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.
The effects of sugar alcohols such as erythritol, xylitol, and sorbitol on periodontopathic biofilm are poorly understood, though they have often been reported to be non-cariogenic sweeteners. In the present study, we evaluated the efficacy of sugar alcohols for inhibiting periodontopathic biofilm formation using a heterotypic biofilm model composed of an oral inhabitant Streptococcus gordonii and a periodontal pathogen Porphyromonas gingivalis. Confocal microscopic observations showed that the most effective reagent to reduce P. gingivalis accumulation onto an S. gordonii substratum was erythritol, as compared with xylitol and sorbitol. In addition, erythritol moderately suppressed S. gordonii monotypic biofilm formation. To examine the inhibitory effects of erythritol, we analyzed the metabolomic profiles of erythritol-treated P. gingivalis and S. gordonii cells. Metabolome analyses using capillary electrophoresis time-of-flight mass spectrometry revealed that a number of nucleic intermediates and constituents of the extracellular matrix, such as nucleotide sugars, were decreased by erythritol in a dose-dependent manner. Next, comparative analyses of metabolites of erythritol- and sorbitol-treated cells were performed using both organisms to determine the erythritol-specific effects. In P. gingivalis, all detected dipeptides, including Glu-Glu, Ser-Glu, Tyr-Glu, Ala-Ala and Thr-Asp, were significantly decreased by erythritol, whereas they tended to be increased by sorbitol. Meanwhile, sorbitol promoted trehalose 6-phosphate accumulation in S. gordonii cells. These results suggest that erythritol has inhibitory effects on dual species biofilm development via several pathways, including suppression of growth resulting from DNA and RNA depletion, attenuated extracellular matrix production, and alterations of dipeptide acquisition and amino acid metabolism.
Periodontal disease is characterized by chronic inflammation in subgingival areas, where a vast array of inflammation-associated metabolites are likely produced from tissue breakdown, increased vascular permeability, and microbial metabolism and then eventually show a steady flow into saliva. Thus, prolonged periodontal inflammation is a key feature of disease activity. Although salivary metabolomics has drawn attention for its potential use in diagnosis of periodontal disease, few authors have used that to investigate periodontal inflammation detection. In this pilot study, the authors explored the use of salivary metabolites to reflect periodontal inflammation severity with a recently proposed parameter-periodontal inflamed surface area (PISA)-used to quantify the periodontal inflammatory burden of individual patients with high accuracy. Following PISA determination, whole saliva samples were collected from 19 subjects before and after removal of supragingival plaque and calculus (debridement) with an ultrasonic scaler to assess the influence of the procedure on salivary metabolic profiles. Metabolic profiling of saliva was performed with gas chromatography coupled to time-of-flight mass spectrometry, followed by multivariate regression analysis with orthogonal projections to latent structures (OPLS) to investigate the relationship between PISA and salivary metabolic profiles. Sixty-three metabolites were identified. OPLS analysis showed that postdebridement saliva provided a more refined model for prediction of PISA than did predebridement samples, which indicated that debridement may improve detection of metabolites eluted from subgingival areas in saliva, thus more accurately reflecting the pathophysiology of periodontitis. Based on the variable importance in the projection values obtained via OPLS, 8 metabolites were identified as potential indicators of periodontal inflammation, of which the combination of cadaverine, 5-oxoproline, and histidine yielded satisfactory accuracy (area under the curve = 0.881) for diagnosis of periodontitis. The authors' findings identified potential biomarkers that may be useful for reflecting the severity of periodontal inflammation as part of monitoring disease activity in periodontitis patients.
Background: Arginine and its derivatives are key factors for inter-bacterial communication in periodontal microflora.Results: ArcD-mediated export of ornithine by oral commensal Streptococcus gordonii facilitates biofilm development of Fusobacterium nucleatum.Conclusion: Ornithine cross-feeding from S. gordonii to F. nucleatum mediates dental biofilm maturation.Significance: This is the first report of metabolic cross-feeding between S. gordonii and F. nucleatum.
Onset of chronic periodontitis is associated with an aberrant polymicrobial community, termed dysbiosis. Findings regarding its etiology obtained using high-throughput sequencing technique suggested that dysbiosis holds a conserved metabolic signature as an emergent property. The purpose of this study was to identify robust biomarkers for periodontal inflammation severity. Furthermore, we investigated disease-associated metabolic signatures of periodontal microbiota using a salivary metabolomics approach. Whole saliva samples were obtained from adult subjects before and after removal of supragingival plaque (debridement). Periodontal inflamed surface area (PISA) was employed as an indicator of periodontal inflammatory status. Based on multivariate analyses using pre-debridement salivary metabolomics data, we found that metabolites associated with higher PISA included cadaverine and hydrocinnamate, while uric acid and ethanolamine were associated with lower PISA. Next, we focused on dental plaque metabolic byproducts by selecting salivary metabolites significantly decreased following debridement. Metabolite set enrichment analysis revealed that polyamine metabolism, arginine and proline metabolism, butyric acid metabolism, and lysine degradation were distinctive metabolic signatures of dental plaque in the high PISA group, which may be related to the metabolic signatures of disease-associated communities. Collectively, our findings identified potential biomarkers of periodontal inflammatory status and also provide insight into metabolic signatures of dysbiotic communities.
Porphyromonas gingivalis forms communities with antecedent oral biofilm constituent streptococci. P. gingivalis major fimbriae bind to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) present on the streptococcal surface, and this interaction plays an important role in P. gingivalis colonization. This study identified the binding domain of Streptococcus oralis GAPDH for P. gingivalis fimbriae. S. oralis recombinant GAPDH (rGAPDH) was digested with lysyl endopeptidase. Cleaved fragments of rGAPDH were applied to a reversephase high-pressure liquid chromatograph equipped with a C 18 column. Each peak was collected; the binding activity toward P. gingivalis recombinant fimbrillin (rFimA) was analyzed with a biomolecular interaction analysis system. The fragment displaying the strongest binding activity was further digested with various proteinases, after which the binding activity of each fragment was measured. The amino acid sequence of each fragment was determined by direct sequencing, mass spectrometric analysis, and amino acid analysis. Amino acid residues 166 to 183 of S. oralis GAPDH exhibited the strongest binding activity toward rFimA; confocal laser scanning microscopy revealed that the synthetic peptide corresponding to amino acid residues 166 to 183 of S. oralis GAPDH (pep166-183, DNFGVVEGLMTTIHAYTG) inhibits S. oralis-P. gingivalis biofilm formation in a dose-dependent manner. Moreover, pep166-183 inhibited interbacterial biofilm formation by several oral streptococci and P. gingivalis strains with different types of FimA. These results indicate that the binding domain of S. oralis GAPDH for P. gingivalis fimbriae exists within the region encompassing amino acid residues 166 to 183 of GAPDH and that pep166-183 may be a potent inhibitor of P. gingivalis colonization in the oral cavity.
Our results suggest that FimA variations have effects on the structures of biofilms formed by P. gingivalis, which may be an important factor in the pathogenesis of periodontitis.
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