Evidence of mutualism between two periodontal pathogens: co‐operative haem acquisition by the HmuY haemophore of Porphyromonas gingivalis and the cysteine protease interpain A (InpA) of Prevotella intermedia

Haem (iron protoporphyrin IX) is both an essential growth factor and a virulence regulator of the periodontal pathogens Porphyromonas gingivalis and Prevotella intermedia, which acquire it through the proteolytic degradation of haemoglobin and other haem‐carrying plasma proteins. The haem‐binding lipoprotein HmuY haemophore and the gingipain proteases of P. gingivalis form a unique synthrophic system responsible for capture of haem from haemoglobin and methaemalbumin. In this system, methaemoglobin is formed from oxyhaemoglobin by the activities of gingipain proteases and serves as a facile substrate from which HmuY can capture haem. This study examined the possibility of cooperation between HmuY and the cysteine protease interpain A (InpA) of Pr. intermedia in the haem acquisition process. Using UV‐visible spectroscopy and polyacrylamide gel electrophoresis, HmuY was demonstrated to be resistant to proteolysis and so able to cooperate with InpA to extract haem from haemoglobin, which was proteolytically converted to methaemoglobin by the protease. Spectroscopic pH titrations showed that both the iron(II) and iron(III) protoporphyrin IX–HmuY complexes were stable over the pH range 4–10, demonstrating that the haemophore could function over a range of pH that may be encountered in the dental plaque biofilm. This is the first demonstration of a bacterial haemophore working in conjunction with a protease from another bacterial species to acquire haem from haemoglobin and may represent mutualism between P. gingivalis and Pr. intermedia co‐inhabiting the periodontal pocket.     

Extracellular deoxyribonuclease production by periodontal bacteria

Palmer LJ, Chapple ILC, Wright HJ, Roberts A, Cooper PR. Extracellular deoxyribonuclease production by periodontal bacteria. J Periodont Res 2012; 47: 439–445. © 2011 John Wiley & Sons A/S Background and Objective: Whilst certain bacteria have long been known to secrete extracellular deoxyribonuclease (DNase), the purpose in microbial physiology was unclear. Recently, however, this enzyme has been demonstrated to confer enhanced virulence, enabling bacteria to evade the host’s immune defence of extruded DNA/chromatin filaments, termed neutrophil extracellular traps (NETs). As NETs have recently been identified in infected periodontal tissue, the aim of this study was to screen periodontal bacteria for extracellular DNase activity. Material and Methods: To determine whether DNase activity was membrane bound or secreted, 34 periodontal bacteria were cultured in broth and on agar plates. Pelleted bacteria and supernatants from broth cultures were analysed for their ability to degrade DNA, with relative activity levels determined using an agarose gel electrophoresis assay. Following culture on DNA‐supplemented agar, expression was determined by the presence of a zone of hydrolysis and DNase activity related to colony size. Results: Twenty‐seven bacteria, including red and orange complex members Porphyromonas gingivalis, Tannerella forsythia, Fusobacterium nucleatum, Parvimonas micra, Prevotella intermedia, Streptococcus constellatus, Campylobacter rectus and Prevotella nigrescens, were observed to express extracellular DNase activity. Differences in DNase activity were noted, however, when bacteria were assayed in different culture states. Analysis of the activity of secreted DNase from bacterial broth cultures confirmed their ability to degrade NETs. Conclusion: The present study demonstrates, for the first time, that DNase activity is a relatively common property of bacteria associated with advanced periodontal disease. Further work is required to determine the importance of this bacterial DNase activity in the pathogenesis of periodontitis.     

