Identification of a Streptococcus gordonii SspB domain that mediates adhesion to Porphyromonas gingivalis.

Porphyromonas gingivalis, a primary pathogen in adult periodontitis, may establish itself in the oral cavity by adhering to early plaque bacteria such as Streptococcus gordonii. Our previous studies (R. J. Lamont et al., Microbiology 140:867-872, 1994) suggested that this interaction is mediated by the SspB polypeptide, a member of the antigen I/II family of streptococcal surface proteins. S. gordonii was recently shown to express a second Ssp polypeptide (SspA) that resembles SspB and the structurally homologous antigen I/II polypeptide (Pac) of Streptococcus mutans. To determine if all of these related antigen I/II proteins interacted with P. gingivalis, SspA, SspB, and Pac were tested for adhesion to P. gingivalis cells. Both of the S. gordonii Ssp proteins bound labeled target cells, whereas the S. mutans Pac polypeptide did not, suggesting that antigen I/II-mediated binding of P. gingivalis by streptococci may be species specific. To investigate the molecular basis for this functional difference, the P. gingivalis binding domain of SspB was mapped. The binding properties of a family of truncated SspB polypeptides lacking C-terminal sequences were determined. In addition, the lack of binding activity exhibited by the Pac protein was exploited to construct and analyze chimeric SspB-Pac polypeptides. Both approaches revealed that the region defined by residues 1167 to 1250 of SspB was essential for P. gingivalis binding. This region of SspA and SspB is entirely conserved, consistent with the binding properties determined for these proteins. However, the corresponding region of Pac differs in both the primary sequence and predicted secondary structure, suggesting that the overall structure of this domain may define its functional activity.     

Identification of a Porphyromonas gingivalis receptor for the Streptococcus gordonii SspB protein

Colonization of the plaque biofilm by the oral pathogen Porphyromonas gingivalis is favored by the presence of antecedent organisms such as Streptococcus gordonii. Coadhesion between P. gingivalis and S. gordonii can be mediated by the SspB protein of S. gordonii; however, the P. gingivalis cognate receptor for this protein has not been identified. In this study, we identified a surface protein of P. gingivalis that interacts with the SspB protein. Coprecipitation between P. gingivalis outer membrane proteins and purified SspB protein demonstrated that a 100-kDa P. gingivalis protein bound to SspB. The 100-kDa protein also bound to an engineered strain of Enterococcus faecalis that expresses the SspB protein on the cell surface. Monospecific polyclonal antibodies to the 100-kDa protein inhibited the binding between P. gingivalis and S. gordonii in a dose-dependent manner up to 86%. Amino acid sequencing of the 100-kDa protein showed homology to a protein previously identified as the P. gingivalis minor fimbria. The minor fimbrial protein may exist as a complex with a hemagglutinin-like protein since the genes encoding these proteins are adjacent on the chromosome and are cotranscribed. Thus, the P. gingivalis receptor for S. gordonii SspB is a 100-kDa protein that structurally may be a minor fimbria-protein complex and functionally effectuates coadhesion.     

Inhibition of Porphyromonas gingivalis adhesion to Streptococcus gordonii by human submandibular-sublingual saliva.

Porphyromonas gingivalis W50 adheres in vitro to biofilms of Streptococcus gordonii G9B. This phenomenon is believed to facilitate the initial colonization of the oral cavity by P. gingivalis and to contribute to the maturation of dental plaque. In this report, we describe the modulating effects of human submandibular-sublingual saliva (HSMSL) on this in vitro model of intergeneric bacterial adhesion (coaggregation). HSMSL inhibited P. gingivalis adhesion to S. gordonii by 50% at a concentration of 57 micrograms of protein per ml. Maximum inhibitory activity was associated with a 43-kDa protein obtained by sequential Sephadex G200 gel filtration and CM52 ion-exchange chromatography of HSMSL. Pools of other column fractions of HSMSL showed no effect or were slightly stimulatory for bacterial adhesion. The binding of radioiodinated column fractions containing the 43-kDa protein by P. gingivalis was accompanied by their rapid enzymatic degradation. Treating P. gingivalis at 60 degrees C for 30 min or with protease inhibitors (phenylmethylsulfonyl fluoride and sodium iodoacetate) reduced adherence to streptococcal biofilms. These treatments did not prevent P. gingivalis from binding soluble HSMSL saliva components, although subsequent proteolysis was nearly eliminated. These observations indicate that surface-associated proteases of P. gingivalis, either independently or in concert with adjacent surface adhesins, interact with surfaces of oral streptococci to facilitate interbacterial adhesion. The adhesion-blocking properties of HSMSL, particularly the 43-kDa protein, may represent an important host defense mechanism in the oral cavity.     

