Blackwell Science, LtdOxford, UKMMIMolecular Microbiology0950-382X© 2005 The Authors; Journal compilation © 2005 Blackwell Publishing Ltd? 2005591152167Original ArticleP. gingivalis-induced platelet aggregationM. Naito et al.
Molecular Microbiology (2006) 59(1), 152–167
doi:10.1111/j.1365-2958.2005.04942.x First published online 24 October 2005
Porphyromonas gingivalis-induced platelet aggregation in plasma depends on Hgp44 adhesin but not Rgp proteinase Mariko Naito,1 Eiko Sakai,2 Yixin Shi,3† Hiroshi Ideguchi,4 Mikio Shoji,1 Naoya Ohara,1 Kenji Yamamoto5 and Koji Nakayama1* Divisions of 1Microbiology and Oral Infection and 2Oral Molecular Pharmacology, Department of Developmental and Reconstructive Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan. 3 Department of Oral Infectious Diseases and Immunology, Graduate School of Dental Science, Kyushu University, Fukuoka, Japan. 4 Department of Laboratory Medicine, School of Medicine, Fukuoka University, Fukuoka, Japan. 5 Department of Pharmacology, Graduate School of Dental Science, Kyushu University, Fukuoka, Japan. Summary Evidence from recent epidemiological studies suggests a link between periodontal infections and increased risk of atherosclerosis and related cardiovascular and cerebrovascular events in human subjects. One of the major pathogens of periodontitis, Porphyromonas gingivalis, has the ability to aggregate human platelets in platelet-rich plasma (PRP). Mechanism of P. gingivalis-induced platelet aggregation in PRP was investigated. Proteinase inhibitors toward Arg-gingipain (Rgp) and Lys-gingipain (Kgp) did not suppress P. gingivalis-induced platelet aggregation in PRP, whereas the Rgp inhibitor markedly inhibited P. gingivalis-induced platelet aggregation using washed platelets. Mutant analysis revealed that P. gingivalis-induced platelet aggregation in PRP depended on Rgp-, Kgp- and haemagglutinin A (HagA)-encoding genes that intragenically coded for adhesins such as Hgp44. Hgp44 adhesin on the bacterial cell surface, which was processed by Rgp and Kgp proteinases, was essential for P. gingivalisinduced platelet aggregation in PRP. P. gingivalis cell-
Accepted 29 September, 2005. *For correspondence. E-mail
[email protected]; Tel. (+81) 95 849 7649; Fax (+81) 95 849 7650. †Present address: Department of Molecular Microbiology, Washington University, St Louis, MO 63110, USA.
© 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd
reactive IgG in plasma, and FcγγRIIa receptor and to a α receptor on platelets were found lesser extent GPIbα to be a prerequisite for P. gingivalis-induced platelet aggregation in PRP. These results reveal a novel mechanism of platelet aggregation by P. gingivalis. Introduction Microorganisms in dental plaque or gingival crevicular biofilm have a chance to disseminate into the bloodstream as a consequence of damage and/or injury of periodontal tissue by periodontal inflammation, dental surgery and dental hygienic treatments. A number of epidemiological studies have shown significant relations between periodontal diseases and cardiovascular diseases (Mattila et al., 1995; Beck et al., 1996; Joshipura et al., 1996; Morrison et al., 1999), however, other studies have not confirmed such relations (Hujoel et al., 2000; Howell et al., 2001). Recently, Taniguchi et al. (2003) have found that infection by one of the periodontal pathogens, Porphyromonas gingivalis, which is also causative of brain abscess (Iida et al., 2004), is markedly associated with carotid atherosclerosis in non-obese Japanese type 2 diabetic patients. Presence of P. gingivalis in atherosclerotic plaques has been reported (Chiu, 1999; Kozarov et al., 2005). Moreover, in a mouse model of atherosclerosis using apolipoprotein E-deficient mice, intravenous and oral inoculations of P. gingivalis cells were found to accelerate atherogenic plaque progression (Li et al., 2002) and to enhance vascular activation (Lalla et al., 2003) respectively. Although the significance of P. gingivalis in aetiology of cardiovascular diseases is still unidentified, these studies have indicated an association between P. gingivalis and cardiovascular diseases. Porphyromonas gingivalis is an anaerobic Gramnegative rod that is implicated as one of the most important aetiological agents of adult periodontal disease. It is asaccharolytic and highly proteolytic. P. gingivalis cysteine proteinases with specificity of cleavage after Arg or Lys residue, which are major extracellular and cell surfaceassociated proteinases, are now termed gingipains. They consist of Arg-specific gingipain (Arg-gingipain, Rgp) and Lys-specific gingipain (Lys-gingipain, Kgp). Rgp is encoded by two genes, rgpA and rgpB, and Kgp is
P. gingivalis-induced platelet aggregation 153 encoded by a single gene, kgp. rgpA and kgp have similar gene structures consisting of an N-terminal propeptide region including a putative signal peptide region, a proteolytic domain, and C-terminal adhesin domains (HbR (Hgp15), Hgp17, Hgp27 and Hgp44). These adhesin domains are responsible for haemoglobin binding (HbR) and haemagglutination (Hgp44 and Hgp17). The adhesin domains are also encoded by the haemagglutinin gene hagA of P. gingivalis (Han et al., 1996). These adhesin proteins encoded by rgpA, kgp and hagA are generated from their proproteins by cleavage with Rgp and Kgp. Rgp appears to be responsible for processing of P. gingivalis surface proteins such as fimbrilin (Nakayama et al., 1996; Kadowaki et al., 1998), 75 kDa protein (Nakayama et al., 1996; Kadowaki et al., 1998), and prolyl tripeptidyl peptidase (Banbula et al., 1999a). Rgp and Kgp degrade a number of host proteins including collagens (type I and IV), serum albumin, haemoglobin, fibronectin, laminin and fibrinogen. Moreover, Rgp and Kgp have the ability to disrupt the host defence systems to degrade and inactivate immunoglobulin (Ig) (Kadowaki et al., 1994; Abe et al., 1998), cytokines (Calkins et al., 1998; Banbula et al., 1999b) and cell surface receptor molecules (Jagels et al., 1996; Scragg et al., 1999; Sugawara et al., 2000; Oleksy et al., 2002). On the other hand, Rgp and Kgp contribute to development and maintenance of inflammation through activation of the kallikrein-kinin cascade (Imamura et al., 1994), impairment of the complement cascade (Wingrove et al., 1992), and activation of the coagulation cascade (Imamura et al., 1997). Animal infection study using various mutant strains has revealed that gingipains are closely associated with virulence of P. gingivalis (Yoneda et al. 2001). Porphyromonas gingivalis cells have the ability to aggregate human platelets in platelet-rich plasma (PRP) (Herzberg et al., 1994). A proteinase preparation from P. gingivalis (protease I), which seemed to be Rgp and Kgp, was able to induce platelet aggregation with washed platelets (Curtis et al., 1993). Lourbakos et al. (2001) have found that purified Rgp has the ability to induce platelet aggregation with washed platelets through activation of the protease-activated receptors (PAR)-1 and -4, expressed on the surface of platelets. The extracellular Nterminal regions of PARs contain thrombin cleavage sites and cleavage of the N-terminal regions by thrombin causes activation of PAR receptors. Rgp-induced PAR activation appears to be similar to that of thrombin (Lourbakos et al., 2001). In this study, we examined whether P. gingivalisinduced platelet aggregation in PRP was attributable to Rgp and/or Kgp by using proteinase inhibitors specific for Rgp and Kgp, various P. gingivalis mutant strains including the rgpA rgpB kgp and rgpA kgp hagA triple mutants and the rgpA rgpB kgp hagA quadruple mutant constructed in
the present study. The results obtained here indicate that P. gingivalis cells have a novel mechanism of platelet aggregation in which P. gingivalis adhesins, glycoprotein (GP) Ibα receptor, FcγRIIa receptor and plasma IgG are involved.