The pga gene cluster in Aggregatibacter actinomycetemcomitans is necessary for the development of natural competence in Ca2+‐promoted biofilms

Natural competence is the ability of bacteria to incorporate extracellular DNA into their genomes. This competence is affected by a number of factors, including Ca²⁺ utilization and biofilm formation. As bacteria can form thick biofilms in the presence of extracellular Ca²⁺, the additive effects of Ca²⁺‐promoted biofilm formation on natural competence should be examined. We evaluated natural competence in Aggregatibacter actinomycetemcomitans, an important periodontal pathogen, in the context of Ca²⁺‐promoted biofilms, and examined whether the pga gene cluster, required for bacterial cell aggregation, is necessary for competence development. The A. actinomycetemcomitans cells grown in the presence of 1 mm CaCl₂ exhibited enhanced cell aggregation and increased levels of cell‐associated Ca²⁺. Biofilm‐derived cells grown in the presence of Ca²⁺ exhibited the highest levels of natural transformation frequency and enhanced expression of the competence regulator gene, tfoX. Natural competence was enhanced by the additive effects of Ca²⁺‐promoted biofilms, in which high levels of pga gene expression were also detected. Mutation of the pga gene cluster disrupted biofilm formation and competence development, suggesting that these genes play a critical role in the ability of A. actinomycetemcomitans to adapt to its natural environment. The Ca²⁺‐promoted biofilms may enhance the ability of bacteria to acquire extracellular DNA.     

Selective bacterial degradation of the extracellular matrix attaching the gingiva to the tooth

The junctional epithelium (JE) is a specialized portion of the gingiva that seals off the tooth‐supporting tissues from the oral environment. This relationship is achieved via a unique adhesive extracellular matrix that is, in fact, a specialized basal lamina (sBL). Three unique proteins – amelotin (AMTN), odontogenic ameloblast‐associated (ODAM), and secretory calcium‐binding phosphoprotein proline‐glutamine rich 1 (SCPPPQ1) – together with laminin‐332 structure the supramolecular organization of this sBL and determine its adhesive capacity. Despite the constant challenge of the JE by the oral microbiome, little is known of the susceptibility of the sBL to bacterial degradation. Assays with trypsin‐like proteases, as well as incubation with Porphyromonas gingivalis, Prevotella intermedia, and Treponema denticola, revealed that all constituents, except SCPPPQ1, were rapidly degraded. Porphyromonas gingivalis was also shown to alter the supramolecular network of reconstituted and native sBLs. These results provide evidence that proteolytic enzymes and selected gram‐negative periodontopathogenic bacteria can attack this adhesive extracellular matrix, intimating that its degradation could contribute to progression of periodontal diseases.     

Erythritol alters microstructure and metabolomic profiles of biofilm composed of Streptococcus gordonii and Porphyromonas gingivalis

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.     

Secreted adenosine triphosphate from Aggregatibacter actinomycetemcomitans triggers chemokine response

Extracellular ATP (eATP) is an important intercellular signaling molecule secreted by activated immune cells or released by damaged cells. In mammalian cells, a rapid increase of ATP concentration in the extracellular space sends a danger signal, which alerts the immune system of an impending danger, resulting in recruitment and priming of phagocytes. Recent studies show that bacteria also release ATP into the extracellular milieu, suggesting a potential role for eATP in host–microbe interactions. It is currently unknown if any oral bacteria release eATP. As eATP triggers and amplifies innate immunity and inflammation, we hypothesized that eATP secreted from periodontal bacteria may contribute to inflammation in periodontitis. The aims of this study were to determine if periodontal bacteria secrete ATP, and to determine the function of bacterially derived eATP as an inducer of inflammation. Our results showed that Aggregatibacter actinomycetemcomitans, but not Porphyromonas gingivalis, Prevotella intermedia, or Fusobacterium nucleatum, secreted ATP into the culture supernatant. Exposure of periodontal fibroblasts to filter sterilized culture supernatant of A. actinomycetemcomitans induced chemokine expression in an eATP‐dependent manner. This occurred independently of cyclic adenosine monophosphate and phospholipase C, suggesting that ionotrophic P2X receptor is involved in sensing of bacterial eATP. Silencing of P2X7 receptor in periodontal fibroblasts led to a significant reduction in bacterial eATP‐induced chemokine response. Furthermore, bacterial eATP served as a potent chemoattractant for neutrophils and monocytes. Collectively, our findings provide evidence for secreted ATP of A. actinomycetemcomitans as a novel virulence factor contributing to inflammation during periodontal disease.     

