Determination and Characterization of the Hemagglutinin-associated Short Motifs Found in Porphyromonas gingivalis Multiple Gene Products*

Yasuko Shibata, Mitsuo Hayakawa, Hisashi Takiguchi, Teruaki Shiroza, and Yoshimitsu AbikoDagger

From the Department of Biochemistry, Nihon University School of Dentistry at Matsudo, 2-870-1, Sakaecho-Nishi, Matsudo, Chiba 271-8587, Japan

    ABSTRACT
Top
Abstract
Introduction
References

Porphyromonas gingivalis is a Gram-negative anaerobic bacterial species implicated as an important pathogen in the development of adult periodontitis. In our studies of P. gingivalis and ways to protect against periodontal disease, we have prepared the monoclonal antibody mAb-Pg-vc and its recombinant antibody, which are capable of inhibiting the hemagglutinating activity of P. gingivalis (Shibata, Y., Kurihara, K., Takiguchi, H., and Abiko, Y. (1998) Infect. Immun. 66, 2207-2212). To clarify the antigenically related hemagglutinating domains, we attempted to determine the minimum motifs responsible for P. gingivalis hemagglutinin. Initially, the 9-kilobase EcoRI fragment encoding the 130-kDa protein was cloned from the P. gingivalis chromosome using mAb-Pg-vc. Western blot analysis of nested deletion clones, the competition experiments using synthetic peptides, and the binding assay of the phage-displayed peptides using the mAb-Pg-vc allowed us to identify the minimum motifs, PVQNLT. Furthermore, the presence of multi-gene family coding for this epitope was confirmed via Southern blot analysis and PCR using the primers complementary to the domain corresponding to this epitope. It is suggested that the hemagglutinin-associated motif may be PVQNLT and that the gene families specifying this motif found in P. gingivalis chromosome encode many hemagglutinin and/or hemagglutinin-related proteases.

    INTRODUCTION
Top
Abstract
Introduction
References

It is recognized that the adherence of bacteria to host tissues is a prerequisite for colonization and one of the causative factors of bacterial pathogenesis. Porphyromonas gingivalis is a Gram-negative anaerobic bacteria that is isolated primarily from infectious periodontal pockets and considered to be the major pathogen for adult periodontitis (1, 2). Colonization of this bacterium in gingival tissues is critical in the pathogenic process of periodontal disease resulting in tissue destruction. Therefore, a number of molecules including fimbriae (3), potential molecular adhesins such as hemagglutinins (4), and lipopolysaccharides (5) responsible for colonization have been identified as the virulence factors.

On the other hand, the cysteine proteases from this pathogen have been extensively investigated because these enzymes could play an important role in tissue destruction, activating host proenzymes, neutralizing host defense mechanisms, and providing essential amino acids for growth as well (6). Some of the enzymes are specific for cleavage of peptide bonds containing arginine (7-12), others are specific for lysine residues (13, 14), whereas still others exhibit specificity for both amino acid residues (15, 16). Moreover, it has been proposed that the adherence of P. gingivalis to erythrocytes (13), fibrinogen (17), fibronectin (18), collagenous substrata (19), and other bacteria (20) is mediated, at least in part, by such proteases at the cell surface. Several investigators have indicated interesting results for the correlation of the protease and hemagglutinin activities in P. gingivalis (8, 21-23): arginine-specific cysteine protease (arginine-gingipain)-deficient mutants exhibited decreased hemagglutinating activity, which suggests notable properties of this protease (8, 23). Recent enzymatic and molecular cloning analysis directly revealed that these proteases are composed of two distinct domains: one for proteolytic activity and the other for hemagglutinating activity (9, 10, 14, 16, 22, 24).

The presence of hemagglutinating activity on the P. gingivalis cell surface was first reported by Okuda and Takazoe (25). Indeed, the specific genes for P. gingivalis hemagglutinin, such as hagA, which have four repeat units of the hemagglutinin domain without the protease activity, have been isolated (26). It is reasonable to expect that some hemagglutinin molecules or other hemagglutinin domains encoded by many protease genes in P. gingivalis possess the ability to degrade a broad range of host proteins. Additionally, protoheme is an absolute requirement for the growth of P. gingivalis (27), and it is probably derived from erythrocytes in the natural niche of the organism (28). Thus, the hemagglutinin molecule may be particularly important for the organism not only for the attachment to the gingival tissues but also to agglutinate and lyse erythrocytes to survive in vivo. Although it is now well known that genes coding for hemagglutinins and other proteases may share the domain specifying hemagglutinin and hence consisting of a multigene family, the minimum motif responsible for hemagglutinating activity in this family has only been speculated by some investigators (29, 30).