Results Effect of proteinase inhibitors on P. gingivalis-induced platelet aggregation Porphyromonas gingivalis cells can aggregate human platelets in PRP (Herzberg et al., 1994). To determine whether P. gingivalis cells were present in platelet aggregates in PRP, P. gingivalis cells were labelled by FITC and mixed with PRP. Fluorescence microscopy revealed that platelet aggregates contained a number of P. gingivalis cells and there were very few free bacterial cells (Fig. 1A). P. gingivalis-induced platelet aggregates in PRP was found to be caused by platelet activation, as revealed by no platelet aggregate formation when using prostaglandin E1-pretreated PRP (Fig. S1). Several studies have demonstrated that P. gingivalis cysteine proteinase, Rgp, has the ability to activate platelets using washed platelets (Curtis et al., 1993; Lourbakos et al., 2001). To examine whether P. gingivalis-induced platelet aggregation in PRP was also attributable to Rgp and/or Kgp activities of the microorganism, a proteinase inhibitor that had the potential to inhibit Rgp or Kgp was added to the mixture of P. gingivalis cell suspension and PRP. Addition of leupeptin (Rgp-specific inhibitor, 1 mM) and KYT36 (Kgp-specific inhibitor, 0.1 mM), the concentrations of which could completely inactivate Rgp and Kgp, respectively, showed no significant effect on P. gingivalisinduced platelet aggregation (Fig. 1B and Table S1). Addition of both inhibitors also showed no effect, indicating that platelet-aggregating factors other than gingipains might be present in P. gingivalis. Localization of the platelet aggregation factors in the outer membrane fraction To determine subcellular localization of the plateletaggregating factors of P. gingivalis, cells of P. gingivalis wild-type strain 33277 were fractionated into the cytoplasmic/periplasmic fraction, the inner membrane fraction and the outer membrane fraction. The outer membrane fraction as well as the whole cell lysates could induce platelet aggregation in PRP (86.8% ± 8.6% aggregation and 78.3% ± 10.9% aggregation, respectively), whereas there was no activity in the cytoplasmic/periplasmic fraction and the inner membrane fraction (2.8% ± 1.3% aggregation and 3.5% ± 1.3% aggregation, respectively), indicating that the aggregating factors were localized on
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Fig. 1. Platelet aggregation in PRP induced by P. gingivalis cells and no effect of proteinase inhibitors on it. A. Microscopic images of P. gingivalis-induced platelet aggregation complex. FITC-labelled P. gingivalis 33277 cells were added to PRP. After maximum aggregation was induced, the reacted mixture was subjected to fluorescence microscopy. B. P. gingivalis 33277 cells were treated with leupeptin, KYT36, or both for 10 min before mixed with PRP. The final concentrations of leupeptin and KYT36 in the mixture were 1 mM and 0.1 mM respectively. Aggregation traces were presented.
the outer membrane fraction. After heat treatment at 100°C for 10 min, the whole cell lysates and the outer membrane fraction lost the activity (−4.3% ± 0.6% aggregation and 2.0% ± 1.0% aggregation, respectively), implying that the aggregating factors were heat labile. Mutant analysis of P. gingivalis-induced platelet aggregation Various mutant strains concerning gingipains have been constructed from P. gingivalis 33277 by insertion of antibiotic resistance gene DNA into gingipain-related genes on the P. gingivalis chromosome using the homologous recombination technique as previously described (Shi et al., 1999) (Table 1 and Fig. 2A). KDP129 has no Kgp activity and KDP133 has no Rgp activity; however, these mutants have adhesins on their cell surfaces that are processed by Rgp and Kgp respectively. KDP136 has no Kgp or Rgp activity, but an unprocessed hagA-derived adhesin proprotein with a high molecular mass on the cell
surface. KDP137 has rgpB-derived Rgp activity but no Kgp activity or adhesins. These strains were examined for platelet aggregation (Fig. 2B and C and Table S1). When using PRP, the Rgp-null mutant KDP133 (rgpA rgpB) showed platelet aggregation with no significant difference in the lag time to onset and the maximum aggregation rate from those of the wild-type parent strain, that was consistent with the fact that the Rgp-specific proteinase inhibitor failed to inhibit P. gingivalis-induced platelet aggregation in PRP. The Kgp-null mutant KDP129 (kgp) also showed platelet aggregation in PRP, whereas the Rgp/Kgp-null mutant KDP136 (rgpA rgpB kgp) showed no platelet aggregation up to 50 min after initiation. The rgpA and kgp genes encode the adhesin domain proteins at their 3′terminal regions in addition to encoding Rgp and Kgp respectively. The adhesin-null mutant KDP137 (rgpA kgp hagA) that had rgpB-derived Rgp activity showed no platelet aggregation. These results suggested that the adhesin proteins might be involved in P. gingivalis-induced platelet aggregation in PRP.
© 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 59, 152–167
P. gingivalis-induced platelet aggregation 155 Table 1. P. gingivalis strains used in this study. Strain
Genotype
Relevant property
Source or reference
33277 KDP129 KDP133 KDP136 KDP137 KDP150 KDP153
Wild type kgp::cat rgpA::tetQ rgpB::erm rgpA::erm rgpB::tetQ kgp::cat rgpA::erm kgp::cat hagA::tetQ fimA::erm rgpA::erm rgpB::tetQ kgp::cat hagA::nimC
Wild type Kgp-null Rgp-null Rgp/Kgp-null Adhesin-null FimA fimbria-less Rgp/Kgp/adhesin–null
ATCC Shi et al. (1999) Shi et al. (1999) Shi et al. (1999) Shi et al. (1999) Shoji et al. (2004) This study
On the other hand, when using washed platelets, KDP137 showed platelet aggregation, which was markedly suppressed by addition of leupeptin. Suppressive effect of leupeptin was also observed in platelet aggregation by KDP129 and to the lesser extent, in that by 33277. KDP133 showed markedly lower aggregation with washed platelets than in PRP. These results indicated that Rgp had the ability to induce aggregation with washed platelets, consistent with the previous studies (Curtis et al., 1993; Lourbakos et al., 2001); however, Rgp failed to activate platelets in PRP. Porphyromonas gingivalis fimbriae was reported to be involved in murine platelet aggregation (Sharma et al., 2000). The fimbria-less mutant KDP150 (fimA) showed platelet aggregation in human PRP (Fig. 2B and 91.6% ± 4.0% aggregation), indicating that fimbriae may not contribute to P. gingivalis-induced platelet aggregation in human PRP. Interbacterial complementation of platelet aggregation between the Rgp/Kgp-null mutant and the adhesin-null mutant Neither the Rgp/Kgp-null mutant nor the adhesin-null mutant showed platelet aggregation. We attempted to determine whether P. gingivalis cells from a mixed culture of these two mutant strains had the ability to aggregate human platelets. Surprisingly, the bacterial cells from the mixed culture showed platelet aggregation (Fig. 3A). The Rgp/Kgp-null mutant expresses the unprocessed adhesin proproteins derived from hagA on the cell surface, whereas the adhesin-null mutant has Rgp activity derived from rgpB. The hagA-derived adhesin proproteins of the Rgp/Kgp-null mutant may be processed after co-culture with the Rgp-producing adhesin-null mutant (Fig. 3B). To determine the possibility, immunoblot analysis using antiadhesin antibodies were performed (Fig. 3C). The Rgp/ Kgp-null mutant cells showed anti-HGP44-immunoreactive proteins with molecular masses larger than 120 kDa, whereas the bacterial cells of the mixed culture showed several anti-HGP44-immunoreactive proteins with low molecular masses that were observed in the wild-type
parent cells but not in the Rgp/Kgp-null mutant cells. These results indicated that Rgp activity of the adhesinnull mutant might process the hagA-derived adhesin proproteins on the cell surface of the Rgp/Kgp-null mutant, resulting in recovery of platelet aggregation. Recovery of platelet aggregation of the Rgp/Kgp-null mutant by treatment with trypsin The interbacterial complementation of platelet aggregation tempted us to determine whether treatment of KDP136 with trypsin that had the same cleavage sites as Rgp/Kgp showed a similar effect. KDP136 cells that were treated with trypsin induced platelet aggregation in PRP (Fig. S2). In this experiment, a trypsin inhibitor was added to the mixture of the bacterial cells and PRP to avoid a direct effect of trypsin on platelets. Trypsin alone could not induce platelet aggregation in PRP at the same concentration as used in this treatment (data not shown). Furthermore, KD136 cells that had been treated with trypsin, harvested and washed with phosphatebuffered saline (PBS) also induced platelet aggregation in PRP, whereas the supernatants containing trypsin showed no aggregation. KDP137 cells treated with trypsin could not induce platelet aggregation (2.7% ± 1.5% aggregation). Immunoblot analysis using anti-Hgp44 and anti-HbR antibodies revealed that after trypsin treatment the immunoreactive proteins derived from hagA were processed into proteins with low molecular masses (Fig. S2C). The immunoreactive proteins with low molecular masses were still associated with the bacterial cells. To confirm whether the proteinaseprocessed hagA adhesin molecules are responsible for P. gingivalis-induced platelet aggregation, we constructed a Rgp/Kgp/adhesin-null mutant (rgpA rgpB kgp hagA; KDP153) and examined trypsin-treated and non-treated KDP153 cells for platelet aggregation, resulting in no aggregation (2.5% ± 1.3% aggregation and 7.0% ± 2.4% aggregation respectively). These results indicated that the proteinase-processed adhesin molecules were a prerequisite for P. gingivalis cells to induce platelet aggregation in PRP.
