Satoshi Shizukuishi

Streptococcus gordonii utilizes several distinct gene functions to recruit Porphyromonas gingivalis into a mixed community

Dental plaque biofilm formation proceeds through a developmental pathway initiated by the attachment of pioneer organisms, such as Streptococcus gordonii, to tooth surfaces. Through a variety of synergistic interactions, pioneer organisms facilitate the colonization of later arrivals including Porphyromonas gingivalis, a potential periodontal pathogen. We have investigated genes of S. gordonii required to support a heterotypic biofilm community with P. gingivalis. By screening a plasmid integration library of S. gordonii, genes were identified that are crucial for the accumulation of planktonic P. gingivalis cells into a multispecies biofilm. These genes were further investigated by specific mutation and complementation analyses. The biofilm‐associated genes can be grouped into broad categories based on putative function as follows: (i) intercellular or intracellular signalling (cbe and spxB), (ii) cell wall integrity and maintenance of adhesive proteins (murE, msrA and atf), (iii) extracellular capsule biosynthesis (pgsA and atf), and (iv) physiology (gdhA, ccmA and ntpB). In addition, a gene for a hypothetical protein was identified. Biofilm visualization and quantification by confocal microscopy confirmed the role of these genes in the maturation of the multispecies community, including biofilm architectural development. The results suggest that S. gordonii governs the development of heterotypic oral biofilms through multiple genetic pathways.

Glyceraldehyde-3-Phosphate Dehydrogenase of Streptococcus oralis Functions as a Coadhesin for Porphyromonas gingivalis Major Fimbriae

ABSTRACT 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.

P. gingivalis accelerates gingival epithelial cell progression through the cell cycle

P. gingivalis, an opportunistic pathogen in periodontal disease, can reside within the epithelial cells that line the gingival crevice. A proteomic analysis revealed that infection of gingival epithelial cells with P. gingivalis induces broadly based changes in the level and phosphorylation status of proteins that exert multi-level control on the eukaryotic cell cycle. Pathways that were impacted by P. gingivalis included those involving cyclins, p53 and PI3K. The predicted infection-dependent phenotype was confirmed by cytofluorimetry that showed an enhanced proliferation rate of gingival epithelial cells infected with P. gingivalis associated with accelerated progression through the S-phase. Elevated cell proliferation was dependent on the presence of the long fimbriae of P. gingivalis. The ability of P. gingivalis, a common inhabitant of the subgingival crevice, to accelerate cell cycling could have biological consequences for barrier and signaling functions, and for physiological status, of the gingival epithelium.

Distinct roles of long/short fimbriae and gingipains in homotypic biofilm development by Porphyromonas gingivalis.

BackgroundPorphyromonas gingivalis, a periodontal pathogen, expresses a number of virulence factors, including long (FimA) and short (Mfa) fimbriae as well as gingipains comprised of arginine-specific (Rgp) and lysine-specific (Kgp) cysteine proteinases. The aim of this study was to examine the roles of these components in homotypic biofilm development by P. gingivalis, as well as in accumulation of exopolysaccharide in biofilms.ResultsBiofilms were formed on saliva-coated glass surfaces in PBS or diluted trypticase soy broth (dTSB). Microscopic observation showed that the wild type strain formed biofilms with a dense basal monolayer and dispersed microcolonies in both PBS and dTSB. A FimA deficient mutant formed patchy and small microcolonies in PBS, but the organisms proliferated and formed a cohesive biofilm with dense exopolysaccharides in dTSB. A Mfa mutant developed tall and large microcolonies in PBS as well as dTSB. A Kgp mutant formed markedly thick biofilms filled with large clumped colonies under both conditions. A RgpA/B double mutant developed channel-like biofilms with fibrillar and tall microcolonies in PBS. When this mutant was studied in dTSB, there was an increase in the number of peaks and the morphology changed to taller and loosely packed biofilms. In addition, deletion of FimA reduced the autoaggregation efficiency, whereas autoaggregation was significantly increased in the Kgp and Mfa mutants, with a clear association with alteration of biofilm structures under the non-proliferation condition. In contrast, this association was not observed in the Rgp-null mutants.ConclusionThese results suggested that the FimA fimbriae promote initial biofilm formation but exert a restraining regulation on biofilm maturation, whereas Mfa and Kgp have suppressive and regulatory roles during biofilm development. Rgp controlled microcolony morphology and biovolume. Collectively, these molecules seem to act coordinately to regulate the development of mature P. gingivalis biofilms.