Our approach to protect from periodontal diseases is to develop a passive immunization system whereby the colonization of P. gingivalis cells onto human host tissues could be blocked. Toward this goal, we first prepared a monoclonal antibody (mAb-Pg-vc) and a recombinant single chain variable fragment antibody that inhibited the hemagglutinating activity of P. gingivalis (31). Using these antibodies, P. gingivalis genomic library was screened, and the gene coding for the 130-kDa protein was isolated. The present investigation will describe the detailed properties of the 130-kDa protein, and its possible role in agglutination with erythrocytes will be discussed.

    EXPERIMENTAL PROCEDURES

Bacteria Culture Conditions and Preparation of the Vesicle Fraction-- P. gingivalis 381 was grown in Todd-Hewitt broth (Difco Laboratories, Detroit, MI) supplemented with hemin (0.2 µg/ml) and vitamin K1 (5 µg/ml) in an anaerobic atmosphere (80% N2, 10% H2, 10% CO2) for 24-48 h. Escherichia coli XL-1 Blue (Stratagene) was grown on LB (1% trypton, 0.5% yeast extract, 0.5% NaCl). For plasmid selection, 60 mg/liter of ampicillin was added to LB agar plates. Vesicles were isolated basically by the method of Grenier and Mayrand (32), with a slight modification as previously reported (33).

Cloning and DNA Sequencing of the Gene Coding for 130-kDa Protein-- P. gingivalis 381 genomic DNA was prepared, digested with EcoRI, and inserted into the EcoRI site of lambda zapII. The constructed phage library was screened by mAb-Pg-vc, and the initial phage clone, HEM9, was isolated. This clone produced an immunoreactive 130-kDa protein (130-kDa hemagglutinating domain; 130k-HMGD). To obtain the minimum coding region for a 130k-HMGD, phage HEM9 DNA was digested with SacI. DNA fragments were subcloned into pBluescript II KS(+) vector (Stratagene), and the mAb-Pg-vc-reactive E. coli transformant harboring pHEM6 was isolated.

For forward DNA sequencing, pHEM6 was digested with ApaI and EcoRI and incubated with exonucleases III and mung bean nuclease. Nested deletion clones were prepared following recircularization and transformation into E. coli cells. For reverse DNA sequencing, the SmaI fragment of pHEM6 was subcloned, and the nested deletion clones were constructed in the same way.

DNA sequencing was performed with the Taq Dyedeoxy system (Applied Biosystems, Inc.) and analyzed on an ABI 373A DNA sequencer. Complete coverage of both strands and the contiguous linkage were achieved by Long Ranger gel solution (FMC Bioproducts) using the LI-COR 4000L infrared automated sequencer (ALOKA). The DNA sequence data were analyzed using the DNASIS programs (TaKaRa) and searched for in the DNA data bank. Sequence grade plasmid DNA was purified by the automatic DNA isolation system PI-50 (KURABO).

Preparation of Antibodies-- Preparation of the rabbit polyclonal antibody raised against P. gingivalis vesicles and its monoclonal derivative, mAb-Pg-vc, has been described previously (31), both of which neutralize the hemagglutinating activity of P. gingivalis vesicles. Recombinant 130k-HMGD was subjected to SDS-polyacrylamide gel electrophoresis, and the protein was purified by excising the gel corresponding to the recombinant 130-kDa protein band. A rabbit serum antibody against the recombinant 130k-HMGD was prepared after three intramuscular injections of the purified protein. Prior to usage, the rabbit serum was absorbed with sonicated E. coli cells to minimize nonspecific immunoreaction.

