Porphyromonas gingivalis fimbriae binds to neoglycoproteins: evidence for a lectin-like interaction

Porphyromonas gingivalis is a likely major pathogen in adult periodontitis. Fimbriae in particular have been suggested as playing an important role in facilitating the initial interaction between the bacteria and the host and triggers host responses. Murakami et al. [Biochem. Biophys. Res. Commun. 192 (1993) 826] have shown that fimbriae of P. gingivalis strongly induced TNF-alpha gene expression in macrophages and expression of TNF-alpha was inhibited by N-acetyl-D-galactosamine, but not inhibited by other sugars. Studies by Sojar et al. [FEBS Lett. 422 (1998) 205] suggested that the oligosaccharide moiety of lactoferrin is involved in the interaction of P. gingivalis fimbriae and human lactoferrin. In the present study, purified fimbriae from P. gingivalis and neoglycoproteins were used to assess lectin-like interaction of fimbriae. In dot blot and overlay assays, iodinated purified P. gingivalis fimbriae as well as biotinylated purified P. gingivalis fimbriae bound strongly to albumin-fucosylamide (albumin-1-amido-1-deoxy-L-fucose) and by lesser extent to albumin-N-acetyl-D-galactosamine (albumin-p-aminophenyl-N-acetyl-beta-D-galactosaminide). However, fimbriae failed to bind carbohydrate free bovine serum albumin, which was used in preparation of the neoglycoproteins. These results suggests that P. gingivalis fimbriae bind to glycoconjugates through lectin-like interaction with carbohydrate. This protein-carbohydrate interactions may be important for triggering events in these cells, which mediate the host response of this pathogen.     

Adherence of Porphyromonas gingivalis to matrix proteins via a fimbrial cryptic receptor exposed by its own arginine-specific protease

Porphyromonas gingivalis , a Gram-negative anaerobe, is known to be involved in the pathogenesis of periodontitis. P. gingivalis fimbriae, which are proteinaceous appendages extending from the cell surface, may contribute to the adherence of the organism to the host cell surface. We previously suggested that arginine-specific protease produced by P. gingivalis enhanced the adherence of purified fimbriae to fibroblasts or matrix proteins. In this study, we have revealed the mechanism of the enhanced binding of fimbriae by the protease in more detail. Arg-specific protease and fimbriae were obtained from P. gingivalis 381 cells and purified. We then analysed the interaction of fimbriae and immobilized fibronectins (intact or partially degraded fibronectin by the purified protease) by using the real-time biomolecular interaction analysis (BIAcore) system with an optical biosensor based on the principles of surface plasmon resonance. BIAcore profiles demonstrated an enhanced interaction between fimbriae and protease-degraded fibronectin. We also showed specific binding of fimbriae to the degraded fibronectin by means of BIAcore analysis. The binding of biotinylated fimbriae to immobilized fibronectin was examined by enzyme-linked biotin–avidin assay. The purified protease enhanced the fimbrial binding to the immobilized fibronectin. The enhancement was inhibited by the addition of l -Arg, or oligopeptides containing the Arg residue at the C-terminus in the fimbrial binding reaction, suggesting that the P. gingivalis fimbriae may potentially have an ability to bind tightly to the Arg residue at C-terminus. Taken together, these studies indicate that P. gingivalis arginine-specific protease can expose a cryptitope in the matrix protein molecules, i.e. the C-terminal Arg residue of the host matrix proteins, so that the organism can adhere to the surface layer in the oral cavity through fimbriae–Arg interaction (a novel host–parasite relationship).     