Relationship of Porphyromonas gingivalis with glycemic level in patients with type 2 diabetes following periodontal treatment

INTRODUCTION The aim of this study was to assess the relationship between serum glycemic levels and subgingival microbial profile alteration following periodontal treatment in patients with type 2 diabetes mellitus. METHODS We studied 30 periodontitis patients with type 2 diabetes mellitus who received full-mouth subgingival debridement by analyzing their subgingival microbial profiles using a polymerase chain reaction method at baseline and various time-points for 12 months following treatment. Concurrently, probing pocket depth, bleeding on probing, and metabolic parameters, including glycated hemoglobin A1c (HbA1c), blood sugar level, C-reactive proteins, total cholesterol, triglyceride, and high-density and low-density lipoprotein cholesterol, were recorded. RESULTS Periodontal conditions were significantly improved after treatment, and the occurrence rates of periodontal bacterial species, including Porphyromonas gingivalis, Tannerella forsythensis, Treponema denticola, and Prevotella intermedia, were also reduced. Interestingly, P. gingivalis was detected more frequently in subjects with increased HbA1c values after periodontal treatment than in those patients with decreased HbA1c values. Furthermore, P. gingivalis with type II fimbriae was detected only in HbA1c-increased subjects, while improvements in HbA1c values were observed only in subjects without type II clones. CONCLUSIONS These results suggest that glycemic level in diabetes is affected by the persistence of P. gingivalis, especially clones with type II fimbriae, in periodontal pockets.

Porphyromonas gingivalis Fimbriae Mediate Coaggregation with Streptococcus oralis through Specific Domains

Fimbriae are major adhesive components on the cell surface of Porphyromonas gingivalis. In this study, we evaluated the role of fimbriae in coaggregation with Streptococcus oralis. Fimbriae purified from P. gingivalis competitively inhibited the coaggregation by 100% at a concentration of 50 ug/mL. On the other hand, the same amount of lipopolysaccharide isolated from P. gingivalis was inhibited by only 25% of the level of the fimbriae. A fimA-inactivated mutant of P. gingivalis failed to show distinct coaggregation activity. Fimbriae added to a solution of various strains of streptococci caused their self-aggregation at a concentration of 10 to 30 ug/mL. The self-aggregation induced by fimbriae was inhibited by \-arginine (20 to 40 mM/L). Iodinated fimbriae reacted with S. oralis cells immobilized on the nitrocellulose membrane, and 100°C heating of the cells diminished the binding abilities. Recombinant fimbrillin (r-Fim, corresponding to whole residues 1 to 337 of native fimbrillin) of P. gingivalis also showed 100% inhibition of the coaggregation. The r-Fim variant (residues 1 to 286) lacking the C-terminal 51 residues was as inhibitory as r-Fim. However, the variant (residues 1 to 265) without the C-terminal 72 residues lost 77% of the inhibitory activity. These findings suggested that residues 266 to 286 contain a domain involved in the coaggregation of P. gingivalis with S. oral is. Inhibition by three polypeptides corresponding to residues 266 to 286, 266 to 337, and 287 to 337 was studied. Peptides 266 to 286 and 266 to 337 inhibited by 96 and 100%, respectively, at a concentration of 1.5 nmol/mL. Peptide 287 to 337 also showed a significant inhibitory effect but to a slightly lesser extent than that of peptide 266 to 286. P. gingivalis fimbriae appear to be involved in coaggregation with streptococci, probably through an adhesive protein molecule(s) of the latter, and the fimbriae possess several domains in the C-terminal residues 266 to 337 for interaction with S. oralis.