Phage-displayed Peptide Library-- The Ph.D.TM ligand screening system (7 peptides) was used to identify the epitopes recognized by mAb-Pg-vc. The sterile 65 × 15 mm polystyrene Petri dishes were coated with 1.5 ml of 0.01 M NaHCO3 containing mAb-Pg-vc and stored overnight at 4 °C in humidified container. After the antibody solution was poured off, blocking buffer (0.1 M NaHCO3, pH 8.6, 5 mg/ml bovine serum albumin, 0.02% NaN3) was added and incubated for 1 h at 4 °C. Then these dishes were washed with TBST buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Tween 20) six times, and 2 × 1011 phage in 1 ml of TBST buffer was added and rocked gently for 1 h at room temperature. These dishes were vigorously washed 10 times with TBST, and the bound phage was then eluted by lowering the pH with 1 ml of 0.2 M glycine-HCl (pH 2.2) containing 1 mg/ml bovine serum albumin. After neutralization with 150 µl of 1 M Tris-HCl (pH 9.1), the phages were amplified by infecting with E. coli NM522. Recovered phage particles were subjected to a repeated biopanning procedure in the same manner. After the third round of biopanning, a total of 10 plaques were selected, and the phages were amplified. To obtain the single-strand phage DNA, each culture supernatant was added to polyethylenglycol/NaCl, and the recovered pellets were incubated with iodide buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 4 M NaI) to remove the phage protein. For sequencing, the primer (5'-CCC TCA TAG TTA GCG TAA CG) was used. To quantify the amounts of phages bound to the coated mAb-Pg-vc, enzyme-linked immunosorbent assay was carried out using horseradish peroxidase-conjugated anti-M13 antibody.

Inhibition of Hemagglutinating Activity by Synthetic Peptides-- The hemagglutinating activity of the vesicle fraction of P. gingivalis 381 was assayed with washed rabbit erythrocytes in round-bottomed microtiter plates. 70 µl of vesicle solution (0.625 µg/ml) and 20 µl of antibody solution were transferred into microtiter wells and incubated for 30 min at room temperature. Then, 100 µl of 2% rabbit erythrocyte was added and incubated for 2 h at room temperature. The inhibition of hemagglutinating activity induced by adding synthetic peptides was carried out under the same conditions as mentioned above.

Cloning of Other Genes Coding for mAb-Pg-vc-reactive Proteins-- To clone other genes coding for the epitope sites recognized by mAb-Pg-vc, the primers F (5'-TCC AAT GAA TTT GCT CCT) and R (5'-ATT TTC GAA TGA TTC GGA) were designed. These two primers were able to amplify the region corresponding to the base positions 1298-1447 in pHEM6. PCR1 was carried out by employing Pfu turbo (Stratagene) DNA polymerase and P. gingivalis genomic DNA as a template, and a reaction was performed for 30 cycles (94 °C for 1 min, 55 °C for 2 min, and 72 °C for 2 min). The PCR products were precipitated with 0.1 M of LiCl, 20 µg of glycogen, and 70% ethanol, ligated into the pCR-Script SK(+) vector (Stratagene), cloned, and then sequenced.

Southern Blot Hybridization-- P. gingivalis genomic DNA was digested with endonucleases, and the fragments were separated in a 0.8% agarose gel. After alkaline denaturation, DNA fragments were transferred onto a Hybond N+ membrane (Amersham). Southern blot analysis was carried out using ECL labeling and indirect detection kits (Amersham) according to supplier's recommendation.

SDS-Polyacrylamide Gel Electrophoresis and Western Blot Analysis-- For SDS-polyacrylamide gel electrophoresis, P. gingivalis whole cells, vesicles, and other recombinant samples were solubilized in 0.1% SDS solution, and subjected to 12% acrylamide-bisacrylamide gels as described by Laemmli (34). Western blots were prepared by electroblotting samples onto nitrocellulose for 1.5 h at 6 V in a transfer buffer (50 mM Tris-glycine, 20% methanol) using a semi-dry transfer cell system (Bio-Rad).