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Fig. 2. Platelet aggregation induced by the various gingipain-related mutants. A. Structures of the gingipain-related genes. rgpA and kgp consist of propeptide, mature proteinase and adhesin domain regions (Hgp44, HbR, Hgp17 and Hgp27). rgpB has propeptide and Rgp proteinase domain regions but lacks most of the adhesin domain region. hagA has a repeated structure consisting of the adhesin domains. R and K indicate a cleavage site by Rgp and Kgp respectively. B and C. Aggregation traces (B) and maximum aggregation rates (C) of various gingipain-related mutants. PRP was used as a source of platelets in (B). L in (C) indicates 1 mM leupeptin. *P < 0.01 related to the maximum aggregation rate of 33277. © 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 59, 152–167
P. gingivalis-induced platelet aggregation 157 Fig. 3. Interbacterial complementation. A. Aggregation traces of the bacterial cells of co-culture of the Rgp/Kgp-null mutant (KDP136) and the adhesin-null mutant (KDP137) were presented. B. Schematic drawing of interbacterial complementation. C. SDS-PAGE analysis. Bacterial cell lysates were subjected to SDS-PAGE. Gels were stained with CBB (lanes 1–4) and blotted onto Immobilon membranes. The membrane was immunoreacted with anti-Hgp44 serum (lanes 5–8). Lanes: 1 and 5, 33277; 2 and 6, KDP136; 3 and 7, KDP137; 4 and 8, co-cultured KDP136 and KDP137. The relative molecular masses are shown to the left.
© 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 59, 152–167
158 M. Naito et al. Induction of platelet aggregation in PRP by the Rgp/Kgp/adhesin-null mutant cells pretreated with the rHgp44 adhesin KDP153 cells were pretreated with the rHgp44 adhesin at 37°C for 50 min. The resulting mixture was subjected to platelet aggregation in PRP. KDP153 cells or the rHgp44 adhesin alone failed to induce platelet aggregation in PRP, whereas rHgp44-pretreated KDP153 induced it (Fig. 4A). rHgp44-pretreated KDP136 and KDP137 also induced platelet aggregation (Fig. S3). Immunoblot analysis revealed that rHgp44 was associated with the bacterial cells after treatment of the bacterial cells with rHgp44 for 50 min (Fig. 4B). When rHgp44 and KDP153 cells were added to PRP at the same time without pretreatment, platelet aggregation did not occur. These results demonstrated that presence of rHgp44 on the bacterial cell surface was required for P. gingivalis-induced platelet aggregation in PRP. This effect of rHgp44 was also seen in KDP136 (data not shown). On the other hand, rHbR did not have such activity.
Roles of GPIIb/IIIa and GPIbα receptors in P. gingivalisinduced platelet aggregation In Streptococcus sanguis-induced platelet aggregation, platelets are stimulated through GPIbα receptor on the platelet surface (Kerrigan et al., 2002). After that, GPIIb/ IIIa is activated by inside-out signalling. Addition of monoclonal antibody (mAb) P2, an antagonist of GPIIb/IIIa, to the mixtures of P. gingivalis cells (33277 and trypsintreated KDP136) and PRP completely inhibited platelet aggregation (Fig. 5A). To determine whether GPIbα receptor was involved in P. gingivalis-induced platelet aggregation in PRP, various mAbs directed against different N-terminal regions of GPIbα were examined for their ability to inhibit the aggregation (Fig. 5B). The mAb SZ-2 significantly inhibited P. gingivalis-induced platelet aggregation (Fig. 5C). Effects of other mAbs on platelet aggregation varied among trials. To confirm the interaction of GPIbα with P. gingivalis-induced platelet aggregation, PRP were pretreated with mocarhagin, a cobra venom metalloproteinase that specifically cleaved an N-terminal region of GPIbα and abolished signals via GPIb/IV/V complex. Aggregation of mocarhagin-treated PRP could be induced by ADP, but not by ristocetin that elicited association of human von Willebrand Factor (vWF) with GPIbα. Platelet aggregation in PRP with 33277 and trypsin-treated KDP136 cells was partially inhibited by treatment with mocarhagin (Fig. 5D). These results indicated that platelet activation via GPIbα contributed to P. gingivalis-induced platelet aggregation in PRP.
Fig. 4. Effect of rHgp44 on platelet aggregation of the Rgp/Kgp/ adhesin-null mutant in PRP. A. Maximum aggregation rates. One portion (10 µl) of the cell suspension of the Rgp/Kgp/adhesin-null mutant (KDP153) was mixed with rHbR (50 µg in 40 µl of PBS), rHgp44 (50 µg in 40 µl of PBS), or PBS at 37°C for 50 min. After that, a whole volume (50 µl) of the mixture was mixed with PRP (180 µl) and subjected to platelet aggregometry. Another portion (10 µl) of the cell suspension was mixed with PRP (180 µl) plus rHbR (50 µg in 40 µl of PBS), PRP (180 µl) plus rHgp44 (50 µg in 40 µl of PBS) or PRP (180 µl) plus PBS (40 µl) without pretreatment. Hatched and closed bars indicate the experiments with and without the pretreatment respectively. Open bars indicate maximum aggregation rates of PRP treated with rHbR and rHgp44. B. SDS-PAGE analysis. KDP153 cells were treated with rHgp44 for 50 min as above. The treated bacterial cells were separated by centrifugation, washed twice with PBS, and subjected to SDS-PAGE analysis. Gels were stained with CBB (lanes 1–4) and blotted onto Immobilon membranes. The membrane was immunoreacted with anti-Hgp44 serum (lanes 5–8). Lanes: 1 and 5, untreated KDP153 cells; 2 and 6, the mixture of KDP153 cells with rHgp44; 3 and 7, the supernatants after centrifugation; 4 and 8, the washed bacterial cells of KDP153 treated with rHgp44. The relative molecular masses are shown to the left.
© 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 59, 152–167
P. gingivalis-induced platelet aggregation 159
Fig. 5. Roles of GPIIb/IIIa and GPIbα in P. gingivalis-induced platelet aggregation. PRP was pretreated with anti-GPIIb/IIIa mAb P2 (15 µg ml−1), control mAb (15 µg ml−1) or PBS at 37°C for 10 min before mixed with P. gingivalis cells. A. Maximum aggregation rates were presented. B. Location of the epitopes of anti-GPIbα monoclonal antibodies (Shen et al., 2000) and the cleavage site of mocarhagin (Ward et al., 1996) on GPIbα. C. Effects of anti-GPIbα mAbs on P. gingivalis-induced platelet aggregation. PRP was pretreated with anti-GPIbα mAbs (AN51, AK2, Hip-1, VM16d and SZ2; 15 µg ml−1), control mAb (15 µg ml−1) or PBS at 37°C for 10 min before mixed with P. gingivalis cells. Maximum aggregation rates were presented. D. Effect of mocarhagin treatment on P. gingivalis-induced platelet aggregation. PRP was pretreated with mocarhagin (5 µg ml−1) or PBS at 37°C for 45 min. Open and closed bars indicate the maximum aggregation rates of control PRP and mocarhagin-treated PRP respectively. Mocarhagintreated PRP was examined for the ability to aggregate in response to ADP (10 µM), ristocetin (1.5 mg ml−1), 33277 with or without leupeptin, and trypsin-treated KDP136 (rgpA rgpB kgp). Leupeptin was added at the final concentration of 1 mM. *P < 0.01 related to the maximum aggregation rate of untreated PRP.