Induction of calprotectin release by Porphyromonasgingivalis lipopolysaccharide in human neutrophils

Calprotectin, a major cytosolic protein of leukocytes, is detected in neutrophils, monocytes/macrophages, and epithelial cells. This protein is known to be a marker for several inflammatory diseases and is detected in inflammatory gingival tissue with periodontal disease. Recently, we found that the calprotectin level in gingival crevicular fluid from periodontitis patients was significantly higher than that of healthy subjects. However, the regulation of calprotectin in periodontal disease is unclear. In the present study, we investigated the effect of lipopolysaccharides of periodontopathic bacteria on calprotectin release from human neutrophils. Neutrophils from healthy donors were treated with lipopolysaccharides from Porphyromonas gingivalis (P‐LPS), Actinobacillus actinomycetemcomitans, Prevotella intermedia, Fusobacterium nucleatum, and Escherichia coli. Calprotectin of neutrophil was identified by immunoblotting and calprotectin amount was determined by ELISA. Two subunits (10 and 14 kDa) of calprotectin were observed in the cell and medium fractions from neutrophils. P‐LPS increased calprotectin release from seven to 16 times the control level after 30 min and its effect appeared in a dose‐dependent manner (10–1000 ng/ml). Lipopolysaccharides from A. actinomycetemcomitans, P. intermedia, F. nucleatum, and E. coli also induced calprotectin release from neutrophils. These results suggest that lipopolysaccharides from periodontopathic bacteria induce calprotectin release from human neutrophils.     

Aggregatibacter actinomycetemcomitans,a potent immunoregulator of the periodontal host defense system and alveolar bone homeostasis

Aggregatibacter actinomycetemcomitans is a perio‐pathogenic bacteria that has long been associated with localized aggressive periodontitis. The mechanisms of its pathogenicity have been studied in humans and preclinical experimental models. Although different serotypes of A. actinomycetemcomitans have differential virulence factor expression, A. actinomycetemcomitans cytolethal distending toxin (CDT), leukotoxin, and lipopolysaccharide (LPS) have been most extensively studied in the context of modulating the host immune response. Following colonization and attachment in the oral cavity, A. actinomycetemcomitans employs CDT, leukotoxin, and LPS to evade host innate defense mechanisms and drive a pathophysiologic inflammatory response. This supra‐physiologic immune response state perturbs normal periodontal tissue remodeling/turnover and ultimately has catabolic effects on periodontal tissue homeostasis. In this review, we have divided the host response into two systems: non‐hematopoietic and hematopoietic. Non‐hematopoietic barriers include epithelium and fibroblasts that initiate the innate immune host response. The hematopoietic system contains lymphoid and myeloid‐derived cell lineages that are responsible for expanding the immune response and driving the pathophysiologic inflammatory state in the local periodontal microenvironment. Effector systems and signaling transduction pathways activated and utilized in response to A. actinomycetemcomitans will be discussed to further delineate immune cell mechanisms during A. actinomycetemcomitans infection. Finally, we will discuss the osteo‐immunomodulatory effects induced by A. actinomycetemcomitans and dissect the catabolic disruption of balanced osteoclast–osteoblast‐mediated bone remodeling, which subsequently leads to net alveolar bone loss.     

In vivo expression of proteases and protease inhibitor,a serpin,by periodontal pathogens at teeth and implants