    RESULTS

Cloning of the Gene Coding for 130k-HMGD-- Cloning of the gene coding for 130k-HMGD and isolation of chimeric plasmid, pHEM6, is outlined under "Experimental Procedures." To examine the antigenic properties of the recombinant protein produced by HEM6, Western blot analysis was carried out. As shown in Fig. 1, both the rabbit polyclonal antibody and mAb-Pg-vc recognized the recombinant protein whose molecular mass was 130 kDa (A and B, lanes 3, respectively). This indicated that the subcloned 4.6-kb SacI fragment originating from the recombinant phage, HEM9, retained the same gene that encodes a recombinant 130-kDa protein responsible for hemagglutinin. The rabbit polyclonal antibody raised against the 130k-HMGD exhibited the superimposable Western blot profile (Fig. 1C) as that of mAb-Pg-vc (Fig. 1B), suggesting that these two antibodies recognized the same epitopes of the hemagglutinin-related molecules of P. gingivalis.


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Fig. 1.   Hemagglutinin-associated immunochemical properties of P. gingivalis whole cells, vesicles, and 130k-HMGD. P. gingivalis whole cells, vesicles, and 130k-HMGD (lanes 1-3, respectively) were separated on SDS-polyacrylamide gel electrophoresis. Transferred proteins were analyzed with anti-vesicle rabbit serum, mAb-Pg-vc, and rabbit anti-130k-HMGD (A-C, respectively).

Sequence Analysis of the 4.6-kb SacI Fragment-- To fully characterize the 130k-HMGD, the 4.6-kb SacI fragment was completely sequenced. The GC composition of this fragment was 48%, a typical value for this organism (data not shown). Fig. 2 (A and B) shows the gene organization and both nucleotide and deduced amino acid sequences, respectively. It was revealed that the 4.6-kb SacI fragment contained two open reading frames (ORFs).


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Fig. 2.   The gene organization (A) and nucleotide and deduced amino acid sequences (B) of the 4.6-kb EcoRI-SacI fragment. In B, the two potential epitope sites, ARS-I and ARS-II, recognized by mAb-Pg-vc are boxed. The proposed hemoglobin-binding domain (39) is underlined.

The upstream ORF contained no ATG initiation codon, which is preceded by a functional ribosome-binding site, ACATT, in this bacterium (35). This ORF did possess the TAA termination codon, suggesting that the upstream ORF might be a 3' portion of a putative gene, and expressed under the control of the plasmid-borne lacZ' gene. The upstream ORF consisted of 1223 codons capable of coding for the 131.5-kDa protein, which is in good agreement with the experimentally determined molecular mass of recombinant 130k-HMGD. Detailed characterization of this ORF will be discussed later.

The downstream ORF was composed of 212 codons coding for the 24.4-kDa protein. Following computer analysis, it was revealed that this gene product exhibited a high homology with the transposase of IS1126 from P. gingivalis W83 (36).

Identification of the mAb-Pg-vc Binding Site in 130k-HMGD-- During the course of the sequencing experiments, a series of nested deletion clones were constructed. Using these clones, we effectively examined the region responsible for mAb-Pg-vc binding (Fig. 3). Western blot analysis of the E. coli lysate harboring each of the deletion plasmids permitted us to specify the antigenically active domains (data not shown). mAb-Pg-vc recognized Asn1-Gly734, Asn1-Arg602, Asn1-Asn482, and Asn1-Thr475. In addition to these clones, E. coli lysate harboring a 5' portion of EcoRI-BamHI fragment, which encompasses amino acid residues Asn1-Ser432, was also analyzed. No immunopositive signals were observed in this clone (data not shown). These results suggested that the epitope might reside within amino acid residues from Ser432 to Thr475. We designate this region as antibody recognition site-I (ARS-I).


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Fig. 3.   Identification of the mAb-Pg-vc binding site in 130k-HMGD. The horizontal line at the top represents the amino acid residues of 130k-HMGD. The bold horizontal lines show the peptide domains expressed in each deletion clone. Presence (+) or absence (-) of immunoreactivity was scored based on the Western blot analysis using a series of nested deletion clones (data not shown).

In 5' deletion clones, both Ile603 and Pro783 exhibited positive signals; however, no signals were observed in Ser989, suggesting that an additional epitope might exist in the C-terminal portion (783-988) of 130k-HMGD. This second epitope region is referred to as ARS-II.