Roles of IgG and FcγRIIa receptor in P. gingivalis-induced platelet aggregation Because the plasma factor vWF is a ligand for GPIbα, we examined whether plasma factors were a prerequisite for P. gingivalis-induced platelet aggregation in PRP (Fig. 6A). Trypsin-treated KDP136 could induce aggregation with washed platelets in the presence of platelet-poor plasma (PPP) but not in the absence of PPP. Addition of human vWF, fibrinogen, or fibronectin to the mixture of trypsin-treated KDP136 and washed platelets failed to induce platelet aggregation. The results indicated that a plasma factor(s) other than vWF, fibrinogen, and fibronec-
tin was a requisite for platelet aggregation induced by trypsin-treated KDP136 cells. The previous reports concerning Helicobacter pylori and S. sanguis suggested contribution of Ig to induction of platelet aggregation (Kerrigan et al., 2002; Byrne et al., 2003). FcγRIIa is the sole Ig receptor on human platelets, and can bind polymeric IgG. GPIb/IX/V complex and FcγRIIa may associate in physical proximity, resulting in functional interplay on human platelet activation (Sullam et al., 1998; Wu et al., 2001). We determined whether P. gingivalis-induced platelet aggregation in PRP depended on IgG and FcγRIIa receptor. Cells of 33277 and trypsin-treated KDP136 could induce aggregation with
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Fig. 6. Roles of plasma factors, FcγRIIa and P. gingivalis cell-reactive IgG in P. gingivalis-induced platelet aggregation. A. Involvement of plasma factors in P. gingivalis-induced platelet aggregation. KDP136 cells with or without trypsin treatment were mixed with PRP, washed platelets, washed platelets plus vWF (10 µg ml−1), washed platelets plus fibronectin (30 µg ml−1), washed platelets plus fibrinogen (0.5 mg ml−1), or washed platelets plus PPP (1/10 volume of the original), and subjected to platelet aggregometry. Trypsin inhibitor was added to the mixture in the experiment with trypsin-treated bacterial cells. Ristocetin (1.5 mg ml−1), trypsin plus trypsin inhibitor, and PBS were used as a control. B. Role of plasma IgG in P. gingivalis-induced platelet aggregation. Washed platelets supplemented with human IgG (1.3 mg ml−1), PPP (1/10 volume of the original) or IgG-depleted PPP (1/10 volume of the original) were examined for aggregation in response to cells of 33277 and trypsintreated KDP136. Trypsin inhibitor and leupeptin were added to the mixtures in the experiments with trypsin-treated KDP136 and 33277 respectively. C. Effect of anti-FcγRIIa mAb in P. gingivalis-induced platelet aggregation. PRP was treated with anti-FcγRIIa mAb (IV.3; 20 µg ml−1), control mAb (20 µg ml−1), or PBS at 37°C for 10 min and mixed with P. gingivalis cells or ristocetin (1.5 mg ml−1). Trypsin inhibitor and leupeptin were added to the mixtures in the experiments with trypsin-treated KDP136 and 33277 respectively. D. Role of P. gingivalis cell-reactive IgG in P. gingivalis-induced platelet aggregation. Human IgG fraction (80 µl, 13 mg ml−1) was mixed with cells of trypsin-treated KDP136 (80 µl, 785 mg ml−1) at room temperature for 10 min and centrifuged at 15 000 r.p.m. for 2 min. The resulting supernatant was used as human IgG lacking P. gingivalis cell-reactive IgG (Pg-treated IgG). The cell precipitate was washed twice with PBS, suspended in PBS and used as trypsin-treated KDP136 coated with IgG. Washed platelets (160 µl) supplemented with human IgG (40 µl, 6.5 mg ml−1) or Pgtreated IgG (40 µl, 6.5 mg ml−1), or with PBS (40 µl) as a control were mixed with cells (20 µl) of KDP136 or trypsin-treated KDP136. Cells of trypsin-treated KDP136 coated with IgG were added to washed platelets. E. Specificity of P. gingivalis cell-reactive IgG. Human IgG fraction (80 µl, 13 mg ml−1) was mixed with cells of trypsin-treated P. gingivalis KDP136, E. coli MC4100 or Prevotella intermedia ATCC 25611 (80 µl, 785 mg ml−1) at room temperature for 10 min and centrifuged at 15 000 r.p.m. for 2 min. The resulting supernatants were used as human IgG treated with the bacterial cells (Pg-treated IgG, Ec-treated IgG and Pi-treated IgG). Washed platelets (160 µl) supplemented with human IgG (40 µl, 6.5 mg ml−1) or bacterial cell-treated IgG (40 µl, 6.5 mg ml−1) were mixed with cells (20 µl) of trypsin-treated KDP136. © 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 59, 152–167
P. gingivalis-induced platelet aggregation 161 washed platelets in the presence of IgG but not IgGdepleted PPP, suggesting that IgG was essential for P. gingivalis cells to induce platelet aggregation (Fig. 6B). Anti-FcγRIIa mAb was examined for the ability to inhibit the aggregation. mAb IV.3 (Rosenfeld et al., 1985) completely inhibited P. gingivalis-induced platelet aggregation in PRP, while it did not affect ristocetin-induced platelet aggregation (Fig. 6C). These results strongly indicated that plasma IgG and FcγRIIa receptor were indispensable for P. gingivalis-induced platelet aggregation in plasma. Involvement of P. gingivalis cell-reactive IgG in P. gingivalis-induced platelet aggregation We determined a titre of P. gingivalis-reactive IgG in serum Ig of the healthy subjects used in this study. The titres of the healthy subjects were in the range of 26–56. Comparing with titres of anti-P. gingivalis antibodies of patients with adult periodontitis, the titres of the healthy subjects were very low, but detectable. To determine whether P. gingivalis cell-reactive antibody contributes to platelet aggregation, P. gingivalis cell-reactive antibody was removed from IgG of the healthy subjects by treatment with cells of trypsin-treated KDP136. IgG lacking P. gingivalis cell-reactive antibody failed to induce platelet aggregation with washed platelets and trypsin-treated KDP136 cells (Fig. 6D). Platelet aggregation with washed platelets, trypsin-treated KDP136, and IgG pretreated with cells of Escherichia coli or Prevotella intermedia was performed, resulting in no statistically significant suppression of platelet aggregation (Fig. 6E), suggesting that sera of the healthy subjects contain anti-P. gingivalis antibodies responsible for platelet aggregation although the titres are very low. Interestingly, IgG pretreated with cells of KDP137 or KDP136 also lost the aggregation-inducing ability (−6.1% ± 0.85% aggregation and 4.9% ± 9.6% aggregation respectively). These results indicate that P. gingivalis cell-reactive IgG was required for P. gingivalis-induced platelet aggregation, but it was not necessary for the IgG to be reactive to adhesins such as Hgp44. Discussion It has been reported that Rgp purified from P. gingivalis can activate washed platelets through PAR-1 and −4 (Lourbakos et al., 2001). However, the mechanism of platelet activation by P. gingivalis was not elucidated completely, especially in in vivo conditions, as like in plasma. In this study, we provide strong evidence to reveal a novel mechanism of P. gingivalis cell-induced platelet aggregation in plasma for which processed adhesins of P. gingivalis, plasma IgG and FcγRIIa receptor and to a lesser extent GPIbα receptor of platelets were required.