Involvement of integrins in fimbriae-mediated binding and invasion by Porphyromonas gingivalis

Interaction between the major fimbriae of Porphyromonas gingivalis and gingival epithelial cells is important for bacterial adhesion and invasion. In this study, we identified integrins as an epithelial cell cognate receptor for P. gingivalis fimbriae. Immunoprecipitation and direct binding assays revealed a physical association between recombinant fimbrillin and beta1 integrins. In vitro adhesion and invasion assays demonstrated inhibition of binding and invasion of P. gingivalis by beta1 integrin antibodies. In contrast, invasion of a fimbriae-deficient mutant of P. gingivalis was not affected by integrin antibodies. Infection of gingival epithelial cells with wild-type P. gingivalis induced tyrosine phosphorylation of the 68 kDa focal adhesion protein paxillin, whereas the fimbriae-deficient mutant failed to evoke similar changes. Interestingly, activation of paxillin was not accompanied by an increase in the phosphorylation of focal adhesion kinase (FAK). These results provide evidence that P. gingivalis fimbriae promote adhesion to gingival epithelial cells through interaction with beta1 integrins, and this association represents a key step in the induction of the invasive process and subsequent cell responses to P. gingivalis infection.     

Inactivation of the Porphyromonas gingivalis fimA gene blocks periodontal damage in gnotobiotic rats.

Fimbrial production by Porphyromonas gingivalis was inactivated by insertion-duplication mutagenesis, using the cloned gene for the P. gingivalis major fimbrial subunit protein, fimA. by several criteria, this insertion mutation rendered P. gingivalis unable to produce fimbrilin or an intact fimbrial structure. A nonfimbriated mutant, DPG3, hemagglutinated sheep erythrocytes normally and was unimpaired in the ability to coaggregate with Streptococcus gordonii G9B. The cell surface hydrophobicity of DPG3 was also unaffected by the loss of fimbriae. However, DPG3 was significantly less able to bind to saliva-coated hydroxyapatite than wild-type P. gingivalis 381. This suggested that P. gingivalis fimbriae are important for adherence of the organism to saliva-coated oral surfaces. Further, DPG3 was significantly less able to cause periodontal bone loss in a gnotobiotic rat model of periodontal disease. These observations are consistent with other data suggesting that P. gingivalis fimbriae play an important role in the pathogenesis of human periodontal disease.     

Identification of glyceraldehyde-3-phosphate dehydrogenase of epithelial cells as a second molecule that binds to Porphyromonas gingivalis fimbriae

Binding of Porphyromonas gingivalis to the host cells is an essential step in the pathogenesis of periodontal disease. P. gingivalis binds to and invades epithelial cells, and fimbriae are thought to be involved in this process. In our earlier studies, two major epithelial cell components of 40 and 50 kDa were identified as potential fimbrial receptors. Sequencing of a cyanogen bromide digestion fragment of the 50-kDa component resulted in an internal sequence identical to keratin I molecules, and hence this cytokeratin represents one of the epithelial cell receptors for P. gingivalis fimbriae. In this study, the 40-kDa component of KB cells was isolated and its amino-terminal sequence determined. The N-terminal amino sequence was found to be GKVKVGVNGF and showed perfect homology with human glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Furthermore, purified P. gingivalis fimbriae were found to bind to rabbit muscle GAPDH. Antibodies directed against internal peptide 49-68 and 69-90 of fimbrillin were shown to inhibit the binding of P. gingivalis and of fimbriae to epithelial cells. Antibodies against these peptides also inhibited the binding of fimbriae to GAPDH. Our results confirmed that the amino-terminal domain corresponding to amino residues 49-68 of the fimbrillin protein is the major GAPDH binding domain. These studies point to GAPDH as a major receptor for P. gingivalis major fimbriae and, as such, GAPDH likely plays a role in P. gingivalis adherence and colonization of the oral cavity, as well as triggering host cell processes involved in the pathogenesis of P. gingivalis infections.     