Structural dissection and in vivo effectiveness of a peptide inhibitor of Porphyromonas gingivalis adherence to Streptococcus gordonii

The interaction of the minor fimbrial antigen (Mfa) with streptococcal antigen I/II (e.g., SspB) facilitates colonization of the dental biofilm by Porphyromonas gingivalis. We previously showed that a 27-mer peptide derived from SspB (designated BAR) resembles the nuclear receptor (NR) box protein-protein interacting domain and potently inhibits this interaction in vitro. Here, we show that the EXXP motif upstream of the NR core α-helix contributes to the Mfa-SspB interaction and that BAR reduces P. gingivalis colonization and alveolar bone loss in vivo in a murine model of periodontitis. Substitution of Gln for Pro(1171) or Glu(1168) increased the α-helicity of BAR and reduced its inhibitory activity in vitro by 10-fold and 2-fold, respectively. To determine if BAR prevents P. gingivalis infection in vivo, mice were first infected with Streptococcus gordonii and then challenged with P. gingivalis in the absence and presence of BAR. Animals that were infected with either 10(9) CFU of S. gordonii DL-1 or 10(7) CFU of P. gingivalis 33277 did not show a statistically significant increase in alveolar bone resorption over sham-infected controls. However, infection with 10(9) CFU of S. gordonii followed by 10(7) CFU of P. gingivalis induced significantly greater bone loss (P < 0.01) than sham infection or infection of mice with either organism alone. S. gordonii-infected mice that were subsequently challenged with 10(7) CFU of P. gingivalis in the presence of BAR exhibited levels of bone resorption similar to those of sham-infected animals. Together, these results indicate that both EXXP and the NR box are important for the Mfa-SspB interaction and that BAR peptide represents a potential therapeutic that may limit colonization of the oral cavity by P. gingivalis.     

Short fimbriae of Porphyromonas gingivalis and their role in coadhesion with Streptococcus gordonii

Porphyromonas gingivalis, one of the causative agents of adult periodontitis, attaches and forms biofilms on substrata of Streptococcus gordonii. Coadhesion and biofilm development between these organisms requires the interaction of the short fimbriae of P. gingivalis with the SspB streptococcal surface polypeptide. In this study we investigated the structure and binding activities of the short fimbriae of P. gingivalis. Electron microscopy showed that isolated short fimbriae have an average length of 103 nm and exhibit a helical structure with a pitch of ca. 27 nm. Mfa1, the major protein subunit of the short fimbriae, bound to SspB protein, and this reaction was inhibited by purified recombinant Mfa1 and monospecifc anti-Mfa1 serum in a dose-dependent manner. Complementation of a polar Mfa1 mutant with the mfa1 gene restored the coadhesion phenotype of P. gingivalis. Hence, the Mfa1 structural fimbrial subunit does not require accessory proteins for binding to SspB. Furthermore, the interaction of Mfa1 with SspB is necessary for optimal coadhesion between P. gingivalis and S. gordonii.     

Secretion of Functional Salivary Peptide by Streptococcus gordonii Which Inhibits Fimbria-Mediated Adhesion of Porphyromonas gingivalis