This finding suggests the tight interaction of vaso-invasive P. gingivalis cells with host immune reaction and thrombosis system. The experiments using the proteinase inhibitors specific for Rgp and Kgp, and the Rgp-null mutant in this study indicated that platelet-aggregating factors other than Rgp might induce aggregation independent of PAR activation. Several lines of evidence show that the adhesin domain proteins such as Hgp44 encoded by rgpA, kgp and hagA are responsible for Rgp-independent platelet aggregation. First, the platelet-aggregating factors were present in the outer membrane fraction. Second, the adhesin-null mutant (rgpA kgp hagA) that possessed Rgp activity failed to aggregate platelets in PRP. Third, the Rgp/Kgp-null mutant (rgpA rgpB kgp) that failed to aggregate platelets in PRP recovered the aggregating ability after co-culture with the Rgp-producing adhesin-null mutant or treatment with trypsin. These treatments caused processing of the hagA-derived adhesin proteins on the surface of the rgpA rgpB kgp mutant cells, revealed by immunoblot analysis with anti-Hgp44 and anti-HbR antibodies. Fourth, the rgpA kgp hagA triple and rgpA rgpB kgp hagA quadruple mutants lacking adhesins failed to aggregate platelets in PRP even after treatment with trypsin. Lastly, the rgpA rgpB kgp hagA quadruple mutant pretreated with rHgp44 showed platelet aggregation in PRP, whereas the quadruple mutant or rHgp44 alone failed to aggregate platelets in PRP. These results also indicate that processing of Hgp44 domain from the proproteins is essential for P. gingivalis cell-mediated platelet aggregation. It can be explained by that a putative binding site of Hgp44 domain of the proproteins essential for platelet aggregation may be masked by other domains of the proproteins or that three-dimensional conformational change of Hgp44 domain by processing may be essential for platelet aggregation. In addition, it is worthy of note that the rgpA kgp hagA triple mutant possessing rgpB-derived Rgp activity showed no platelet aggregation in PRP but with washed platelets, indicating that plasma may inhibit Rgp-mediated platelet activation probably because concentration of total proteins in human plasma is 60–80 mg ml−1 and a number of plasma proteins are targets for Rgp digestion. In this context, Rgp activity was reduced to 53% when plasma was added to the reaction mixture at a final concentration of 1.25% (M. Naito and K. Nakayama, unpubl. data). Roles of the adhesin domain proteins have been characterized in several aspects. Hgp44 and Hgp17 seem to have a haemagglutinating activity (Kelly et al., 1997; Shibata et al., 1999) because an mAb suppressing P. gingivalis-induced haemagglutination reacts to Hgp44 and Hgp17. HbR, also termed Hgp15 or HA2, is a haemoglobin-binding protein (Nakayama et al., 1998). P. gingivalis requires haem for its growth (Gibbons and MacDonald, 1960; Lev et al., 1971). P. gingivalis cells
© 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 59, 152–167
162 M. Naito et al. adhere to erythrocytes by Hgp44 and Hgp17 proteins and may obtain haem from erythrocytes. Hgp44 is also responsible for coaggregation of P. gingivalis with one of the early colonizing microorganisms in gingival crevicular biofilm, P. intermedia (Kamaguchi et al. 2003). HbR can bind lactoferrin. (Shi et al., 2000). The present study may add a novel role, i.e. platelet aggregation, to the adhesin proteins. rHgp44 alone failed to aggregate platelets in PRP although the protein appears to be required for P. gingivalis-induced platelet aggregation. As shown in this study, the adhesin generated from hagA is located on the cell surface. The Rgp/Kgp/adhesin-null mutant (rgpA rgpB kgp hagA) pretreated with rHgp44 induced platelet aggregation in PRP, whereas without the pretreatment the mutant failed to aggregate platelets in PRP even in the presence of rHgp44. During the pretreatment, rHgp44 attached to the cell surface of the mutant, as revealed by immunoblot analysis. These results indicate that presence of adhesins on the outer membrane may be important for platelet aggregation. rHgp44 still had the ability to attach to KDP153 bacterial cells that had been incubated at 80°C or treated with proteinase K (M. Naito and K. Nakayama, unpubl. data), suggesting that a binding target on the bacterial cell surface for Hgp44 is a nonproteinous molecule such as lipid or sugar. Identification of the binding site is under way. What receptor molecule on the surface of platelets contributes to Rgp-independent platelet aggregation caused by P. gingivalis cells? A GPIIb/IIIa antagonist, mAb P2, completely abolished platelet aggregation by the trypsinpretreated Rgp/Kgp-null mutant as well as the intact wildtype strain, suggesting that Rgp-independent aggregation requires activation of GPIIb/IIIa. Anti-FcγRIIa mAb completely abolished P. gingivalis-induced platelet aggregation in PRP. Deprivation of IgG from plasma also abolished it. On the other hand, the anti-GPIbα mAb (SZ2) partially suppressed P. gingivalis-induced platelet aggregation in plasma. Mocarhagin-pretreated platelets showed a reduction in P. gingivalis-induced platelet aggregation. These results strongly indicate that plasma IgG, FcγRIIa and to a lesser extent GPIbα are required for Rgp-independent platelet aggregation in PRP caused by P. gingivalis cells. Deprivation of P. gingivalis cell-reactive IgG from plasma IgG abolished platelet aggregation, suggesting activation of FcγRIIa receptor on platelets by IgG-P. gingivalis cell immune complex. Streptococcus sanguis (Kerrigan et al., 2002) and Helicobactor pylori (Byrne et al., 2003) have been found to use GPIbα and Ig for activation of platelets as like here we elucidated about P. gingivalis. H. pylori and S. sanguis require plasma factors other than Ig for induction of platelet aggregation; vWF and fibrinogen in H. pylori and fibrinogen in S. sanguis. Platelet aggregation using washed
platelets reveals that P. gingivalis requires Ig as a plasma factor for platelet aggregation, but not vWF, fibrinogen or fibronectin. However, involvement of GPIIb/IIIa in P. gingivalis cell-induced platelet aggregation implies responsibility of vWF and extracellular matrix (ECM) proteins such as fibrinogen and fibronectin for the aggregation because it is well known that platelet aggregates is formed by binding of these proteins to activated GPIIb/IIIa on the cell surface of platelets. Secretion of fibrinogen, fibronectin and vWF from activated platelets may account for no requirement of vWF, fibrinogen or fibronectin as a plasma factor for P. gingivalis cell-induced platelet aggregation using washed platelets. The rHgp44 adhesin has the ability to bind to ECM proteins such as fibrinogen and fibronectin (E. Sakai, M. Naito, and K. Nakayama, unpubl. data). Interaction of ECM proteins with adhesins such as Hgp44 and cluster formation of P. gingivalis-reactive Ig may take place on the same P. gingivalis cell surface when the bacterial cells are mixed with PRP. Clustered Ig on the bacterial cell surface may bind to FcγRIIa and activate platelets, and ECM proteins anchored to the same bacterial cell by adhesins may bind to activated GPIIb/IIIa and further activate platelets, resulting in development of platelet aggregation. Because we failed to find any effect of finbrinogen or fibronectin on platelet aggregation with washed platelets by trypsin-treated KDP136 cells and various amounts of Ig (Fig. S4), we cannot rule out another possibility that adhesins on the bacterial cell surface bind to a receptor on platelets and arrest the bacterial cells on them, and next, P. gingivalis-reactive Ig clusters on the same bacterial surface and activates platelets through FcγRIIa, leading to platelet aggregation although we have not identify the platelet receptor for adhesins. A similar scenario was previously suggested in Streptococcus pyogenes- and Staphylococcus aureus-induced platelet aggregation (Sjöbring et al., 2002). Full aggregation of platelets in plasma induced by P. gingivalis cells takes place within several min, suggesting that microaggregation of platelets with P. gingivalis cells probably occurs very rapidly after invasion of the bacterial cells to the bloodstream. Aggregates of platelets and P. gingivalis cells can be subjected to phagocytosis by peripheral blood monocytes. As P. gingivalis has been detected in atherosclerotic plaque (Chiu, 1999; Kozarov et al., 2005), P. gingivalis cells might reach atherogenic lesions using monocytes as a vector. P. gingivalis-carrying monocytes might alter their function of scavenging damaged lipoproteins and accelerate inflammation, which might result in enlargement of atherogenic lesions. The findings in the present study might contribute to elucidate novel pathogenesis of P. gingivalis and provide a basis for development of prevention of atherosclerosis and/or thrombosis particularly for patients with periodontal diseases.
© 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 59, 152–167
P. gingivalis-induced platelet aggregation 163 Experimental procedures Bacterial strains Porphyromonas gingivalis strains used in this study are listed in Table 1. All the mutant strains used in this study are derivatives of P. gingivalis wild-type strain 33277 (ATCC). E. coli strain BL21 (DE3) was used for production of recombinant proteins (Studier et al., 1990). E. coli MC4100 and P. intermedia 25611 (ATCC) were also used.
Chemicals and proteins ADP was purchased from MCM. mAb against human GPIbα (Shen et al., 2000) AN51, was purchased from Dako A/S, VM16-d from Sanbio, SZ-2 from Immunotech Coulter, Hip1 from BD Bioscience, and AK2 from Cymbus Biotechnology. Anti-GPIIb/IIIa mAb, P2, was purchased from Immunotech Coulter. Prostaglandin E1 was purchased from Wako pure chemicals (Osaka, Japan). Anti-FcγRIIa mAb, mAb IV.3, was purified from the culture medium of hybridoma HB-217 (American Type Culture Collection) using protein G affinity chromatography as recommended by the manufacturer (Amersham Biosciences AB). Mouse monoclonal IgG isotype control was purchased from R and D System. Rabbit antibodies raised against HbR and Hgp44 were prepared as previously described (Nakayama et al., 1998; Sato et al., 2005). Peroxidase-conjugated swine anti-rabbit Ig and peroxidase-conjugated rabbit anti-human IgG were purchased from Dako. Ristocetin and FITC were purchased from Sigma. Trypsin was purchased from Gibco BRL and soybean trypsin inhibitor from Wako pure chemicals. Leupeptin (Peptide Institute) and KYT36 (Baba et al., 2002) were used for Rgp and kgp inhibitors respectively. Mocarhagin (Ward et al., 1996) was a gift from Dr Michael Berndt (Baker Research Medical Institute, Prahran, Victoria, Australia). Human vWF was purchased from Haematologic Technologies and human plasma fibronectin and fibrinogen were from Sigma.
Preparation of standardized bacterial suspensions Porphyromonas gingivalis cells were cultured anaerobically (10% CO2, 10% H2, 80% N2) in Todd-Hewitt broth (BD Bioscience) supplemented with 5 µg of haemin and 0.5 µg of menadione per ml at 37°C. Bacterial cells were harvested and washed twice in cold PBS, pH 7.5. The washed bacterial cells were suspended in PBS to give an optical density of 1.5 at 620 nm for platelet aggregometry.
