Porphyromonas gingivalis initiates coagulation and secretes polyphosphates - A mechanism for sustaining chronic inflammation?

BACKGROUND: Periodontitis is a chronic inflammation resulting in destruction of tooth-supporting bone. Chronic inflammation is characterized by extravascular fibrin deposition. Fibrin is central to destruction of bone; monocytes bind to fibrin and form osteoclasts, thus providing a link between coagulation and the tissue destructive processes in periodontitis. The oral microbiome is essential to oral health. However, local ecological changes, such as increased biofilm formation, result in a dysbiotic microbiome characterized by an increase of protease-producing species e.g. Porphyromonas gingivalis. Proteases initiate inflammation and may cleave coagulation factors. Polyphosphates (polyP) may also provide bacteria with procoagulant properties similar to platelet-released polyP. P. gingivalis has also been found in remote locations related to vascular pathology and Alzheimer's disease. OBJECTIVES: The aim of this study was to investigate procoagulant activity of ten different species of oral bacteria present in oral health and disease as well as presence of polyP and fibrin formation in planktonic and biofilm bacteria. METHODS: Oral bacteria were studied for protease production and procoagulant activity. The presence of polyP and formation of fibrin was observed using confocal microscopy. RESULTS: P. gingivalis showed strong protease activity and was the only species exerting procoagulant activity. Confocal microscopy showed polyP intracellularly in planktonic bacteria and extracellularly after biofilm formation. Fibrin formation emanated from planktonic bacteria and from both bacteria and polyP in biofilm cultures. CONCLUSIONS: The procoagulant activity of P. gingivalis could explain its role in chronic inflammation, locally in oral tissues as well as in remote locations.

Polymicrobial synergy stimulates Porphyromonas gingivalis survival and gingipain expression in a multi-species subgingival community.

BACKGROUND: Dysbiosis in subgingival microbial communities, resulting from increased inflammatory transudate from the gingival tissues, is an important factor in initiation and development of periodontitis. Dysbiotic communities are characterized by increased numbers of bacteria that exploit the serum-like transudate for nutrients, giving rise to a proteolytic community phenotype. Here we investigate the contribution of interactions between members of a sub-gingival community to survival and development of virulence in a serum environment-modelling that in the subgingival pocket. METHODS: Growth and proteolytic activity of three Porphyromonas gingivalis strains in nutrient broth or a serum environment were assessed using A600 and a fluorescent protease substrate, respectively. Adherence of P. gingivalis strains to serum-coated surfaces was studied with confocal microscopy and 2D-gel electrophoresis of bacterial supernatants used to investigate extracellular proteins. A model multi-species sub-gingival community containing Fusobacterium nucleatum, Streptococcus constellatus, Parvimonas micra with wild type or isogenic mutants of P. gingivalis was then created and growth and proteolytic activity in serum assessed as above. Community composition over time was monitored using culture techniques and qPCR. RESULTS: The P. gingivalis strains showed different growth rates in nutrient broth related to the level of proteolytic activity (largely gingipains) in the cultures. Despite being able to adhere to serum-coated surfaces, none of the strains was able to grow alone in a serum environment. Together in the subgingival consortium however, all the included species were able to grow in the serum environment and the community adopted a proteolytic phenotype. Inclusion of P. gingivalis strains lacking gingipains in the consortium revealed that community growth was facilitated by Rgp gingipain from P. gingivalis. CONCLUSIONS: In the multi-species consortium, growth was facilitated by the wild-type and Rgp-expressing strains of P. gingivalis, suggesting that Rgp is involved in delivery of nutrients to the whole community through degradation of complex protein substrates in serum. Whereas they are constitutively expressed by P. gingivalis in nutrient broth, gingipain expression in the model periodontal pocket environment (serum) appeared to be orchestrated through signaling to P. gingivalis from other members of the community, a phenomenon which then promoted growth of the whole community.

Plasminogen coating increases initial adhesion of oral bacteria in vitro.