Porphyromonas gingivalis, a putative periodontopathogen, can bind to human salivary components with its fimbriae. We have previously shown that fimbriae specifically bind to a peptide domain shared by a major salivary component, i.e., proline-rich (glyco)proteins (PRPs). The synthetic domain peptide PRP-C (pPRP-C) significantly inhibits the fimbrial binding to PRPs. In this study, a recombinant strain of Streptococcus gordonii secreting pPRP-C was generated as a model of a possible approach to prevent the oral colonization by the pathogen. A duplicate DNA fragment (prpC) encoding pPRP-C was obtained by self-complementary annealing of synthetic oligonucleotides. prpC was connected downstream to a promoter and a gene encoding a signal peptide of Streptococcus downei glucosyltransferase I in frame. The linked fragments were inserted into the plasmid pMNK-4 derived from pVA838. The constructed plasmid was inserted to produce the transformant S. gordonii G9B, which then successfully secreted recombinant pPRP-C (r-pPRP-C) of the expected size. The concentrated bacterial culture supernatant containing r-pPRP-C inhibited the binding of P. gingivalis cells and fimbriae to PRP1 in a dose-dependent manner up to 72 and 77%, respectively. The r-pPRP-C concentrate also inhibited the coaggregation of P. gingivalis with various streptococcal strains as effectively as synthetic pPRP-C in a dose-dependent manner. Collectively, pPRP-C was found to be able to prevent P. gingivalis adherence to salivary receptor protein and plaque-forming bacteria. These results suggest that this recombination approach with a nonperiodontopathic bacterium may be suitable for the therapeutic prevention of P. gingivalis adherence to the oral cavity.     

Identification of murine protective epitopes on the Porphyromonas gingivalis fimbrillin molecule.

Fimbriae from Porphyromonas gingivalis are believed to play an important role in the pathogenesis of periodontal diseases. The aim of the present study was to identify the fimbrial protective T-cell epitopes in CBA/J mice. A truncated protein corresponding to amino acids 1 to 198, PgF1-198, was generated and allowed us to demonstrate that the N terminus of the protein contains T-cell epitopes. With synthetic peptides, an immunodominant sequence was identified between amino acids 103 and 122. The corresponding peptide, PgF-P8, induced T-cell proliferation after in vitro restimulation of in vivo-primed cells, giving a stimulation index comparable to the one obtained with r-fimbrillin, and induced production of both Th1 and Th2 cytokines. Growth supernatant contained significant levels of interleukin 2 (IL-2), gamma interferon, IL-4 (28 pg/ml), and tumor necrosis factor alpha. Immunization of mice with r-fimbrillin, PgF1-198, and PgF-P8 induced production of antibodies specific to r-fimbrillin and PgF-P8. In addition, by using the mouse chamber model we found that mice immunized with PgF-P8 were dramatically protected against a normally lethal injection of P. gingivalis. Animals immunized with PgF-P8 40 days prior to challenge showed a 60% survival rate when challenged with P. gingivalis, compared with just 25% survival in control animals and just 5% survival in mice immunized with PgF-P8 only 21 days prior to challenge. Although the protection depended on the time of immunization before the bacterial challenge, it did not correlate with in vivo local cytokine production (IL-2, IL-4, IL-6, tumor necrosis factor alpha, and gamma interferon), specific antibody levels, or the isotype of anti-PgF-P8 antibodies produced.     

Oral immunization with recombinant Streptococcus gordonii expressing porphyromonas gingivalis FimA domains

Porphyromonas gingivalis, a gram-negative anaerobe, is implicated in the etiology of adult periodontitis. P. gingivalis fimbriae are one of several critical surface virulence factors involved in both bacterial adherence and inflammation. P. gingivalis fimbrillin (FimA), the major subunit protein of fimbriae, is considered an important antigen for vaccine development against P. gingivalis-associated periodontitis. We have previously shown that biologically active domains of P. gingivalis fimbrillin can be expressed on the surface of the human commensal bacterium Streptococcus gordonii. In this study, we examined the effects of oral coimmunization of germfree rats with two S. gordonii recombinants expressing N (residues 55 to 145)- and C (residues 226 to 337)-terminal epitopes of P. gingivalis FimA to elicit FimA-specific immune responses. The effectiveness of immunization in protecting against alveolar bone loss following P. gingivalis infection was also evaluated. The results of this study show that the oral delivery of P. gingivalis FimA epitopes via S. gordonii vectors resulted in the induction of FimA-specific serum (immunoglobulin G [IgG] and IgA) and salivary (IgA) antibody responses and that the immune responses were protective against subsequent P. gingivalis-induced alveolar bone loss. These results support the potential usefulness of the S. gordonii vectors expressing P. gingivalis fimbrillin as a mucosal vaccine against adult periodontitis.     