Porphyromonas gingivalis and Tannerella forsythia secrete proteases, gingipains and KLIKK‐proteases. In addition, T. forsythia produces a serpin (miropin) with broad inhibitory spectrum. The aim of this pilot study was to determine the level of expression of miropin and individual proteases in vivo in periodontal and peri‐implant health and disease conditions. Biofilm and gingival crevicular fluid (GCF)/ peri‐implant sulcular fluid (PISF) samples were taken from healthy tooth and implant sites (n = 10), gingivitis and mucositis sites (n = 12), and periodontitis and peri‐implantitis sites (n = 10). Concentration of interleukin‐8 (IL‐8), IL‐1β and IL‐10 in GCF was determined by enzyme‐linked immunosorbent assay. Loads of P. gingivalis and T. forsythia and the presence of proteases and miropin genes were assessed in biofilm by quantitative PCR, whereas gene expression was estimated by quantitative RT‐PCR. The presence of P. gingivalis and T. forsythia, as well as the level of IL‐8 and IL‐1β, were associated with disease severity in the periodontal and peri‐implant tissues. In biofilm samples harboring T. forsythia, genes encoding proteases were found to be present at 72.4% for karilysin and 100% for other KLIKK‐protease genes and miropin. At the same time, detectable mRNA expression of individual genes ranged from 20.7% to 58.6% of samples (for forsylisin and miropsin‐1, respectively). In comparison with the T. forsythia proteases, miropin and the gingipains were highly expressed. The level of expression of gingipains was associated with those of miropin and certain T. forsythia proteases around teeth but not implants. Cumulatively, KLIKK‐proteases and especially miropin, might play a role in pathogenesis of both periodontal and peri‐implant diseases.     

The role of phagocytosis,oxidative burst and neutrophil extracellular traps in the interaction between neutrophils and the periodontal pathogen Porphyromonas gingivalis

Neutrophils are regarded as the sentinel cells of innate immunity and are found in abundance within the gingival crevice. Discovery of neutrophil extracellular traps (NETs) within the gingival pockets prompted us to probe the nature of the interactions of neutrophils with the prominent periopathogen Porphyromonas gingivalis. Some of the noted virulence factors of this Gram‐negative anaerobe are gingipains: arginine gingipains (RgpA/B) and lysine gingipain (Kgp). The aim of this study was to evaluate the role of gingipains in phagocytosis, formation of reactive oxygen species, NETs and CXCL8 modulation by using wild‐type strains and isogenic gingipain mutants. Confocal imaging showed that gingipain mutants K1A (Kgp) and E8 (RgpA/B) induced extracellular traps in neutrophils, whereas ATCC33277 and W50 were phagocytosed. The viability of both ATCC33277 and W50 dwindled as the result of phagocytosis and could be salvaged by cytochalasin D, and the bacteria released high levels of lipopolysaccharide in the culture supernatant. Porphyromonas gingivalis induced reactive oxygen species and CXCL8 with the most prominent effect being that of the wild‐type strain ATCC33277, whereas the other wild‐type strain W50 was less effective. Quantitative real‐time polymerase chain reaction revealed a significant CXCL8 expression by E8. All the tested P. gingivalis strains increased cytosolic free calcium. In conclusion, phagocytosis is the primary neutrophil response to P. gingivalis, although NETs could play an accessory role in infection control. Although gingipains do not seem to directly regulate phagocytosis, NETs or oxidative burst in neutrophils, their proteolytic properties could modulate the subsequent outcomes such as nutrition acquisition and survival by the bacteria.     

Escape from the competence state in Streptococcus mutans is governed by the bacterial population density

Horizontal gene transfer through natural DNA transformation is an important evolutionary mechanism among bacteria. Transformation requires that the bacteria are physiologically competent to take and incorporate free DNA directly from the environment. Although natural genetic transformation is a remarkable feature of many naturally competent bacteria, the process is energetically expensive for the cells. Consequently, a tight control of the competence state is necessary. The objective of the present work was to help decipher the molecular mechanisms regulating the escape from the competence state in Streptococcus mutans, the principal etiological agent responsible for tooth decay in humans. Our results showed that the cessation of competence in S. mutans was abrupt, and did not involve the accumulation of a competence inhibitor nor the depletion of a competence activator in the extracellular environment. The competence state was repressed at high cell population density via concomitant repression of sigX gene encoding the master regulator of the competence regulon. Co‐culture experiments performed with oral and non‐oral bacteria showed that S. mutans assesses its own population density and also the microbial density of its surroundings to regulate its competence escape. Interestingly, neither the intra‐species and extra‐species quorum‐sensing systems nor the other 13 two‐component regulatory systems identified in S. mutans were involved in the cell‐density‐dependent escape of the competence state. Altogether, our results suggest a complex mechanism regulating the competence shut‐off involving cell‐density‐dependent repression of sigX through an as yet undefined system, and possibly SigX protein stability.     