Inhibition of Vesicle-associated Hemagglutinating Activity with Synthetic Peptides-- As mentioned above, two immunoreactive regions, ARS-I and ARS-II, were identified in recombinant 130k-HMGD. Comparison of the deduced amino acid sequences corresponding to these regions revealed the presence of three nearly resembling amino acid stretches, PVQNLT, LKWD(N)AP, and LS(N)ES(D)FEN. The first two are within ARS-I, whereas the remainder is outside of immunoreactive region.

To confirm the exact epitope domain, a series of overlapping peptides, encompassing the amino acid residues from Lys423 to Thr473, were synthesized (Fig. 4A). The monoclonal antibody, mAb-Pg-vc, recognized the peptide-437AP. Using these peptides, inhibition of the vesicle-associated hemagglutinating activity was examined. As shown in Fig. 4B, peptide-431EG (EGSNEFAPVQNL) and peptide-437AP (APVQNLTGSSVG) indicated an inhibitory effect on the vesicle-associated hemagglutinating activity. Although the other peptides KVTLKWDAPNGT, APNGTPNPNPNP, and NPNPGTT showed only limited inhibition, KVCKDVTVEGSN and SSVGQKVTLKWD had no effect. This strongly suggests that the region around the APVQNL structure possesses the ability to compete with vesicles for binding to erythrocytes.


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Fig. 4.   Inhibition of vesicle-associated hemagglutinating activity with synthetic peptides. A, immunoreactivity of the synthetic peptides by mAb-Pg-vc. In this experiment, a total of seven synthetic peptides were prepared. To exemplify the relative position of these peptides, two amino acid sequences (419Gly to 470Trp and 873Gly to 933Trp) corresponding to ARS-I (boxed sequences) and a portion of ARS-II, respectively, were shown at the top and bottom of seven peptides. The vertical dashed lines denote the common stretches present in both ARSs. 50 µl of the synthetic peptide (0.2 mM) were slot-blotted onto the nitrocellulose membrane. The boxed sequence indicates the ARS-I site. B, inhibition of hemagglutination by synthetic peptides spanning residues 423-475 of 130k-HMGD. Synthetic peptides (12-mers overlapping by 5-6 residues) were assayed for inhibition of hemagglutination of rabbit erythrocytes mediated by purified vesicle fraction from P. gingivalis. E, erythrocytes; V, vesicles; A, antibody (mAb-Pg-vc); P, peptide (0.1 mM). C, comparison of the putative epitope residue of mAb-Pg-vc from the phage-displayed peptide library with the residue of A.

Phage-displayed Epitope Mapping-- To confirm the epitope structure recognized by mAb-Pg-vc, phage clones expressing random hepta-residues were screened by biopanning as outlined under "Experimental Procedures." As a result, a total of seven independent phage clones were isolated. Predicted amino acid sequences are: FPVSQEL, HPVGNTS, KPLTIDT, THGPLSP, KHPTYRQ, YKLNPTR, and YTIGPPS. Among these, quantification of phages bound to coated mAb-Pg-vc by enzyme-linked immunosorbent assay indicated that following four hepta-peptides were immuno-positive; FPVSQEL, HPVGNTS, KPLTIDT, and YKLNPTR (data not shown). Comparison of these peptide sequences with that of 130k-HMGD suggested that PVQNLT (438-443) seemed to be the structure most responsible for the epitope site of mAb-Pg-vc (Fig. 4C). This conclusion was supported by the results shown in Figs. 3 and 4 (A and B).

Southern Blot Analysis of P. gingivalis Chromosome Specific for the mAb-Pg-vc Epitope-- Both the monoclonal antibody mAb-Pg-vc and the rabbit polyclonal antibody raised against 130k-HMGD recognized several proteins (Fig. 1). This suggested that P. gingivalis might possess gene families that encode for mAb-Pg-vc-reactive proteins. Alternatively, it was possible to explain that these multiple bands were degradation products of progenitor proteins. To confirm either possibility, Southern blot analysis was carried out. The P. gingivalis chromosome was digested with EcoRI or SacI, digested doubly with EcoRI and SacI, and transferred onto membrane. For a positive control, the 9-kb EcoRI fragment from HEM9 was also used. These blots were analyzed using the 4.6-kb SacI and the 150-bp (epi-150) fragments as probes. The latter fragment corresponds to Ser433-Asn482, which contained the proposed epitope structure, PVQNLT. As shown in Fig. 5, both probes exhibited superimposable blotting profiles. As expected, the 9- and 4.6-kb bands were visualized in genomic EcoRI and EcoRI-SacI digests. In addition to these bands, several DNA fragments, ranging from 4.3 to 19.3 kb in size, could also be detected in genomic SacI digest. These data suggested that the P. gingivalis chromosome might contain multiple discrete loci coding for the mAb-Pg-vc epitope.