Platelet preparation Blood was drawn from healthy human volunteers with no symptoms of periodontitis who had abstained from taking any anti-platelet medication for 2 weeks. The blood was immediately mixed with 3.8% sodium citrate (9:1 v/v) and centrifuged at 100 g for 20 min at room temperature. PRP layer was transferred to another tube. The remaining blood was recentrifuged at 500 g for 20 min to obtain PPP. Washed platelets were prepared as previously described (Kariyazono et al., 2001). Platelet precipitates obtained from PRP were washed
twice with acid-citrated dextrose (Terumo), and resuspended in 5 mM HEPES-buffered Tyrode solution, pH 7.4 (Sigma). To prepare human IgG fraction and IgG-depleted PPP, PPP was applied to protein G affinity column chromatography. Each purified IgG and IgG-depleted PPP was used with platelets derived from the same donor.
Platelet aggregometry Platelet aggregation was measured using a 12-channel automated aggregometer (HEMA TRACER, MC Medical) set to 1000 r.p.m. at 37°C. Stirred suspensions (200 µl, 6 × 107 particles) of PRP or washed platelets were challenged with bacterial suspensions (20 µl, 6.8 × 107 cfu) except for the experiments specially indicated. The rate and extent of platelet aggregation were monitored by the percentage of light transmission and presented as aggregation traces. Aggregation traces presented in this study are from typical data of at least three experiments using platelets from different individuals. The maximum aggregation rate was presented with the mean and standard deviation of three independent trials.
Fluorescence microscopy Bacterial cells were harvested, washed twice in cold PBS, pH 7.5 and labelled by incubation with 1 mg ml−1 FITC in 1% DMSO-PBS at 25°C for 30 min. The labelled cells were washed twice with PBS and subjected to platelet aggregation and fluorescence microcopy (Axiovert 200 M, Zeiss).
Subcellular fractionation of P. gingivalis Culture of P. gingivalis 33277 at mid-log phase was subjected to the following fractionation as described previously (Murakami et al., 2002). Briefly, the cells were harvested by centrifugation (10 000 g, 20 min), washed twice with 10 mM HEPES buffer (pH 7.4), suspended with the same buffer and sonicated on ice for 1 min. Unbroken cells and large debris were removed by centrifugation (1000 g, 10 min), and the supernatants (whole cell lysates) were subjected to ultracentrifugation (100 000 g, 1 h), yielding precipitates containing bacterial membrane fractions and supernatants containing cytoplasmic and periplasmic fractions. The precipitates were suspended in 10 mM HEPES buffer (pH 7.4), supplemented with Triton X-100 at the final concentration of 1% and mixed gently at room temperature for 30 min. The solution was subjected to ultracentrifugation at 100 000 g for 1 h to yield the bacterial outer membrane fraction and the inner membrane fraction as precipitates and supernatants respectively. Each fraction corresponding to 1 mg of bacterial cells was subjected to platelet aggregometry.
Interbacterial complementation The Rgp/Kgp-null mutant (KDP136) was co-cultured with the adhesin-null mutant (KDP137) in supplemented Todd Hewitt broth anaerobically at 37°C for 1–2 days. After that, the cocultured cells were harvested as described above and subjected to platelet aggregometry and immunoblot analysis.
© 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 59, 152–167
164 M. Naito et al. Trypsin treatment The Rgp/Kgp-null mutant (KDP136) was cultured at 37°C for 1 day. The bacterial cells were harvested, washed with PBS and suspended in PBS to give an optical density of 1.5 at 620 nm. Trypsin was added to the suspension at the final concentration of 0.5 mg ml−1 and incubated at 37°C for 2 h, followed by addition of trypsin inhibitor at 2.5 mg ml−1. The trypsin-treated bacterial solution was subjected to platelet aggregometry and immunoblot analysis. The trypsin-treated bacterial solution was further separated to cell precipitates and supernatants by centrifugation. The cell precipitates were then washed with PBS and suspended in the original volume of PBS. The supernatants and the washed cell suspension were subjected to platelet aggregometry and immunoblot analysis.
Immunoblot analysis Before being dissolved in the Laemmli sample buffer, samples were treated with 10% trichloroacetic acid to inactivate endogenous proteinases. The resulting samples in the sample buffer were separated by SDS-PAGE, and the gels were stained with Coomassie brilliant blue (CBB) or transblotted onto Immobilon membranes (Millipore). The membrane was blocked with 5% skim milk in PBS containing 0.05% Tween 20 (PBST) for 1 h at 37°C, and reacted with anti-HbR or antiHgp44 rabbit antiserum in 5% skim milk in PBST for 1 h at 37°C. The membrane was washed with PBST and subjected to immunodetection using peroxidase-conjugated swine antirabbit Ig and ECL plus detection system (Amersham Biosciences AB).
Treatment of human platelets with mocarhagin Platelet-rich plasma was treated with a proteinase-disintegrin, mocarhagin (5 µg ml−1) at 37°C for 45 min and used for platelet aggregometry.
Construction of the rgpA rgpB kgp hagA quadruple mutant A DNA fragment corresponding to a region (540 bp) upstream of the hagA gene was polymerase chain reaction (PCR)-amplified from the chromosomal DNA of P. gingivalis 33277 with two primers (HagA-F; 5′-TACCTGTTATCTAC CGATATCCGGC-3′ and BamH-R; 5′-AAAAGGATCCTCCTT GGCCTTGGATGTTCGAATAC-3′). A DNA fragment corresponding to a region (699 bp) in the 5′-end region of the hagA gene was PCR-amplified with two primers (BamH-F; 5′-AGGAGGATCCTTTTGGTTTGCCAACGGAACGATCC-3′ and HagA-R; 5′-CGTTTCCGGTTGTGGACAAGAACAC-3′). These two DNA fragments were annealed and subjected to PCR amplification with the primers HagA-F and HagA-R. The resulting amplified fragment (1239 bp) was inserted to pGEM-T easy vector (Promega) by using a T-vector system, giving rise to pKD400. A 2 kb Sau3AI restriction fragment from pFK111 plasmid that contained the metronidazole resistance gene nimC of pIP419 plasmid of Bacteroides thetaiotaomicron (Trinh et al., 1995) was then inserted into the BamHI site of pKD400, giving rise to pKD401. KDP136 was
transformed to be metronidazole-resistant by electroporation with linearized pKD401 plasmid DNA to yield KDP153 (rgpA::erm rgpB::tetQ kgp::cat hagA::nimC). Correct gene replacement of KDP153 was verified by PCR and Southern blot analysis. Purification of recombinant Hgp44 (rHgp44) and HbR (rHbR) adhesin proteins rHbR was prepared as described previously (Nakayama et al., 1998). The DNA fragment coding for the Hgp44 domain protein was amplified from the rgpA gene of P. gingivalis 33277 with two primers (44-F; 5′-CCATATGAGCGGTCAGGCCGAG-3′ and 44-R; 5′-GCTC GAGTGCCGTAATCGTCTCTTC-3′). The resulting fragment was then inserted into the NdeI-XhoI site of pET22b plasmid (Novagen) to yield pKD402. E. coli BL21 (DE3) harbouring pKD402 was grown in L broth. Isopropyl β-D-thiogalactoside was added to the culture at 0.1 µM followed by incubation for 2 h to overproduce the rHgp44. rHgp44 was purified by the Ni-NTA purification system (Invitrogen).
Acknowledgements We thank Dr Michael Berndt, Baker Research Medical Institute, for giving us mocarhagin. Drs Kuniaki Okamoto and Mihoko Nonaka, Nagasaki University Graduate School of Biomedical Science, and Ms. Junko Kato, Department of Laboratory Medicine, Fukuoka University School of Medicine, are appreciated for technical assistance. We also thank Dr Yukio Ozaki, Department of Clinical and Laboratory Medicine, University of Yamanashi, Dr Hiroko Kariyazono, Kagoshima University Faculty of Medicine, and members of the Division of Microbiology and Oral Infection for helpful suggestion. This work was supported by Grants-in-Aid (15019078 and 16017282) for scientific research from the Ministry of Education, Science, Sports, Culture and Technology, Japan and a grant from Yakult.