Inhibitory effects of Porphyromonas gingivalis fimbriae on interactions between extracellular matrix proteins and cellular integrins

Porphyromonas gingivalis is a predominant periodontal pathogen, whose fimbriae are considered to be a major virulence factor, especially for bacterial adherence and invasion of host cells. In the present study, we investigated the influence of fimbriae on the interactions between alphavbeta3- and alpha5beta1-integrins and their ligand extracellular matrix (ECM) proteins (vitronectin and fibronectin), using human alphavbeta3- and alpha5beta1-integrin-overexpressing CHO cell lines (CHOalphavbeta3 and CHOalpha5beta1, respectively). P. gingivalis was found to have significantly greater binding to CHOalphavbeta3 and CHOalpha5beta1 than to control cells, whereas a fimbria-deficient mutant showed negligible binding to any of the tested cell lines. CHOalphavbeta3 and CHOalpha5beta1 cells attached to the polystyrene culture dishes in the presence of their ligand ECM proteins, while fimbriae markedly inhibited those attachments in a dose-dependent manner, with the highest dose of fimbriae achieving complete inhibition. In addition, the binding of vitronectin and fibronectin to CHOalphavbeta3 and CHOalpha5beta1 was inhibited by P. gingivalis cells. These results suggest that P. gingivalis fimbriae compete with ECM proteins for alphavbeta3- and alpha5beta1-integrins, and inhibit integrin/ECM protein-related cellular functions.     

Evidence that Porphyromonas (Bacteroides) gingivalis fimbriae function in adhesion to Actinomyces viscosus.

Porphyromonas (Bacteroides) gingivalis adheres to gram-positive bacteria, such as Actinomyces viscosus, when colonizing the tooth surface. However, little is known of the adhesins responsible for this interaction. A series of experiments were performed to determine whether P. gingivalis fimbriae function in its coadhesion with A. viscosus. Fimbriae typical of P. gingivalis were isolated from strain 2561 (ATCC 33277) by the method of Yoshimura et al. (F. Yoshimura, K. Takahashi, Y. Nodasaka, and T. Suzuki, J. Bacteriol. 160:949-957, 1984) in fractions enriched with a 40-kDa subunit, the fimbrillin monomer, P. gingivalis-A. viscosus coaggregation was inhibited by purified rabbit antifimbrial immunoglobulin G (IgG) at dilutions eightfold higher than those of preimmune IgG, providing indirect evidence implicating P. gingivalis fimbriae in coadhesion. Three types of direct binding assays further supported this observation. (i) Mixtures of isolated P. gingivalis fimbriae and A. viscosus WVU627 cells were incubated for 1 h, washed vigorously with phosphate-buffered saline (pH 7.2), and subjected to electrophoresis. Transblots onto nitrocellulose were probed with antifimbrial antiserum. Fimbrillin labeled positively on these blots. No reaction occurred with the control protein, porcine serum albumin, when blots were exposed to anti-porcine serum albumin, (ii) A. viscosus cells incubated with P. gingivalis fimbriae were agglutinated only after the addition of antifimbrial antibodies. (iii) Binding curves generated from an enzyme immunoassay demonstrated concentration-dependent binding of P. gingivalis fimbriae to A. viscosus cells. From these lines of evidence, P. gingivalis fimbriae appear to be capable of binding to A. viscosus and mediating the coadhesion of these species.     

Binding of Porphyromonas gingivalis fimbriae to Treponema denticola dentilisin

Treponema denticola has been reported to coaggregate with Porphyromonas gingivalis and localize closely together in matured subgingival plaque. In this study of the interaction of T. denticola with P. gingivalis, the P. gingivalis fimbria-binding protein of T. denticola was identified by two-dimensional electrophoresis followed by a ligand overlay assay with P. gingivalis fimbriae, and was determined to be dentilisin, a chymotrypsin-like proteinase of T. denticola. The binding was further demonstrated with a ligand overlay assay using an isolated GST fusion dentilisin construct. Our results suggest that P. gingivalis fimbriae and T. denticola dentilisin are implicated in the coaggregation of these bacteria.     

Fimbria-mediated coaggregation between human oral anaerobes Treponema medium and Porphyromonas gingivalis.