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Fig. 5.   Southern blot hybridization analysis of P. gingivalis chromosomal DNA with the epitope coding DNA probe. P. gingivalis chromosomal DNA was digested with EcoRI (E) or SacI (S) or doubly digested with EcoRI and SacI (ES). For positive control, phage HEM9 DNA was digested with EcoRI. These restriction fragments were separated on a 0.8% agarose gel, transferred onto membrane, and analyzed with the 4.6-kb EcoRI-SacI fragment (pHEM6 insert) or epi-150 as probes.

PCR Amplification of Genes Coding for Epitope-specific Site-- Because Southern blot analysis suggested the presence of multiple discrete loci coding for the mAb-Pg-vc recognizable epitope, we attempted to clone these genes using a PCR technique (Fig. 6). The two primers were designed so that the 150-bp fragment (epi-150), which was used as the probe in the Southern blot analysis, could be amplified when the 4.6-kb SacI fragment was used as a template (Fig. 6C, lanes 2 and 4). When the P. gingivalis chromosome was used as a template, several discrete DNA fragments were detected on the agarose gel (Fig. 6C, lane 1). These PCR products were ligated with plasmid and transformed onto E. coli cells. We randomly picked 48 white colonies, and all inserts were sequenced. As a result, following five independent clones were isolated with duplications (Fig. 6D): epi-6 (165 bp), epi-11 (135 bp), epi-12 (150 bp), epi-41 (165 bp), and epi-22 (1.4 kb). The deduced amino acid sequence of epi-12 is exactly the same as that of 130k-HMGD and several other registered gene products with or without protease activity (GenBankTM accession numbers PGTLAGEN, PGU42210, and PGU75366; Swisprot accession number AF017059). In the other four clones, no identical sequences were found in the registered genes; however, each clone exhibited the following similarity; epi-06 (PGPRPR1), epi-11 (PGU41807), epi-41 (PGPRPR1), and epi-22 (PGU41807). Comparison of DNA sequences between each clone and that of the computer-aided counterpart protein indicated the presence of conservative (underlined) and nonconservative base-changes or deletion (boxed). In epi-06, for example, DNA sequence corresponding to underlined region (PNPNP) in counterpart protein PGPRPR1 is different from that of epi-06; however, translated amino acids were conserved. In addition, boxed NP sequence found in epi-06 was missing in PGPRP1. That the similar nucleotide disturbances were also observed in the remaining three clones rules out the possibility of contamination of strains used in the present study.


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Fig. 6.   Evidence of multigenicity of epitope site in P. gingivalis by PCR analysis. A, diagrammatic representation of the PCR experiment. Both F and R primers were designed so that when P. gingivalis genomic DNA, one of deletion clones (base positions 1-1806), or 4.6-kb EcoRI-SacI was used as template, production of the 150-bp fragment could be expected. Because the additional region responsible for epitope was present, another deletion clone (base position 2349-4600) was also used. B, assessment of nonspecific amplification. To avoid misunderstanding due to nonspecific amplification, the following combinations of primer(s) were employed, and PCR was carried out using P. gingivalis chromosome as template: FR, normal PCR; FF, F primer only; RR, R primer only. M, 100-base ladder for size marker. C, comparison of PCR-amplified products from genomic DNA (lane 1), 1-1806 (lane 2), 2349-4600 (lane 3), and 1-4600 (pHEM6; lane 4). D, deduced amino acid sequences of the chromosome-borne PCR products. For details, see "Results."