Molecular pathogenicity of Streptococcus anginosus

Streptococcus anginosus and the closely related species Streptococcus constellatus and Streptococcus intermedius, are primarily commensals of the mucosa. The true pathogenic potential of this group has been under‐recognized for a long time because of difficulties in correct species identification as well as the commensal nature of these species. In recent years, streptococci of the S. anginosus group have been increasingly found as relevant microbial pathogens in abscesses and blood cultures and they play a pathogenic role in cystic fibrosis. Several international studies have shown a surprisingly high frequency of infections caused by the S. anginosus group. Recent studies and a genome‐wide comparative analysis suggested the presence of multiple putative virulence factors that are well‐known from other streptococcal species. However, very little is known about the molecular basis of pathogenicity in these bacteria. This review summarizes our current knowledge of pathogenicity factors and their regulation in S. anginosus.     

Activation of inflammasomes in dendritic cells and macrophages by Mycoplasma salivarium

Interleukin‐1β (IL‐1β) plays crucial roles in the pathogenesis of periodontal disease. It is produced after the processing of pro‐IL‐1β by caspase‐1, which is activated by the inflammasome‐a multiprotein complex comprising nucleotide‐binding domain leucine‐rich repeat‐containing receptor (NLR), the adaptor protein apoptosis‐associated speck‐like protein containing a caspase‐recruitment domain (ASC), and procaspase‐1. Mycoplasma salivarium preferentially inhabits the gingival sulcus and the incidence and number of organisms in the oral cavity increase significantly with the progression of periodontal disease. To initially clarify the association of this organism with periodontal diseases, this study determined whether it induces IL‐1β production by innate immune cells such as dendritic cells or macrophages by using Mycoplasma pneumoniae as a positive control. Both live and heat‐killed M. salivarium and M. pneumoniae cells induced IL‐1β production by XS106 murine dendritic cells as well as pyroptosis. The activities were significantly downregulated by silencing of caspase‐1. Bone‐marrow‐derived macrophage (BMMs) from wild‐type and NLR‐containing protein 3 (NLRP3)‐, ASC‐, and caspase‐1‐deficient mice were examined for IL‐1β production in response to these mycoplasmas. Live M. salivarium and M. pneumoniae cells almost completely lost the ability to induce IL‐1β production by BMMs from ASC‐ and caspase‐1–deficient mice. Their activities toward BMMs from NLRP3‐deficient mice were significantly but not completely attenuated. These results suggest that live M. salivarium and M. pneumoniae cells can activate several types of inflammasomes including the NLRP3 inflammasome. Both M. salivarium and M. pneumoniae cells can activate THP‐1 human monocytic cells to induce IL‐1β production. Hence, the present finding that M. salivarium induces IL‐1β production by dendritic cells and macrophages may suggest the association of this organism with periodontal diseases.     

The GroEL protein of Porphyromonas gingivalis accelerates tumor growth by enhancing endothelial progenitor cell function and neovascularization