Bacterial binding phenomena among different bacterial genera or species play an important role in bacterial colonization in a mixed microbiota such as in the human oral cavity. The coaggregation reaction between two gram-negative anaerobes, Treponema medium and Porphyromonas gingivalis, was characterized using fimbria-deficient mutants of P. gingivalis and specific antisera against purified fimbriae and bacterial whole cells. T. medium ATCC 700273 strongly coaggregated with fimbriate P. gingivalis strains ATCC 33277 and 381, but not with afimbriate strains including transposon-induced fimbria-deficient mutants and KDP98 as a fimA-disrupted mutant of P. gingivalis ATCC 33277. In the P. gingivalis-T. medium coaggregation assay, the presence of rabbit antiserum against the purified fimbriae or the whole cells of P. gingivalis ATCC 33277 produced different "aggregates" consisting predominantly of P. gingivalis cells with few spirochetes, but both preimmune serum and the antiserum against the afimbriate KDP98 cells did not inhibit the coaggregation reaction. Heated P. gingivalis cells lost their ability to bind both heated and unheated T. medium cells. This T. medium-P. gingivalis coaggregation reaction was inhibited by a cysteine proteinase inhibitor, leupeptin, and also by arginine and lysine, but not by EDTA or sugars including lactose. A binding assay on nitrocellulose membranes and immunoelectron microscopy demonstrated that a heat-stable 37 kDa surface protein on the T. medium cell attached to the P. gingivalis fimbriae.     

Specific interactions between Porphyromonas gingivalis fimbriae and human extracellular matrix proteins

The interactions of the extracellular matrix (ECM) proteins (laminin, elastin, fibronectin, type I collagen, thrombospondin and vitronectin) with the fimbriae of Porphyromonas gingivalis were analyzed based on surface plasmon resonance (SPR) spectroscopy using a biomolecular interaction analyzing system (BIAcore). The BIAcore profiles demonstrated that fimbriae specifically bound to all of the ECM proteins with significant association constants (Ka). Vitronectin showed the highest affinity to fimbriae (Ka = 3.79 x 10(6) M-1), while the affinity of laminin was lowest (Ka = 2.15 x 10(6) M-1). A synthetic peptide which is a potent inhibitor of fimbrial binding to salivary proteins was not significantly effective on the fimbrial interactions with the ECM proteins. Using polystyrene microtiter plates revealed that P. gingivalis fimbriae bound markedly to immobilized fibronectin and type I collagen, while the interaction of fimbriae with the other ECM proteins was not clearly demonstrated. These results suggest that interactions between fimbriae and the ECM proteins occur with specific affinities which are not mediated by mechanisms identical to those of salivary proteins. It was also shown that SPR spectroscopy is a useful method to analyze these specific interactions.     

Differential interactions of fimbriae and lipopolysaccharide from Porphyromonas gingivalis with the Toll-like receptor 2-centred pattern recognition apparatus

The lipopolysaccharide (LPS) and fimbriae of Porphyromonas gingivalis play important roles in periodontal inflammation and pathogenesis. We investigated fimbriae and LPS from several P. gingivalis strains in terms of relative dependence on Toll-like receptor (TLR) signalling partners or accessory pattern-recognition molecules mediating ligand transfer to TLRs, and determined induced assembly of receptor complexes in lipid rafts. Fimbriae could utilize TLR1 or TLR6 for cooperative TLR2-dependent activation of transfected cell lines, in contrast to LPS and a mutant version of fimbriae which displayed preference for TLR1. Whether used to activate human cell lines or mouse macrophages, fimbriae exhibited strong dependence on membrane-expressed CD14 (mCD14), which could not be substituted for by soluble CD14 (sCD14). In contrast, sCD14 efficiently substituted for mCD14 in LPS-induced cellular activation. LPS-binding protein was more important for LPS- than for fimbria-induced cell activation, whereas the converse was true for CD11b/CD18. Cell activation by LPS or fimbriae required lipid raft function and formation of heterotypic receptor complexes (TLR1-2/CD14/CD11b/CD18), although wild-type fimbriae additionally recruited TLR6. In summary, TLR2 activation by P. gingivalis LPS or fimbriae involves differential dependence on accessory signalling or ligand-binding receptors, which may differentially influence innate immune responses.     