    DISCUSSION

P. gingivalis is a Gram-negative anaerobic bacterium that has become recognized as a major pathogen for adult periodontitis. One characteristic aspect of this bacterium is the formation of vesicle particles, budding from the parental P. gingivalis cell surface. One of the pathogenic properties resulted from released vesicles is the ability to agglutinate erythrocytes or hemagglutination. Using vesicle particles as antigen, we prepared two antibodies, anti-vesicle rabbit serum and mouse monoclonal antibody mAb-Pg-vc (31), capable of inhibiting hemagglutinating activity. In the present study, one of the antibodies, mAb-Pg-vc, was employed to screen P. gingivalis genomic library, and the 4.6-kb EcoRI-SacI fragment encoding for 130-kDa protein was successfully isolated. Complete nucleotide sequencing revealed that this fragment contained two ORFs; the upstream ORF is a 3' portion of putative gene and is responsible for 130k-HMGD, whereas the downstream ORF specifies IS1126-like (30, 36). Using the purified recombinant 130k-HMGD protein as the "landmark," we carried out three experiments: (i) Western blot analysis of nested deletion clones, (ii) competition experiments using synthetic peptides, and (iii) binding assay of phage-displayed peptide library. As a result, a possible amino acid stretch, PVQNLT, responsible for antigenicity was identified. Southern blot analysis of P. gingivalis genome with DNA fragment corresponded to this antigenic region as probe, the presence of gene family coding for PVQNLT traits was suggested, and further PCR experiment coupled with nucleotide sequencing confirmed this. Curtis et al. (29) and Kelly et al. (37) have reported that the synthetic polypeptide GVSPKVCKDVTVEGSNEFAPVQNLT (residues 907-931 of PrpR1) was recognized in the serum from a patient with periodontitis and that this domain must be related to bacterial colonization. These information gave authenticity to our results.

Unexpectedly, however, 130k-HMGD exhibited no prominent hemagglutinating activity. This observation strongly suggests that vesicle-associated agglutination of erythrocytes consists of many steps. Attachment of vesicles onto the surface of erythrocyte may trigger the molecular cascade toward the hemagglutination, and native 130k-HMGD is involved in one of early events in these steps. To confirm this hypothesis, further experiments will be necessary.

Using the DNA sequence data obtained in the present study, we performed a homology analysis of the registered genes. As a result, several genes could be identified with high homology. Among these, a total of 14 genes possessing the hemagglutinin domains were chosen, and their protein structures are shown in Fig. 7. Based on the protein structure, these proteins could be classified into five groups. The first group is prtK (W50), prtP (W12), and lysine-specific cysteine protease (W83), in which the hemagglutinin domains exhibit the highest homology (99.3, 99.0, and 98.8%, respectively) with that of 130k-HMGD. The second group (kgp and hagD from 381) is the homologue of the lysine gingipain K in which the C-terminal portion of residues (amino acids 890-1150) are different. The third group consists of rgp-1 (H66), prtR (W50), and prpR1 (W50), whose hemagglutinin domain is shorter than that of the first two groups. The fourth group is composed of prtH (W83), prtRII (381), rgp2 (H66), and rgpB and possesses only short hemagglutinin domain. The last protein is the HagA (381), which contains four repeating hemagglutinin domains in a single molecule. In a comparison of the protein structures of 130k-HMGD and the PrtK, it appeared that the "native" gene coding for 130k-HMGD is a homologue of the prtK and that the 3' two-thirds of this gene was cloned in the present study. We predict P. gingivalis strain 381 might retain this gene. Additional computer analysis of the registered genes other than P. gingivalis provided us the interesting hemagglutinin molecules (HA1 and HA2) originating from influenza virus. Of these, HA1 contains PVQNLT homologue residue, PLQNLT (38). This further supports the fact that PVQNLT might contribute to the immunogenicity in 130k-HMGD.