Coinvasion of dentinal tubules by Porphyromonas gingivalis and Streptococcus gordonii depends upon binding specificity of streptococcal antigen I/II adhesin

Cell wall-anchored polypeptides of the antigen I/II family are produced by many species of oral streptococci. These proteins mediate adhesion of streptococci to salivary glycoproteins and to other oral microorganisms and promote binding of cells to collagen type I and invasion of dentinal tubules. Since infections of the root canal system have a mixed anaerobic bacterial etiology, we investigated the hypothesis that coadhesion of anaerobic bacteria with streptococci may facilitate invasive endodontic disease. Porphyromonas gingivalis ATCC 33277 cells were able to invade dentinal tubules when cocultured with Streptococcus gordonii DL1 (Challis) but not when cocultured with Streptococcus mutans NG8. An isogenic noninvasive mutant of S. gordonii, with production of SspA and SspB (antigen I/II family) polypeptides abrogated, was deficient in binding to collagen and had a 40% reduced ability to support adhesion of P. gingivalis. Heterologous expression of the S. mutans SpaP (antigen I/II) protein in this mutant restored collagen binding and tubule invasion but not adhesion to P. gingivalis or the ability to promote P. gingivalis coinvasion of dentin. An isogenic afimbrial mutant of P. gingivalis had 50% reduced binding to S. gordonii cells but was unaffected in the ability to coinvade dentinal tubules with S. gordonii wild-type cells. Expression of the S. gordonii SspA or SspB polypeptide on the surface of Lactococcus lactis cells endowed these bacteria with the abilities to bind P. gingivalis, penetrate dentinal tubules, and promote P. gingivalis coinvasion of dentin. The results demonstrate that collagen-binding and P. gingivalis-binding properties of antigen I/II polypeptides are discrete functions. Specificity of antigen I/II polypeptide recognition accounts for the ability of P. gingivalis to coinvade dentinal tubules with S. gordonii but not with S. mutans. This provides evidence that the specificity of interbacterial coadhesion may influence directly the etiology of pulpal and periapical diseases.     

Identification and Characterization of Porphyromonas gingivalis Client Proteins That Bind to Streptococcus oralis Glyceraldehyde-3-Phosphate Dehydrogenase

Coaggregation of Porphyromonas gingivalis and oral streptococci is thought to play an important role in P. gingivalis colonization. Previously, we reported that P. gingivalis major fimbriae interacted with Streptococcus oralis glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and that amino acid residues 166 to 183 of GAPDH exhibited strong binding activity toward P. gingivalis fimbriae (H. Nagata, M. Iwasaki, K. Maeda, M. Kuboniwa, E. Hashino, M. Toe, N. Minamino, H. Kuwahara, and S. Shizukuishi, Infect. Immun. 77:5130–5138, 2009). The present study aimed to identify and characterize P. gingivalis components other than fimbriae that interact with S. oralis GAPDH. A pulldown assay was performed to detect potential interactions between P. gingivalis client proteins and S. oralis recombinant GAPDH with amino acid residues 166 to 183 deleted by site-directed mutagenesis. Seven proteins, namely, tonB-dependent receptor protein (RagA4), arginine-specific proteinase B, 4-hydroxybutyryl-coenzyme A dehydratase (AbfD), lysine-specific proteinase, GAPDH, NAD-dependent glutamate dehydrogenase (GDH), and malate dehydrogenase (MDH), were identified by two-dimensional gel electrophoresis followed by proteomic analysis using tandem mass spectrometry. Interactions between these client proteins and S. oralis GAPDH were analyzed with a biomolecular interaction analysis system. S. oralis GAPDH showed high affinity for five of the seven client proteins (RagA4, AbfD, GAPDH, GDH, and MDH). Interactions between P. gingivalis and S. oralis were measured by a turbidimetric method and fluorescence microscopy. RagA4, AbfD, and GDH enhanced coaggregation, whereas GAPDH and MDH inhibited coaggregation. Furthermore, the expression of luxS in P. gingivalis was upregulated by RagA4, AbfD, and GDH but was downregulated by MDH. These results indicate that the five P. gingivalis client proteins function as regulators in P. gingivalis biofilm formation with oral streptococci.     