Oral streptococcal glyceraldehyde-3-phosphate dehydrogenase mediates interaction with Porphyromonas gingivalis fimbriae

Interaction of Porphyromonas gingivalis with plaque-forming bacteria is necessary for its colonization in periodontal pockets. Participation of Streptococcus oralis glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and P. gingivalis fimbriae in this interaction has been reported. In this investigation, the contribution of various oral streptococcal GAPDHs to interaction with P. gingivalis fimbriae was examined. Streptococcal cell surface GAPDH activity was measured by incubation of a constant number of streptococci with glyceraldehyde-3-phosphate and analysis for the conversion of NAD+ to NADH based on the absorbance at 340 nm. Coaggregation activity was measured by a turbidimetric assay. Cell surface GAPDH activity was correlated with coaggregation activity (r = 0.854, P < 0.01) with Spearman's rank correlation coefficient. S. oralis ATCC 9811 and ATCC 10557, Streptococcus gordonii G9B, Streptococcus sanguinis ATCC 10556, and Streptococcus parasanguinis ATCC 15909 exhibited high cell surface GAPDH activity and coaggregation activity; consequently, their cell surface GAPDHs were extracted with mutanolysin and purified on a Cibacron Blue Sepharose column. Subsequently, their DNA sequences were elucidated. Purified GAPDHs bound P. gingivalis recombinant fimbrillin by Western blot assay, furthermore, their DNA sequences displayed a high degree of homology with one another. Moreover, S. oralis recombinant GAPDH inhibited coaggregation between P. gingivalis and the aforementioned five streptococcal strains in a dose-dependent manner. These results suggest that GAPDHs of various plaque-forming streptococci may be involved in their attachment to P. gingivalis fimbriae and that they may contribute to P. gingivalis colonization.     

Expression of saliva-binding epitopes of the Porphyromonas gingivalis FimA protein on the surface of Streptococcus gordonii

Porphyromonas gingivalis, a gram-negative oral anaerobic bacterium, has been implicated in the onset and development of periodontitis. The P. gingivalis fimbriae which mediate bacterial adherence to host oral sites and induce host inflammatory responses have been suggested as a potential antigen candidate. for vaccine development. This study was undertaken to generate Streptococcus gordonii vectors expressing the major subunit protein (FimA) of P. gingivalis fimbriae for testing as a potential live vaccine against periodontitis. We report here the expression of the C-terminal saliva-binding epitopes of P. gingivalis FimA on the surface of S. gordonii and demonstrate that domains containing free cysteine residues are poorly expressed on the surface of S. gordonii.     

Interleukin-6 negatively regulates Porphyromonas gingivalis fimbria-stimulated fibronectin expression in human gingival fibroblasts

Our previous study demonstrated that fibronectin (FN) is a negative regulator of Porphyromonas gingivalis fimbria-induced pathogenesis in the initiation and development of chronic periodontal diseases. We show herein the regulatory action of interleukin-6 (IL-6) on FN expression in fimbria-treated human gingival fibroblasts. Interestingly, the decrease in FN expression in the cells treated with fimbriae at a high dose (8 microg of protein ml(-1)) was negated by treatment with anti-IL-6 antibody. Also, the increase in FN expression in cells treated with fimbriae at a low dose (1 microg of protein ml(-1)) was inhibited by exogenous IL-6. These results suggest that P. gingivalis fimbria-stimulated FN expression in human gingival fibroblasts is negatively regulated by endogenous IL-6.     