Plasminogen is a major plasma protein and the zymogen of the broad spectrum protease plasmin. Plasmin activity leads to tissue degradation, direct and through activation of metalloproteinases. Infected tooth root canals, as a consequence of the inflammatory response and eventual necrosis, contain tissue fluid and blood components. These will coat the root canal walls and act as conditioning films that allow bacterial biofilms to grow and be a potential source of hematogenously spreading bacteria. We investigated the effect of in vitro surface conditioning with human plasminogen on the initial adhesion of bacteria. Four bacterial species, L. salivarius, E. faecalis, A. naeslundii, and S. gordonii, isolated from dental root canals, and three other oral streptococci, S. oralis, S. anginosus, and S. sanguinis, were grown in albumin- or plasminogen-coated flow chambers and studied by confocal laser scanning microscopy using the cell viability staining LIVE/DEAD and 16S rRNA fluorescence in situ hybridization (FISH). A. naeslundii, L. salivarius and in particular S. gordonii showed a higher initial adhesion to the plasminogen-coated surfaces. E. faecalis did not show any preference for plasminogen. Four-species biofilms cultured for 96 h showed that streptococci increased their proportion with time. Further experiments aimed at studying different streptococcal strains. All these adhered more to plasminogen-coated surfaces than to albumin-coated control surfaces. The specificity of the binding to plasminogen was verified by blocking lysine-binding sites with epsilon-aminocaproic acid. Plasminogen is thus an important plasma component for the initial adhesion of oral bacteria, in particular streptococci. This binding may contribute to their spread locally as well as to distant organs or tissues.

PAI-2/SerpinB2 inhibits proteolytic activity in a P. gingivalis-dominated multispecies bacterial consortium.

OBJECTIVE: The aim of this study was to investigate the ability of the serine protease inhibitor plasminogen activator inhibitor type 2 (PAI-2/Serpin B2) to inhibit proteases produced by a multispecies bacterial consortium in vitro. BACKGROUND: Gingival and periodontal inflammation is associated with an increased flow of protein-rich gingival fluid. This nutritional change in the microenvironment favors bacteria with a proteolytic phenotype, triggering inflammation and associated tissue breakdown. PAI-2 is produced by macrophages and keratinocytes and is present in very high concentrations in gingival crevicular fluid; the highest level in the body. DESIGN: A multispecies bacterial consortium comprising nine bacterial strains, resembling the conditions in a periodontal pocket, was grown planktonically and as a biofilm. After seven days PAI-2 was added to the consortium and the proteolytic activity was assayed with fluorogenic protease substrates; FITC-labeled casein to detect global protease activity, fluorescent H-Gly-Pro-AMC for serine protease activity and fluorescent BIKKAM-10 for Porphyromonas gingivalis-associated protease activity. Protease activity associated with biofilm cells was examined by confocal scanning laser microscopy. RESULTS: PAI-2 inhibited proteolytic activity of the bacterial consortium, as seen by decreased fluorescence of all substrates. PAI-2 specifically inhibited P. gingivalis proteolytic activity. CONCLUSION: To our knowledge, this is the first time that PAI-2 has been shown to inhibit bacterial proteases. Given the high concentration of PAI-2 in the gingival region, our results indicate that PAI-2 might play a role for the integrity of the epithelial barrier.

Effect of fluoride and chlorhexidine digluconate mouthrinses on plaque biofilms.

OBJECTIVE: To develop a model in which to investigate the architecture of plaque biofilms formed on enamel surfaces in vivo and to compare the effects of anti-microbial agents of relevance for caries on biofilm vitality. Materials and Methodology : Enamel discs mounted on healing abutments in the pre-molar region were worn by three subjects for 7 days. Control discs were removed before subjects rinsed with 0.1% chlorhexidine digluconate (CHX) or 0.2% sodium fluoride (NaF) for 1 minute. Biofilms were stained with Baclight Live/Dead and z-stacks of images created using confocal scanning laser micoscopy. The levels of vital and dead/damaged bacteria in the biofilms, assessed as the proportion of green and red pixels respectively, were analysed using ImageTrak(®) software. Results : The subjects showed individual differences in biofilm architecture. The thickness of the biofilms varied from 28-96µm although cell density was always the greatest in the middle layers. In control biofilms, the overall levels of vitality were high (71-98%) especially in the area closest to the enamel interface. Rinsing with either CHX or NaF caused a similar reduction in overall vitality. CHX exerted an effect throughout the biofilm, particularly on the surface of cell clusters whereas NaF caused cell damage/death mainly in the middle to lower biofilm layers. Conclusion : We describe a model that allows the formation of mature, undisturbed oral biofilms on human enamel surfaces in vivo and show that CHX and NaF have a similar effect on overall vitality but differ in their sites of action.