Role of gallium and silver from phosphate-based glasses on in vitro dual species oral biofilm models of Porphyromonas gingivalis and Streptococcus gordonii

Phosphate-based glasses (PBGs) are excellent controlled delivery agents for antibacterial ions such as silver and gallium. The aim of this study was to assess the potential utility of novel PBGs combining both gallium and silver for use in periodontal therapy. To this end, an in vitro biofilm model with the putative periodontal pathogen, Porphyromonas gingivalis, and an initial colonizer, Streptococcus gordonii, was established. The effect of increasing calcium content in gallium-silver-doped PBG on the susceptibility of P. gingivalis was examined. A decrease in degradation rates (30.34, 25.19, 21.40 μg mm(-2) h(-1)) with increasing PBG calciumcontent (10, 11, 12 mol.% respectively) was observed, correlating well with gallium and silver ion release and antimicrobial activity against planktonic P. gingivalis (approximately 5.4log(10) colony-forming units (CFU) reduction after 24h by the C10 glass compared with controls) and S. gordonii (total growth inhibition after 32h by C10, C11 and C12 glasses compared with controls). The most potent PBG (C10) was evaluated for its ability to inhibit the biofilm growth of P. gingivalis in a newly established constant-depth film fermentor model. The simultaneous release of silver and gallium from the glass reduced P. gingivalis biofilm growth with a maximum effect (1.92log(10) CFU reduction) after 168 h. Given the emergence of antibiotic-resistant bacteria and dearth of new antibiotics in development, the glasses, especially C10, would offer effective alternatives to antibiotics or may complement current therapies through controlled, localized delivery of gallium and silver ions at infected sites in the oral cavity.     

Adherence of Porphyromonas (Bacteroides) gingivalis to Streptococcus sanguis in vitro.

Intergeneric bacterial adherence is responsible for the complexity of the microbiota in human dental plaque and is believed to enable some extraneous bacteria to initially colonize the human oral cavity. Some current evidence indicates that Streptococcus sanguis, an early colonizer of teeth, enhances subsequent colonization by Porphyromonas (Bacteroides) gingivalis, a bacterium associated with advanced adult periodontitis. In this study, selected strains of P. gingivalis and S. sanguis were tested for their adherence activities in vitro. A differential filtration assay was devised in which one member of the test pair was radiolabeled. Heterogeneous aggregates that formed in mixed suspensions were collected on polycarbonate filters (8-microns pore size) and were washed free of individual bacteria and small homologous clumps. P. gingivalis 381, W50, JKG7, and 33277 adhered to S. sanguis G9B, M5, Challis 6, and 38. P. gingivalis A7A1-28 did not adhere well to S. sanguis under these conditions. More precise measurements of intergeneric adherence were obtained with an alternative assay with radiolabeled P. gingivalis and an artificial dental plaque composed of S. sanguis coupled to cyanogen bromide-activated agarose beads. CNBr-agarose was selected as the supporting matrix for the plaque because it was uniformly and permanently coated with S. sanguis and because P. gingivalis had negligible adherence activity for streptococcus-free beads. P. gingivalis W50 grown to the early stationary phase adhered to S. sanguis-coated beads in higher numbers than either midlogarithmic- or late-stationary-phase cells. Intergeneric adherence was not inhibited or reversed by the presence of lactose or other monosaccharides or disaccharides. Pretreatment of either bacterium with trypsin or proteinase K reduced subsequent adherence by 86 to 100%. Neuraminidase treatment of P. gingivalis caused 98% reduction of adherence, whereas similar treatment of S. sanguis caused only a 2% loss. Preincubation of P. gingivalis at 60 degrees C for 30 min decreased subsequent adherence to S. sanguis-coated beads by 94%. Adherence was reduced by 96% when bacteria were assayed while suspended in human whole saliva or when pretreated with saliva and subsequently assayed in buffer. The concentration of whole human saliva required to inhibit 50% adherence in this assay was 23 micrograms per ml (1:200 dilution). Suspension of the bacteria in normal rabbit serum resulted in 94% inhibition of adherence. These data indicate that saliva and serum may be important host defense factors for controlling Porphyromonas-Streptococcus adherence.     