Doxycycline inhibits TREM-1 induction by Porphyromonas gingivalis

The triggering receptor expressed on myeloid cells 1 (TREM-1) is a cell surface receptor of the immunoglobulin superfamily, with the capacity to amplify pro-inflammatory cytokine production. Porphyromonas gingivalis is a Gram-negative anaerobic species highly implicated in inflammatory periodontal disease, with potential involvement in systemic inflammation. Porphyromonas gingivalis positively regulates TREM-1 expression and production in monocytic cells. Subantimicrobial doses of doxycycline (SDD) are used as an adjunct treatment in periodontal therapy, because of their anti-inflammatory properties. The aim of this study was to investigate the effect of SDD on P.?gingivalis-induced TREM-1 expression and secretion by the myelomonocytic cell line MonoMac-6. After 24?h of challenge, P.?gingivalis enhanced TREM-1 gene expression by the cells, with a concomitant increase in soluble TREM-1 release. Nevertheless, SDD concentrations between 2 and 10?μg?mL(-1) abolished TREM-1 expression and release, already after 4?h of administration. Moreover, SDD reduced P.?gingivalis-induced interleukin-8 secretion, confirming its anti-inflammatory effects. In conclusion, SDD inhibits bacterially induced TREM-1, and this effect may partly account for its generalized anti-inflammatory properties. This could partly explain the clinical efficacy of SDD as an adjunctive treatment for periodontal disease, but may also indicate that SDD could serve as a suitable modulator of systemic inflammatory responses.     

Immunochemical characterisation and epitope mapping of a novel fimbrial protein (Pg-II fimbria) of Porphyromonas gingivalis

Abstract Mouse monoclonal antibody (mAb) Pgf-II specific for a 72-kDa major cell-surface protein (72K-CSP) derived from Porphyromonas gingivalis OMZ 409 was prepared. Immunoblotting analysis revealed that mAb Pgf-II reacted with 72K-CSP but not with 41-kDa fimbrial subunit protein (41K-fimbrilin) derived from P. gingivalis 381. Electron microscopic observation revealed that P. gingivalis OMZ 409 possessed peritrichous, thin fimbriae on their surface. Immunogold electron microscopy also demonstrated that mAb Pgf-II bound to the 72K-CSP examined with the gold particles arranged along the fibril array originating from the cell surface of the bacteria. These findings suggested that P. gingivalis 72K-CSP was identifiable as another fimbriae (termed Pg-II fimbriae) different from the fimbriae (termed Pg-I fimbriae) composed of a 41K-fimbrilin. Using multipin peptide synthesis technology, 102 sequential overlapping peptides covering the entire 514 amino-acid stretch of Pg-II fimbriae were synthesised. Seven immunodominant regions within Pg-II fimbrial protein molecule, which definitely reacted with the serum of patients with periodontal diseases, were detected.     

Glutamic protease distribution is limited to filamentous fungi

Porphyromonas gingivalis is an etiologic agent of periodontal disease in humans, which has been linked to an increased risk for atherosclerosis-related events. In this study, we examined the effect of P. gingivalis infection on human macrophages with respect to foam cell formation, the hallmark of early atherogenesis, and the potential of P. gingivalis to induce its uptake by these cells. Human monocyte-derived macrophages were incubated with low density lipoprotein and infected with P. gingivalis FDC381 or its fimbriae deficient mutant, DPG3. Consistent with a role for fimbriae in this process, strain 381 significantly increased foam cell formation as compared to DPG3. Recovery of viable P. gingivalis in antibiotic protection experiments was significantly higher for strain 381 than for DPG3. By transmission electron microscopy, the wild-type strain was shown to adhere to and enter THP-1 cells. These results suggest that properties of P. gingivalis which render it capable of adhering to/invading other cell types may also be operative in macrophages and play an important role in its atherogenic potential.     

Synergistic biofilm formation by Treponema denticola and Porphyromonas gingivalis

Biofilm formation is an important step in the etiology of periodontal diseases. In this study, in vitro biofilm formation by Treponema denticola and Porphyromonas gingivalis 381 displayed synergistic effects. Confocal microscopy demonstrated that P. gingivalis attaches to the substratum first as a primary colonizer followed by coaggregation with T. denticola to form a mixed biofilm. The T. denticola flagella mutant as well as the cytoplasmic filament mutant were shown to be essential for biofilm formation as well as coaggregation with P. gingivalis. The major fimbriae and Arg-gingipain B of P. gingivalis also play important roles in biofilm formation with T. denticola.