Glycoprotein 340 and sialic acid in minor-gland and whole saliva of children, adolescents, and adults.

Glycoprotein 340 (gp-340) is a bacterial-binding glycoprotein found in major-gland and minor-gland saliva. Sialic acid, a common terminal structure of salivary glycoproteins, interacts with microorganisms and host ligands, as well as with free radicals. This study investigated the contents of gp-340 and sialic acid in minor-gland saliva and whole saliva of children (3 yr of age), adolescents (14 yr of age), and adults (20-25 yr of age). Labial-gland saliva and buccal-gland saliva were collected on filter paper, and unstimulated whole saliva was collected by draining into a tube. The relative amount of gp-340 and sialic acid was determined by ELISA and by enzyme-linked lectin assay (ELLA), respectively. In minor-gland saliva, no statistically significant differences in gp-340 and sialic acid were seen between the age-groups. Among adults, significantly lower amounts of gp-340 and sialic acid were seen in labial saliva compared with buccal saliva. In whole saliva, the amount of gp-340 was significantly lower among adults compared with children. No differences between genders were seen. Stable content of gp-340 and sialic acid in minor-gland saliva across the age-groups, and a higher content of gp-340 in the whole saliva of the youngest age-group (3-yr-olds) compared with the adult group, may reflect that those components are vital innate factors of immunity in children's saliva.

Plasminogen binding by oral streptococci from dental plaque and inflammatory lesions.

Plasminogen binding by bacteria is a virulence factor important for the entry and dissemination of bacteria in the body. A wide variety of bacteria bind plasminogen, including both organisms causing disease and components of the normal oral flora. The purpose of this study was to examine the characteristics of plasminogen binding by six clinical isolates of oral streptococci from both dental plaque and inflammatory lesions. All the strains bound plasminogen with approximately the same affinity, and binding was specific and lysine-dependent as evidenced by its inhibition by epsilon-aminocaproic acid. All of the test strains were capable of activating bound plasminogen to plasmin without the addition of a plasminogen activator, and subsequent analysis revealed the presence of streptokinase in all strains. However, the streptococci exhibited fibrinolytic activity only in the presence of plasminogen and this could be inhibited by the addition of epsilon-aminocaproic acid. SDS-PAGE and 2D gel electrophoresis coupled with plasminogen ligand blotting showed that only a subset of the total proteins (2-15) were involved in the binding of plasminogen. Partial identification of the binding proteins revealed that four glycolytic enzymes, enolase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate mutase, were predominant in binding plasminogen. The binding of plasminogen by bacteria from pus did not differ from that of the strains from supragingival plaque. The findings illustrate how apparently innocuous commensal bacteria are capable of utilizing a mechanism that is generally regarded as being of importance to pathogenicity and suggest an additional role of plasminogen binding.

Tissue plasminogen activator (t-PA) and placental plasminogen activator inhibitor (PAI-2) in gingival crevicular fluid from patients with Papillon-Lefèvre syndrome.

OBJECTIVES: Numerous patients with Papillon-Lefèvre syndrome (PLS) express a severe periodontal inflammation that results in premature loss of deciduous and permanent teeth. The plasminogen activating (PA) system is involved in physiological and pathological processes including epithelial healing, extracellular proteolysis and local inflammatory reactions. The aim of the study was to explore a possible role of the PA system in patients with PLS. MATERIAL AND METHODS: Samples of gingival crevicular fluid (GCF) were collected from areas with gingival infection in 20 patients with PLS and in 20 healthy controls. The concentration of tissue plasminogen activator (t-PA) and inhibitor (PAI-2) was measured with ELISA. RESULTS: The median level of PAI-2 was significantly higher (p < 0.01) in PLS patients than in the controls, while the median value of t-PA did not differ between the groups. No difference in t-PA or PAI-2 levels was found regarding age, gender or presence of active periodontal disease. CONCLUSION: The findings indicate an atypical activity of the PA system with a disturbed epithelial function in PLS patients, suggesting that the periodontal destruction seen in patients with PLS is secondary to a hereditary defect in the defense system.