Identification of Porphyromonas gingivalis lipopolysaccharide-binding proteins in human saliva

Porphyromonas gingivalis causes periodontal diseases and its lipopolysaccharide (LPS) is considered as a major virulence factor responsible for pathogenesis. Since initial recognition of P. gingivalis LPS (Pg.LPS) in the oral cavity might be crucial for the host response, we identified Pg.LPS-binding proteins (Pg.LPS-BPs) using Pg.LPS-immobilized beads and a high-resolution mass spectrometry. LPS purified from P. gingivalis was conjugated onto N-hydroxysuccinimidyl-Sepharose(?) 4 Fast Flow beads. Notably, Pg.LPS-conjugated beads could stimulate Toll-like receptor 2 (TLR2) as determined by a TLR2-depdendent reporter expression system using CHO/CD14/TLR2. In addition, the Pg.LPS-conjugated beads induced the production of inflammatory mediators such as nitric oxide and interferon-gamma-inducible protein-10 in the macrophage cell-line, RAW 264.7. These results imply that Pg.LPS retained its immunological properties during the conjugation process. Then, the Pg.LPS-conjugated beads were mixed with a pool of saliva obtained from nine human subjects to capture Pg.LPS-BPs and molecular identities were determined by LTQ-Orbitrap hybrid fourier transform mass spectrometry. Pg.LPS-BPs captured at high frequencies included alpha-amylase, cystatin, prolactin-inducible protein, lysozyme C, immunoglobulin components, serum albumin, lipocalin-1, and submaxillary gland androgen-regulated protein 3B. These proteins are known to be involved in bacterial adhesion and colonization, anti-microbial functions or modulation of immune responses.     

Identification of Porphyromonas gingivalis lipopolysaccharide-binding proteins in human saliva

Porphyromonas gingivalis causes periodontal diseases and its lipopolysaccharide (LPS) is considered as a major virulence factor responsible for pathogenesis. Since initial recognition of P. gingivalis LPS (Pg.LPS) in the oral cavity might be crucial for the host response, we identified Pg.LPS-binding proteins (Pg.LPS-BPs) using Pg.LPS-immobilized beads and a high-resolution mass spectrometry. LPS purified from P. gingivalis was conjugated onto N-hydroxysuccinimidyl-Sepharose^® 4 Fast Flow beads. Notably, Pg.LPS-conjugated beads could stimulate Toll-like receptor 2 (TLR2) as determined by a TLR2-depdendent reporter expression system using CHO/CD14/TLR2. In addition, the Pg.LPS-conjugated beads induced the production of inflammatory mediators such as nitric oxide and interferon-gamma-inducible protein-10 in the macrophage cell-line, RAW 264.7. These results imply that Pg.LPS retained its immunological properties during the conjugation process. Then, the Pg.LPS-conjugated beads were mixed with a pool of saliva obtained from nine human subjects to capture Pg.LPS-BPs and molecular identities were determined by LTQ-Orbitrap hybrid fourier transform mass spectrometry. Pg.LPS-BPs captured at high frequencies included alpha-amylase, cystatin, prolactin-inducible protein, lysozyme C, immunoglobulin components, serum albumin, lipocalin-1, and submaxillary gland androgen-regulated protein 3B. These proteins are known to be involved in bacterial adhesion and colonization, anti-microbial functions or modulation of immune responses.     

Identification and Testing of Porphyromonas gingivalis Virulence Genes with a pPGIVET System

An in vivo expression technology (IVET) system was designed to identify previously unknown virulence genes of Porphyromonas gingivalis. Fourteen ivi (for in vivo induced) genes that are induced during infection in a mouse abscess model were identified in our study. Of these, seven had homology to genes in the NCBI database, and the rest had no homology to reported DNA sequences. In order to determine virulence-related properties of these genes, three mutant strains, deleted of ivi8 (no homology to genes in the database), ivi10 (homologous to a putative TonB-dependent outer membrane receptor protein), and ivi11 (an immunoreactive 33-kDa antigen PG125 in P. gingivalis), were created. The mutants were tested in a mouse abscess model for alterations in virulence relative to the wild type by a competition assay in BALB/c mice. After 5 days we observed the enrichment of the wild-type strain over mutant strains Deltaivi10 and Deltaivi11, which indicated that mutant strains Deltaivi10 and Deltaivi11 are less able to survive in this model than the wild-type strain, while Deltaivi8 survives as well as the wild-type strain. We propose that knockout of these ivi genes reduced the ability of the mutated P. gingivalis to survive and cause infection compared to the wild-type strain at the site of injection. Also, in separate experiments, groups of mice were challenged with subcutaneous injections of each individual mutant strain (Deltaivi8, Deltaivi10, and Deltaivi11) or with the wild-type strain alone and were then examined to assess their general health status. The results showed that knockout of these ivi genes conferred a reduction in virulence. The ability of the mutants to invade KB cells compared to the wild type was also determined. Interestingly, the CFU counts of the mutant strain Deltaivi10 recovered from KB cells were eight times lower than those of the wild type, indicating that this mutant has a lower capacity for invasion. These results demonstrate that IVET is a powerful tool in discovering virulence genes and the significant role that ivi genes play in the pathogenesis of this species.     