Nasal Flt3 Ligand cDNA Elicits CD11c+CD8+ Dendritic Cells for Enhanced Mucosal Immunity

Nasal immunization is an effective way to induce both mucosal and systemic immune responses. In this study, we assessed a cDNA vector for Flt3 ligand (FL) for its potential to enhance mucosal immunity or tolerance. Interestingly, tolerance was avoided and elevated levels of OVA-specific Ab responses were induced in nasal washes, fecal extracts, and saliva as well as in plasma when compared with mice given nasal OVA plus DNA plasmid without the FL gene. In addition, significant levels of OVA-specific CD4+ T cell proliferative responses and OVA-induced IL-4 and IL-2 production were noted in spleen and cervical lymph nodes. Further, marked increases in FL protein occurred in the nasal lamina propria and submandibular glands and the frequencies of CD11c+CD8+ dendritic cells (DCs) significantly increased in the mucosal tissues. Moreover, these DCs expressed high levels of CD40, CD80, CD86, and MHC class II molecules. Nasal delivery of plasmid FL with OVA resulted in FL expression in both mucosal inductive and effector sites and resulted in expanded activated lymphoid DCs. Thus, nasal plasmid FL prevents mucosal tolerance and enhances active immunity when given by a mucosal route.

A Porphyromonas gingivalis tyrosine phosphatase is a multifunctional regulator of virulence attributes

Low Molecular Weight Tyrosine Phosphatases (LMWTP) are widespread in prokaryotes; however, understanding of the signalling cascades controlled by these enzymes is still emerging. Porphyromonas gingivalis, an opportunistic oral pathogen, expresses a LMWTP, Ltp1, that is differentially regulated in biofilm communities. Here we characterize the enzymatic activity of Ltp1 and, through the use of mutants that lack Ltp1 or expresses catalytically defective Ltp1, show that tyrosine phosphatase activity constrains both monospecies biofilm development and community development with the antecedent oral biofilm constituent Streptococcus gordonii. Exopolysaccharide production is downregulated by Ltp1 through transcriptional regulation of multiple genes involved in biosynthesis and transport. Furthermore, Ltp1 regulates transcriptional activity of luxS and thus impacts AI‐2‐dependent signalling in biofilm communities. In the absence of Ltp1 transcription across the hmu haemin uptake locus is reduced, and consequently uptake of haemin is impaired in the Ltp1 mutant. The gingipain proteinases Kgp and RgpA/B remain phosphorylated in the Ltp1 mutant. Phosphorylated Rgps are poorly secreted, whereas cell surface activity of phosphorylated Kgp is enhanced. By controlling the activity of several virulence‐associated properties, Ltp1 may restrain the pathogenic potential of P. gingivalis and maintain a commensal interaction with the host.

Effect of topical application of Coenzyme Q10 on adult periodontitis

Topical application of Coenzyme Q10 (CoQ10) to the periodontal pocket was evaluated with and without subgingival mechanical debridement. Ten male patients with adult periodontitis participated and 30 periodontal pockets were selected. During the first 3 weeks, the patients did not receive any periodontal therapy except the topical application of CoQ10. After the first 3-week period, root planning and subgingival scaling were performed in all sites. CoQ10 was applied in 20 of the pockets once a week for a period of 6 weeks. Soybean oil was applied to the remaining 10 sites as a control. In the first 3-week period, significant reductions in gingival crevicular fluid flow, probing depth and attachment loss were found only at experimental sites. After mechanical subgingival debridement, significant decreases in the plaque index, gingival crevicular fluid flow, probing depth and attachment loss were found both at experimental and control sites. However, significant improvements in the modified gingival index, bleeding on probing and peptidase activity derived from periodontopathic bacteria were observed only at experimental sites. These results suggest that topical application of CoQ10 improves adult periodontitis not only as a sole treatment but also in combination with traditional nonsurgical periodontal therapy.

Characterization of Binding of Streptococcus oralis Glyceraldehyde-3-Phosphate Dehydrogenase to Porphyromonas gingivalis Major Fimbriae

ABSTRACT 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 × 107 M−1, suggesting that S. oralis GAPDH functions as a dominant receptor for P. gingivalis and contributes to P. gingivalis colonization.