Reduced gingival fluid flow: a peripheral marker of the pharmacological effect of roquinimex.

OBJECTIVE: Roquinimex is a drug with effects on inflammation and tumors. The pharmacological effect is not fully understood, and the molecular mechanism most characterized in vitro is an increase of plasminogen activator inhibitor type 2 (PAI-2) in human peripheral blood monocytes. The aims were to investigate peripheral pharmacological effects of roquinimex on peripheral blood monocytes and dog gingival fluid (GCF). DESIGN: Six dogs were used in a cross-over study. The amount of GCF was determined with a Periotron. The PAI-2 concentration in GCF was determined with ELISA. Monocytes were isolated from peripheral blood. RESULTS: Dogs treated with the drug had significantly lower GCF flow values and the PAI-2 concentration in GCF was higher, but no effect was seen on peripheral monocytes. CONCLUSION: Roquinimex treatment led to a consistently decreased flow rate of GCF and a higher local concentration of PAI-2 in GCF.

The plasminogen activating system in periodontal health and disease.

The plasminogen activating system is important for extracellular proteolysis and plays a regulatory role in interactions with other tissue degrading systems. Studies on the plasminogen activating system in gingival crevicular fluid (GCF) as well as gingival tissue are reviewed. t-PA, u-PA, PAI-1 and PAI-2 have all been detected in GCF. Especially t-PA and PAI-2 are found in high concentrations. In tissue studies fibrinolytic activity has been found in the gingival pocket epithelium in humans and in animal studies. t-PA and PAI-2 have been detected there immunohistochemically. Local production of the PAs and PAls has been verified with in situ hybridization. In inflammation, a more intense and widespread immunohistochemical staining of t-PA and PAI-2 is seen. Higher concentrations of t-PA and PAI-2 are found in GCF but the balance between them seems to be constant. A systemically disturbed balance of the plasminogen activating system in GCF has been observed during pregnancy, with a possible protective function of PAI-2. In studies of periodontitis, the production of PAI-2 seemed to be locally lowered at impaired sites. In a study of children, a higher inflammatory response to bacterial plaque was accompanied by a higher fibrinolytic ativity in GCF samples. Bacterial LPS has been found to change the ratio of t-PA to PAI-2 in cultured gingival fibroblasts. Interactions between PAI-2 and a protease in the gingival epithelium has been verified through the immunohistochemical detection of relaxed PAI-2.

Plasminogen activating capacity in gingival fluid from deteriorating and stable periodontal pockets.

The plasminogen activating system plays an important role in tissue proteolysis in physiological as well as pathological processes. Earlier studies have shown high concentrations of the plasminogen activator t-PA as well as its inhibitor PAI-2 in gingival crevicular fluid (GCF). In addition, gingival inflammatory reactions have been related to increases in t-PA and PAI-2. In order to explore the potential role of the plasminogen activating system for the development of destructive periodontal disease, the aim of this study was to assess the balance of the activator t-PA to the inhibitor PAI-2 in GCF from patients, clinically defined to represent different periodontal conditions. The Progression Group consisted of 12 periodontitis patients with 1 or more sites having shown an increased pocket depth of > or = 3 mm during the last 2 years of maintenance care and with > or = 8 unchanged or improving sites during the period. The Non Progression Group consisted of patients who had shown a decreased or unchanged pocket depth of all sites during the last 3 years of maintenance care. Sampling of GCF was done with small disks of Millipore-filter, and t-PA and PAI-2 were analyzed with ELISAs. There was no difference in the t-PA/PAI-2 ratio between the two groups. However, an intra-individual comparison within the Progression Group showed a higher ratio at the deteriorating sites than at the stable sites. Even though no difference was found between the groups, the higher t-PA/PAI-2 ratio at the deteriorating sites in the Progression Group suggests an involvement of the plasminogen activating system in the proteolytic events leading to breakdown of the tooth supporting tissues.