Characterization of binding of Streptococcus oralis glyceraldehyde-3-phosphate dehydrogenase to Porphyromonas gingivalis major fimbriae

Binding of Streptococcus oralis glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to Porphyromonas gingivalis fimbriae was characterized via a biomolecular interaction analysis system. The interaction was specific, and the association constant value was 4.34 x 10(7) M(-1), suggesting that S. oralis GAPDH functions as a dominant receptor for P. gingivalis and contributes to P. gingivalis colonization.     

Negative Correlation of Distributions of Streptococcus cristatus and Porphyromonas gingivalis in Subgingival Plaque

Porphyromonas gingivalis is one of the major causative agents of adult periodontitis. One of the features of this periodontal pathogen is its ability to attach to a variety of oral bacterial surfaces and to colonize subgingival dental plaque. We have shown that Streptococcus cristatus CC5A inhibits expression of fimA, a gene encoding the major protein subunit of long fimbriae in P. gingivalis; as a result, S. cristatus interrupts formation of P. gingivalis biofilms. Here we further demonstrate that the inhibitory activity of S. cristatus affects multiple strains of P. gingivalis and that optimal inhibitory activity correlates with levels of arginine deiminase expression in S. cristatus. More strikingly, the impact of S. cristatus on P. gingivalis colonization was revealed by comparing levels of P. gingivalis and S. cristatus in subgingival dental plaque. Spearman correlation analysis indicated a negative correlation between the distributions of S. cristatus and P. gingivalis (r = −0.57; P < 0.05). These data suggest that some early colonizers of dental plaque, such as S. cristatus, may be beneficial to the host by antagonizing the colonization and accumulation of periodontal pathogens such as P. gingivalis.Periodontitis is a bacterial biofilm (dental plaque)-related infectious disease. Although over 750 oral bacterial taxa can be detected in the oral cavity, three gram-negative species, Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola—known as the red complex—have strong associations with chronic periodontal disease (9, 28). It has been suggested that these gram-negative bacteria are later colonizers of dental plaques and are recruited to microbial communities by earlier colonizers, such as oral streptococci and Actinomyces species, via specific interactions of surface molecules (10). While there is no direct evidence that the earlier colonizers are associated with periodontitis, some of these organisms can provide a favorable environment for periodontal pathogens such as P. gingivalis. A well-studied interaction of earlier colonizers and later colonizers of dental plaque is coadhesion of Streptococcus gordonii and P. gingivalis. Specific adherence of P. gingivalis to S. gordonii strains was demonstrated in in vitro experiments (12). Studies by independent laboratories showed involvement of multiple sets of adhesins in the two bacteria. The first set of adhesins is the P. gingivalis long fimbriae (FimA) and glyceraldehyde-3-phosphate dehydrogenase present on the surfaces of streptococci (17). The second set involves the P. gingivalis short fimbriae (Mfa1) and the streptococcal SspA/B (antigen I/II) adhesins (4, 23). It is likely that these specific protein-protein interactions promote P. gingivalis colonization on existing biofilms consisting of S. gordonii and related oral streptococci (4, 27).We previously reported an antagonistic relationship between P. gingivalis and Streptococcus cristatus CC5A. P. gingivalis was unable to form microcolonies with S. cristatus, due to repression of fimA expression in the presence of S. cristatus (32). Moreover, the long fimbriae are important for aspects of P. gingivalis colonization. Previous studies have shown that a P. gingivalis strain with a fimA deficiency has a diminished capacity to adhere to human gingival fibroblasts and epithelial cells (6) and is deficient in invasion of epithelial cells (35). The fimA mutant also is less able to induce periodontal bone loss in a gnotobiotic rat model (18). Our recent study identified an S. cristatus surface protein, arginine deiminase (ArcA), responsible for eliciting repression of fimA expression in P. gingivalis 33277 (34). The arcA gene is found in a number of oral bacteria, mainly in streptococci (3). However, arcA is differentially expressed among oral streptococcal strains (16). Higher-level expression of arcA was observed in S. cristatus than in S. gordonii, which may contribute to the ability of the organism to prevent P. gingivalis colonization in the oral cavity. In this study, we tested an antagonistic role of S. cristatus in P. gingivalis colonization. We postulate that inhibition of FimA production in P. gingivalis by S. cristatus requires higher expression of ArcA and that colonization of S. cristatus strains expressing an elevated level of ArcA plays an important role in antagonizing P. gingivalis colonization.     

