen & Palva, 2005). Signal sequences for TfsA and TfsB (230 and 270 kDa, respectively) were estimated as having 22 and 28 amino acids (although unintentionally depicted as 19 and 30 amino acids, respectively, in the figures), respectively, in a previous report (Lee et al., 2006). The signal peptide for TfsA was assigned as MNKKVFTLLAASLMLFMTAFMADA (24 amino acids) because we successfully determined the N-terminal sequence of the 230 kDa protein (Table 3). However, the N-terminal sequence of TfsB was unidentified, presumably because of N-terminal blockage. The predicted N-terminal amino acid of the mature TfsB protein is presumably glutamine, 5 amino acids ahead of the N-terminal amino acid of the two digested peptides (270K fragments 2 and 3 in Table 3), since it is known to be convertible to pyroglutamic acid, an undetected amino acid derivative in the sequence reaction (Murakami et al., 2002b). As another possibility, glycosylation might cause N-terminal blockage. Therefore, we estimated here that the signal sequence was MIMIMNKKIFTLLAGAFMGFAAVSSVTA (28 amino acids). We have shown by electron microscopy using the deletion mutants that the 230 and 270 kDa proteins compose the S-layer; however, the precise molecular arrangement of these proteins in the S-layer remains unclear. Lee et al. (2006) also reported that the S-layer proteins from T. forsythensis were glycoproteins. We confirmed this using a commercial glycoprotein staining kit after SDS-PAGE (data not shown). However, the exact contents and compositions of sugars in the S-layer proteins are unknown.
Previous studies indicated that T. forsythensis cells (Munemasa et al., 2000), especially the S-layer (Sabet et al., 2003), had haemagglutinating and adherence activities. We therefore examined whether the S-layer proteins on the T. forsythensis cell surface might contain haemagglutinin, using S-layer protein deletion mutants. Our results indicated that lack of the S-layer had a minor effect on haemagglutination. Moreover, haemagglutination inhibition by the S-layer-specific antisera raised against the 230 and/or 270 kDa proteins was almost the same as that by the control pre-immune serum. Thus the S-layer seemed to have a marginal association with the haemagglutinating activity. Since thin-section microscopy revealed that the cell surfaces of the single mutants (230 and 270) were partially covered with substances like the S-layer, an abnormal state of the cell surface could hinder haemagglutinating activity. On the other hand, in the double mutant (230-270), which lacked the S-layer completely, the putative haemagglutinin might be fully exposed and function. We have previously reported N-acetylneuraminyllactose-sensitive haemagglutinin on the envelope, although its gene has not been identified yet (Murakami et al., 2002a). We could not find any reliable report on other bacteria whose S-layer protein had haemagglutinating activity.
In contrast, the S-layer proteins in T. forsythensis were clearly responsible for adhesion to the host cells. The S-layer protein-deletion mutants almost completely lost adherence. Although only a few bacteria were internalized into the host cells even in the wild-type strain (data not shown), we speculated that invasion of the host cells, which may occur after adhesion, would be lower in the deletion mutants. It has been reported that Lactobacillus S-layers are involved in mediating adhesion to different host tissues (Åvall-Jääskeläinen & Palva, 2005), whereas the S-layer from Campylobacter rectus had little effect on adhesion to HEp-2 epithelial cells (Wang et al., 2000). To examine the pathogenicity of the S-layer in T. forsythensis, a murine abscess lesion model, as described essentially for P. gingivalis (Kumagai et al., 2001), was used; however, the parent and all three mutants caused abscesses to similar degrees and we could not detect any difference between the parent and the mutant strains (data not shown). Mixed infection with other bacteria in the murine abscess model may be needed to detect differences of virulence among them (Yoneda et al., 2001).
The S-layer is an important interface between the bacterial cell and its environment (Sleytr et al., 1993). We used several representative antibiotics to examine whether the S-layer might contribute to antimicrobial susceptibility. MICs for the wild-type were similar to those described by Takemoto et al. (1997). Most of the MICs for the mutants were not different from that for the wild-type. The mutants showed higher MICs to Cp because of cat gene expression. On the other hand, MICs to vancomycin were lower in the mutants. The reason is unclear; however, S-layers in prokaryotes are considered to function as molecular sieves and as ion traps (Sleytr & Messner, 1988; Sleytr et al., 1993; Sleytr & Beveridge, 1999; Sára & Sleytr, 2000). Regarding another function, the T. forsythensis S-layer proteins did not play a role in cell shape determination and maintenance as judged from electron microscopy. Not only thin-sectioned cells but also negatively stained cells showed similar cell shapes for wild-type and the S-layer-deficient strains. Other possible functions of S-layer proteins, molecules related to S-layer synthesis, and molecules for anchoring S-layer proteins to the outer membrane will be elucidated in the near future.
In this study, we have shown a systematic method for construction of gene-deletion mutants in T. forsythensis. Although T. forsythensis S-layer proteins seemed to have a marginal relationship to haemagglutinating activity, these proteins were responsible for adhesion to Ca9-22 gingival epithelial cells and KB cells. In conclusion, S-layer proteins in T. forsythensis may play an important role in the early stage of oral infections, including periodontal disease.
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ACKNOWLEDGEMENTS |
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Edited by: P. E. Kolenbrander
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Received 1 July 2006;
revised 11 November 2006;
accepted 16 November 2006.
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