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Fig. 7.   Comparison of primary structures of the hemagglutinin-associated proteins from P. gingivalis. The primary structures of the 130k-HMGD and computer-accessed following 14 proteins possessing hemagglutinating activity were aligned: prtK (PGU75366, 99.3%), prtP (U42210, 2214278A, 99.0%) (16), (AF017059, 98.8%), lysine gingipain K (2307336A, 82.4%) (40), kgp (D83258, 2219327A, 85.6%) (14), hagD (PGU68468, 85.5%), RGP-1 (A55426, 65.8%) (10), prtR (2219212A, 65.5%) (11), prpR1 (PGPRPR1, 65.1%) (9), prtH (PRTH-PORGI, 61.7%) (41), prtRII (D26470, CPGI-PORG-1, 36.7%) (12), rgp2 (PGU85038, 47.6%), rgpB (2314276A, 47.6%), and hagA (PGU41807) (26). Open rectangles in 14 proteins indicate the regions homologous to the 130k-HMGD where single amino acid changes are shown by dots. Two epitope sites (PVQNLT) are also shown. Dotted vertical lines denote the positions of hemoglobin receptor domains (HGP-15) proposed by Nakayama et al. (39).

Recently, Nakayama et al. (39) have reported that the hemoglobin receptor domain (HGP-15) was found in several P. gingivalis proteases including arginine-gingipain (rgp1), lysine-specific cysteine proteases (prtP, and kgp), and hemagglutinin (hagA), which was located next to the putative hemagglutinin region. Using a lysine-gingipain-deficient mutant in which the uptake of hemin was greatly retarded, Okamoto et al. (42) indicated the defect of hemoglobin adsorption and heme accumulation. Interestingly enough, the duplicating immunoreactive amino acid stretch, PVQNLT, identified in the present study could be found in both sides of the HGP-15 in 130k-HMGD and other hemagglutinin-associated proteins as well. This fact is consistent with the idea that prior to penetration into erythrocyte, two PVQNLT stretches positioned in both side of HGP-15 might facilitate the attachment of P. gingivalis onto the erythrocyte cell surface. Once erythrocytes are coagulated, HGP-15 may function under the aid of the proteolytic activity equipped with the adjacent domain to obtain heme molecules. This gene structure seems to be ideal in this bacterium for the growth in the periodontal pocket to obtain the heme molecules as an iron source.

Deduced amino acid sequence of 130k-HMGD, the identification of short motif present in this protein, and the multiplication process of this motif in P. gingivalis cells shed the light on the two arguments that can explain the pathogenicity of this bacterium. First, deduced amino acid sequence indicated that the PVQNLT stretch is followed by a PN repeat, which is composed of typical structure-braking residues, making immunoresponsible residues outside of the cell wall. In ARS-II, a proline-rich stretch (TTTPPPG), another structure-braking feature, did exist that may again facilitate localization of the PVQNLT toward the molecular surface position. Second, nucleotide sequencing of the 4.6-kb EcoRI-SacI fragment indicated the presence of IS1126-like next to the gene coding for 130k-HMGD. Because the insertion sequence has the ability to move on chromosome concomitant with a particular gene, it is likely that this IS sequence may help spread the gene coding for PVQNLT all around the P. gingivalis chromosome and that it contributes to make this bacterium a more virulent strain.

In the present study, we identified the amino acid stretch, PVQNLT, responsible for hemagglutinating activity. Because the synthetic peptide, APVQNLTGSSVG, exhibited the inhibitory activity toward hemagglutinin and because these motifs are so specific and widely distributed at the P. gingivalis cell surface, this information might provide useful tools to establish a passive immunization system to prevent periodontal disease in human.

    FOOTNOTES

* This work was supported in part by Grants-in-Aid for Scientific Research and Research for the Frontier Science from The Ministry of Education, Science, Sports and Culture of Japan C2 10671780 and A1 10357020, by Fund for Comprehensive Research on Aging and Health from The Ministry of Public Welfare of Japan Grant 96A2303, and by the Suzuki Memorial Grant of Nihon University School of Dentistry at Matsudo Young Researchers Grant 98-2004.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB019363.

Dagger To whom correspondence should be addressed: Dept. of Biochemistry, Nihon University School of Dentistry at Matsudo, 2-870-1, Sakaecho-Nishi, Matsudo, Chiba 271-8587, Japan. Tel.: 81-47-360-9328; Fax: 81-47-360-9329; E-mail: yabiko{at}mascat.nihon-u.ac.jp.

    ABBREVIATIONS

The abbreviations used are: PCR, polymerase chain reaction; kb, kilobase(s); ORF, open reading frame; ARS, antibody recognition site; bp, base pair(s).

    REFERENCES
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Abstract
Introduction
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