Glyceraldehyde-3-phosphate dehydrogenase of Streptococcus oralis functions as a coadhesin for Porphyromonas gingivalis major fimbriae

Cohesive interactions between Porphyromonas gingivalis and plaque-forming bacteria, such as Streptococcus oralis, are considered to play an important role in the colonization of P. gingivalis in periodontal sites. Although P. gingivalis fimbriae have been reported to mediate coaggregation with S. oralis, the S. oralis molecule involved has not been identified. We identified the coadhesin of S. oralis ATCC 9811 and purified it by affinity column chromatography. We found that the molecular mass of the purified protein was approximately 40 kDa. Dot blot and Western blot assays showed binding of the 40-kDa protein to P. gingivalis fimbriae. Further, turbidimetric assays showed that the coadhesin inhibited coaggregation between P. gingivalis and S. oralis in a dose-dependent manner. Analyses of the amino-terminal sequences of the protein and its lysyl endopeptidase-cleaved fragments revealed that the coadhesin was identical to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Next, we cloned the gene that encodes S. oralis GAPDH and found that the sequence had a high degree of homology with the sequences of GAPDHs of various bacteria, including Streptococcus gordonii and Fusobacterium nucleatum. To confirm the contribution of S. oralis GAPDH to the interaction with P. gingivalis, a recombinant GAPDH protein was generated in Escherichia coli; this protein bound to P. gingivalis fimbriae and had an inhibitory effect on coaggregation. These results suggest that S. oralis GAPDH functions as a coadhesin for P. gingivalis fimbriae. In addition, considering the high degree of homology of the GAPDHs of various bacteria, those of other plaque-forming bacteria also may contribute to the colonization of P. gingivalis.     

Purification of a Streptococcus mutans protein that binds to heart tissue and glycosaminoglycans.

Proteins of Streptococcus mutans MT703 were isolated by differential filtration from chemically defined culture medium following growth of the bacteria. Incubation of this preparation with cryostat-cut sections of fresh rabbit cardiac muscle resulted in deposition of streptococcal components on basement membranes of sarcolemmal sheaths and capillary walls, as indicated by indirect immunofluorescence assay. Binding of radioiodinated streptococcal proteins to heart in vitro was time dependent and saturable. Unlabeled S. mutans proteins competitively inhibited 72% of heart binding by the radiolabeled proteins, indicating a high level of binding specificity. A selection of components common to tissue basement membranes was tested for their abilities to inhibit the binding of streptococcal proteins to heart tissue. Of the glycosaminoglycans, heparin was the most effective inhibitor, followed by heparan sulfate and chondroitin sulfate. Hyaluronic acid was not inhibitory. Of the glycoproteins tested, laminin and collagen type IV were weakly inhibitory, whereas fibronectin was ineffective. A single polypeptide was purified to homogeneity by affinity chromatography on a column of heparin-agarose. Gel filtration chromatography of the purified protein under nondissociating conditions showed a single component at 31 kilodaltons (kDa), whereas in sodium dodecyl sulfate-polyacrylamide gel electrophoresis one band appeared at 8 kDa. This indicates that the tissue-binding protein may either be a linear polypeptide or be released into the environment by the bacterium as a tetramer of the 8-kDa polypeptide. The purified protein had an isoelectric point of 9.5 and showed binding activity for basement membranes in thin sections of heart. Chemical analyses of the purified binding protein showed it to have high contents of lysine and alanine and to be devoid of half-cystine, methionine, tyrosine, histidine, and both neutral and amino sugars.