References Abe, N., Kadowaki, T., Okamoto, K., Nakayama, K., Ohishi, M., and Yamamoto, K. (1998) Biochemical and functional properties of lysine-specific cysteine proteinase (Lysgingipain) as a virulence factor of Porphyromonas gingivalis in periodontal disease. J Biochem (Tokyo) 123: 305–312. Baba, A., Kadowaki, T., Asao, T., and Yamamoto, K. (2002) Roles for Arg- and Lys-gingipains in the disruption of cytokine responses and loss of viability of human endothelial cells by Porphyromonas gingivalis infection. Biol Chem 383: 1223–1230. Banbula, A., Mak, P., Bugno, M., Silberring, J., Dubin, A., Nelson, D., et al. (1999a) Prolyl tripeptidyl peptidase from Porphyromonas gingivalis. A novel enzyme with possible pathological implications for the development of periodontitis. J Biol Chem 274: 9246–9252. Banbula, A., Bugno, M., Kuster, A., Heinrich, P.C., Travis, J., and Potempa, J. (1999b) Rapid and efficient inactivation of IL-6 gingipains, lysine- and arginine-specific proteinases from Porphyromonas gingivalis. Biochem Biophys Res Commun 261: 598–602. Beck, J., Garcia, R., Heiss, G., Vokonas, P.S., and Offenbacher, S. (1996) Periodontal disease and cardiovascular disease. J Periodontol 67: 1123–1137.
© 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 59, 152–167
P. gingivalis-induced platelet aggregation 165 Byrne, M.F., Kerrigan, S.W., Corcoran, P.A., Atherton, J.C., Murray, F.E., Fitzgerald, D.J., and Cox, D.M. (2003) Helicobacter pylori binds von Willebrand factor and interacts with GPIb to induce platelet aggregation. Gastroenterology 124: 1846–1854. Calkins, C.C., Platt, K., Potempa, J., and Travis, J. (1998) Inactivation of tumor necrosis factor-alpha by proteinases (gingipains) from the periodontal pathogen, Porphyromonas gingivalis. Implications of immune evasion. J Biol Chem 273: 6611–6614. Chiu, B. (1999) Multiple infections in carotid atherosclerotic plaques. Am Heart J 138: S534–S536. Curtis, M.A., Macey, M., Slaney, J.M., and Howells, G.L. (1993) Platelet activation by protease I of Porphyromonas gingivalis W83. FEMS Microbiol Lett 110: 167–173. Gibbons, R.J., and MacDonald, J.B. (1960) Hemin and vitamin K compounds as required factors for the cultivation of certain strains of Bacteroides melaninogenicus. J Bacteriol 80: 164–170. Han, N., Whitlock, J., and Progulske-Fox, A. (1996) The hemagglutinin gene A (hagA) of Porphyromonas gingivalis 381 contains four large, contiguous, direct repeats. Infect Immun 64: 4000–4007. Herzberg, M.C., MacFarlane, G.D., Liu, P., and Erickson, P.R. (1994) The platelet as an inflammatory cell in periodontal diseases: interancton with Porphyromonas gingivalis. In Molecular Pathogenesis of Periodontal Disease. Genco, R. (eds). Washington, DC: American Society of Microbiology, pp. 247–255. Howell, T.H., Ridker, P.M., Ajani, U.A., Hennekens, C.H., and Christen, W.G. (2001) Periodontal disease and risk of subsequent cardiovascular disease in US male physicians. J Am Coll Cardiol 37: 445–450. Hujoel, P.P., Drangsholt, M., Spiekerman, C., and DeRouen, T.A. (2000) Periodontal disease and coronary heart disease risk. JAMA 284: 1406–1410. Iida, Y., Honda, K., Suzuki, T., Matsukawa, S., Kawai, T., Shimahara, T., and Chiba, H. (2004) Brain abscess in which Porphyromonas gingivalis was detected in cerebrospinal fluid. Br J Oral Maxillofac Surg 42: 180. Imamura, T., Pike, R.N., Potempa, J., and Travis, J. (1994) Pathogenesis of periodontitis: a major arginine-specific cysteine proteinase from Porphyromonas gingivalis induces vascular permeability enhancement through activation of the kallikrein/kinin pathway. J Clin Invest 94: 361–367. Imamura, T., Potempa, J., Tanase, S., and Travis, J. (1997) Activation of blood coagulation factor X by arginine-specific cysteine proteinases (gingipain-Rs) from Porphyromonas gingivalis. J Biol Chem 272: 16062–16067. Jagels, M.A., Travis, J., Potempa, J., Pike, R., and Hugli, T.E. (1996) Proteolytic inactivation of the leukocyte C5a receptor by proteinases derived from Porphyromonas gingivalis. Infect Immun 64: 1984–1991. Joshipura, K.J., Rimm, E.B., Douglass, C.W., Trichopoulos, D., Ascherio, A., and Willett, W.C. (1996) Poor oral health and coronary heart disease. J Dent Res 75: 1631–1636. Kadowaki, T., Yoneda, M., Okamoto, K., Maeda, K., and Yamamoto, K. (1994) Purification and characterization of a novel arginine-specific cysteine proteinase (argingipain) involved in the pathogenesis of periodontal disease from
the culture supernatant of Porphyromonas gingivalis. J Biol Chem 269: 21371–21378. Kadowaki, T., Nakayama, K., Yoshimura, F., Okamoto, K., Abe, N., and Yamamoto, K. (1998) Arg-gingipain acts as a major processing enzyme for various cell surface proteins in Porphyromonas gingivalis. J Biol Chem 273: 29072– 29076. Kamaguchi, A., Ohyama, T., Sakai, E., Nakamura, R., Watanabe, T., Baba, H., and Nakayama, K. (2003) Adhesins encoded by the gingipain genes of Porphyromonas gingivalis are responsible for co-aggregation with Prevotella intermedia. Microbiology 149: 1257–1264. Kariyazono, H., Nakamura, K., Shinkawa, T., Yamaguchi, T., Sakata, R., and Yamada, K. (2001) Inhibition of platelet aggregation and the release of P-selectin from platelets by cilostazol. Thromb Res 101: 445–453. Kelly, C.G., Booth, V., Kendal, H., Slaney, J.M., Curtis, M.A., and Lehner, T. (1997) The relationship between colonization and haemagglutination inhibiting and B cell epitopes of Porphyromonas gingivalis. Clin Exp Immunol 110: 285– 291. Kerrigan, S.W., Douglas, I., Wray, A., Heath, J., Byrne, M.F., Fitzgerald, D., and Cox, D. (2002) A role for glycoprotein Ib in Streptococcus sanguis-induced platelet aggregation. Blood 100: 509–516. Kozarov, E.V., Dorn, B.R., Shelburne, C.E., Dunn, W.A., Jr, and Progulske-Fox, A. (2005) Human atherosclerotic plaque contains viable invasive Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis. Arterioscler Thromb Vasc Biol 25: e17–18. Epub 2005 January 20. Lalla, E., Lamster, I.B., Hofmann, M.A., Bucciarelli, L., Jerud, A.P., Tucker, S., et al. (2003) Oral infection with a periodontal pathogen accelerates early atherosclerosis in apolipoprotein E-null mice. Arterioscler Thromb Vasc Biol 23: 1405–1411. Lev, M., Keudell, K.C., and Milford, A.F. (1971) Succinate as a growth factor for Bacteroides melaninogenicus. J Bacteriol 108: 175–178. Li, L., Messas, E., Batista, E.L., Jr, Levine, R.A., and Amar, S. (2002) Porphyromonas gingivalis infection accelerates the progression of atherosclerosis in a heterozygous apolipoprotein E-deficient murine model. Circulation 105: 861– 867. Lourbakos, A., Yuan, Y.P., Jenkins, A.L., Travis, J., AndradeGordon, P., Santulli, R., et al. (2001) Activation of protease-activated receptors by gingipains from Porphyromonas gingivalis leads to platelet aggregation: a new trait in microbial pathogenicity. Blood 97: 3790–3797. Mattila, K.J., Valtonen, V.V., Nieminen, M., and Huttunen, J.K. (1995) Dental infection and the risk of new coronary events: prospective study of patients with documented coronary artery disease. Clin Infect Dis 20: 588–592. Morrison, H.I., Ellison, L.F., and Taylor, G.W. (1999) Periodontal disease and risk of fatal coronary heart and cerebrovascular diseases. J Cardiovasc Risk 6: 7–11. Murakami, Y., Imai, M., Nakamura, H., and Yoshimura, F. (2002) Separation of the outer membrane and identification of major outer membrane proteins from Porphyromonas gingivalis. Eur J Oral Sci 110: 157–162. Nakayama, K., Yoshimura, F., Kadowaki, T., and Yamamoto, K. (1996) Involvement of arginine-specific cysteine protein-
© 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 59, 152–167
166 M. Naito et al. ase (Arg-gingipain) in fimbriation of Porphyromonas gingivalis. J Bacteriol 178: 2818–2824. Nakayama, K., Ratnayake, D.B., Tsukuba, T., Kadowaki, T., Yamamoto, K., and Fujimura, S. (1998) Haemoglobin receptor protein is intragenically encoded by the cysteine proteinase-encoding genes and the haemagglutininencoding gene of Porphyromonas gingivalis. Mol Microbiol 27: 51–61. Oleksy, A., Banbula, A., Bugno, M., Travis, J., and Potempa, J. (2002) Proteolysis of interleukin-6 receptor (IL-6R) by Porphyromonas gingivalis cysteine proteinases (gingipains) inhibits interleukin-6-mediated cell activation. Microb Pathog 32: 173–181. Rosenfeld, S.I., Looney, R.J., Leddy, J.P., Phipps, D.C., Abraham, G.N., and Anderson, C.L. (1985) Human platelet Fc receptor for immunoglobulin G. Identification as a 40 000-molecular-weight membrane protein shared by monocytes. J Clin Invest 76: 2317–2322. Sato, K., Sakai, E., Veith, P.D., Shoji, M., Kikuchi, Y., Yukitake, H., et al. (2005) Identification of a new membraneassociated protein which influences transport/maturation of gingipains and adhesins of Porphyromonas gingivalis. J Biol Chem 280: 8668–8677. Scragg, M.A., Cannon, S.J., Rangarajan, M., Williams, D.M., and Curtis, M.A. (1999) Targeted disruption of fibronectin– integrin interactions in human gingival fibroblasts by the RI protease of Porphyromonas gingivalis W50. Infect Immun 67: 1837–1843. Sharma, A., Novak, E.K., Sojar, H.T., Swank, R.T., Kuramitsu, H.K., and Genco, R.J. (2000) Porphyromonas gingivalis platelet aggregation activity: outer membrane vesicles are potent activators of murine platelets. Oral Microbiol Immunol 15: 393–396. Shen, Y., Romo, G.M., Dong, J.F., Schade, A., McIntire, L.V., Kenny, D., et al. (2000) Requirement of leucine-rich repeats of glycoprotein (GP) Ibα for shear-dependent and static binding of von Willebrand factor to the platelet membrane GP Ib-IX–V complex. Blood 95: 903–910. Shi, Y., Ratnayake, D.B., Okamoto, K., Abe, N., Yamamoto, K., and Nakayama, K. (1999) Genetic analyses of proteolysis, hemoglobin binding, and hemagglutination of Porphyromonas gingivalis. Construction of mutants with a combination of rgpA, rgpB, kgp, and hagA. J Biol Chem 274: 17955–17960. Shi, Y., Kong, W., and Nakayama, K. (2000) Human lactoferrin binds and removes the hemoglobin receptor protein of the periodontopathogen Porphyromonas gingivalis. J Biol Chem 275: 30002–30008. Shibata, Y., Hayakawa, M., Takiguchi, H., Shiroza, T., and Abiko, Y. (1999) Determination and characterization of the hemagglutinin-associated short motifs found in Porphyromonas gingivalis multiple gene products. J Biol Chem 274: 5012–5020. Shoji, M., Naito, M., Yukitake, H., Sato, K., Sakai, E., Ohara, N., and Nakayama, K. (2004) The major structural components of two cell surface filaments of Porphyromonas gingivalis are matured through lipoprotein precursors. Mol Microbiol 52: 1513–1525. Sjöbring, U., Ringdahl, U., and Ruggeri, Z.M. (2002) Induction of platelet thrombi by bacteria and antibodies. Blood 100: 4470–4477.
Studier, F.W., Rosenberg, A.H., Dunn, J.J., and Dubendorff, J.W. (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185: 60– 89. Sugawara, S., Nemoto, E., Tada, H., Miyake, K., Imamura, T., and Takada, H. (2000) Proteolysis of human monocyte CD14 by cysteine proteinases (gingipains) from Porphyromonas gingivalis leading to lipopolysaccharide hyporesponsiveness. J Immunol 165: 411–418. Sullam, P.M., Hyun, W.C., Szollosi, J., Dong, J., Foss, W.M., and Lopez, J.A. (1998) Physical proximity and functional interplay of the glycoprotein Ib-IX–V complex and the Fc receptor FcγRIIA on the platelet plasma membrane. J Biol Chem 273: 5331–5336. Taniguchi, A., Nishimura, F., Murayama, Y., Nagasaka, S., Fukushima, M., Sakai, M., et al. (2003) Porphyromonas gingivalis infection is associated with carotid atherosclerosis in non-obese Japanese type 2 diabetic patients. Metabolism 52: 142–145. Trinh, S., Haggoud, A., Reysset, G., and Sebald, M. (1995) Plasmids pIP419 and pIP421 from Bacteroides: 5nitroimidazole resistance genes and their upstream insertion sequence elements. Microbiology 141: 927–935. Ward, C.M., Andrews, R.K., Smith, A.I., and Berndt, M.C. (1996) Mocarhagin, a novel cobra venom metalloproteinase, cleaves the platelet von Willebrand factor receptor glycoprotein Ibα. Identification of the sulfated tyrosine/ anionic sequence Tyr-276-Glu-282 of glycoprotein Ibα as a binding site for von Willebrand factor and α-thrombin. Biochemistry 35: 4929–4938. Wingrove, J.A., DiScipio, R.G., Chen, Z., Potempa, J., Travis, J., and Hugli, T.E. (1992) Activation of complement components C3 and C5 by a cysteine proteinase (gingipain-1) from Porphyromonas (Bacteroides) gingivalis. J Biol Chem 267: 18902–18907. Wu, Y., Suzuki-Inoue, K., Satoh, K., Asazuma, N., Yatomi, Y., Berndt, M.C., and Ozaki, Y. (2001) Role of Fc receptor γ-chain in platelet glycoprotein Ib-mediated signaling. Blood 97: 3836–3845. Yoneda, M., Hirofuji, T., Anan, H., Matsumoto, A., Hamachi, T., Nakayama, K., and Maeda, K. (2001) Mixed infection of Porphyromonas gingivalis and Bacteroides forsythus in a murine abscess model: involvement of gingipains in a synergistic effect. J Periodontal Res 36: 237–243.
Supplementary material The following supplementary material is available for this article online: Table S1. Onset time of platelet aggregation. Fig. S1. No aggregation in the mixture of PGE1-pretreated PRP and P. gingivalis cells. PRP was pretreated with PGE1 (0.5 µg ml−1) or PBS at 37°C for 1 h. Pretreated PRP (200 µl, 6 × 107 particles) was examined for the ability to aggregate in response to P. gingivalis 33277 (20 µl, 6.8 × 107 cfu). Aggregation traces were presented. Fig. S2. Recovery of platelet aggregation of the Rgp/Kgpnull mutant by trypsin treatment. A. Aggregation traces were presented. B. Schematic drawing of effect of trypsin treatment.
© 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 59, 152–167
P. gingivalis-induced platelet aggregation 167 C. SDS-PAGE analysis. Bacterial cell lysates, precipitates and supernatants were subjected to SDS-PAGE. Gels were stained with CBB (lanes 1–4) and blotted onto Immobilon membranes. The membrane was immunoreated with antiHgp44 serum (lanes 5–8) and anti-HbR serum (lanes 9–12). Lanes: 1, 5 and 9, KDP136 (no treatment); 2, 6 and 10, trypsin-treated KDP136; 3, 7 and 11, supernatants from trypsin-treated KDP136; 4, 8 and 12, cell precipitates from trypsin-treated KDP136. The relative molecular masses are shown to the left. Fig. S3. Effect of rHgp44 on platelet aggregation of the Rgp/ Kgp-null mutant and the adhesin-null mutant in PRP. Maximum aggregation rates were presented. One portion (10 µl) of the cell suspension of the Rgp/Kgp-null mutant (KDP136) or the adhesin-null mutant (KDP137) with 1 mM leupeptin was mixed with rHbR (50 µg in 40 µl of PBS), rHgp44 (50 µg
in 40 µl of PBS), or PBS (40 µl) at 37°C for 50 min. After that, a whole volume (50 µl) of the mixture was mixed with PRP (180 µl) and subjected to platelet aggregometry. Fig. S4. No significant effect of fibronectin or fibrinogen on platelet aggregation of the mixture of trypsin-treated KDP136, IgG and washed platelets. Washed platelets were mixed with trypsin-treated KDP136, human IgG (1.3 mg ml−1, 0.26 mg ml−1, or 0.05 mg ml−1) and fibronectin (30 µg ml−1) or fibrinogen (0.5 mg ml−1), and subjected to platelet aggregometry. Trypsin inhibitor was added to the mixture. Maximum aggregation rates were presented. FN and Fb indicate fibronectin and fibrinogen respectively. This material is available as part of the online article from http://www.blackwell-synergy.com
© 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 59, 152–167