Porphyromonas gingivalis is a bacterial species that causes destruction of periodontal tissues. Additionally, previous evidence indicates that GroEL from P. gingivalis may possess biological activities involved in systemic inflammation, especially inflammation involved in the progression of periodontal diseases. The literature has established a relationship between periodontal disease and cancer. However, it is unclear whether P. gingivalis GroEL enhances tumor growth. Here, we investigated the effects of P. gingivalis GroEL on neovasculogenesis in C26 carcinoma cell‐carrying BALB/c mice and chick eggs in vivo as well as its effect on human endothelial progenitor cells (EPC) in vitro. We found that GroEL treatment accelerated tumor growth (tumor volume and weight) and increased the mortality rate in C26 cell‐carrying BALB/c mice. GroEL promoted neovasculogenesis in chicken embryonic allantois and increased the circulating EPC level in BALB/c mice. Furthermore, GroEL effectively stimulated EPC migration and tube formation and increased E‐selectin expression, which is mediated by eNOS production and p38 mitogen‐activated protein kinase activation. Additionally, GroEL may enhance resistance against paclitaxel‐induced cell cytotoxicity and senescence in EPC. In conclusion, P. gingivalis GroEL may act as a potent virulence factor, contributing to the neovasculogenesis of tumor cells and resulting in accelerated tumor growth.     

Whole genome sequence and phenotypic characterization of a Cbm+ serotype e strain of Streptococcus mutans

We report the whole genome sequence of the serotype e Cbm⁺ strain LAR01 of Streptococcus mutans, a dental pathogen frequently associated with extra‐oral infections. The LAR01 genome is a single circular chromosome of 2.1 Mb with a GC content of 36.96%. The genome contains 15 phosphotransferase system gene clusters, seven cell wall‐anchored (LPxTG) proteins, all genes required for the development of natural competence and genes coding for mutacins VI and K8. Interestingly, the cbm gene is genetically linked to a putative type VII secretion system that has been found in Mycobacteria and few other Gram‐positive bacteria. When compared with the UA159 type strain, phenotypic characterization of LAR01 revealed increased biofilm formation in the presence of either glucose or sucrose but similar abilities to withstand acid and oxidative stresses. LAR01 was unable to inhibit the growth of Strpetococcus gordonii, which is consistent with the genomic data that indicate absence of mutacins that can kill mitis streptococci. On the other hand, LAR01 effectively inhibited growth of other S. mutans strains, suggesting that it may be specialized to outcompete strains from its own species. In vitro and in vivo studies using mutational and heterologous expression approaches revealed that Cbm is a virulence factor of S. mutans by mediating binding to extracellular matrix proteins and intracellular invasion. Collectively, the whole genome sequence analysis and phenotypic characterization of LAR01 provides new insights on the virulence properties of S. mutans and grants further opportunities to understand the genomic fluidity of this important human pathogen.     

中性粒细胞细胞外陷阱网与牙周炎的相关性研究进展

中性粒细胞是机体防御系统的成员之一,在免疫反应中发挥着重要作用。中性粒细胞细胞外陷阱网(NET)是新近发现的中性粒细胞发挥作用的一种形式,除了能够诱获并杀灭病原体而发挥作用之外,还可能对机体产生直接或者间接的损伤。NET不仅出现在牙周组织中,还与牙周致病菌息息相关。本文对NET与牙周炎的相关性研究进行综述。     

Historical aspects of studies on roles of the inflammasome in the pathogenesis of periodontal diseases

The proinflammatory cytokine interleukin‐1β (IL‐1β) is produced as inactive proIL‐1β and then processed by caspase‐1 to become active. In 2002, it was demonstrated that the intracellular multiprotein complex known as the inflammasome functions as a molecular platform to trigger activation of caspase‐1. Inflammasomes are known to function as intracellular sensors for a broad spectrum of various pathogen‐associated and damage‐associated molecular patterns. In 1985, it was demonstrated that Porphyromonas gingivalis, a representative bacterium causing chronic periodontitis, induces IL‐1 production by murine peritoneal macrophages. Since then, many studies have suggested that IL‐1, particularly IL‐1β plays key roles in the pathogenesis of periodontal diseases. However, the term “inflammasome” was not used until the involvement of inflammasomes in periodontal disease was suggested in 2009. Several subsequent studies on the roles of the inflammasome in the pathogenesis of periodontal diseases have been published. Interestingly, two contradictory reports on the modulation of inflammasomes by P. gingivalis have been published. Some papers have described how P. gingivalis activates the inflammasome to produce IL‐1β whereas some stated that P. gingivalis inhibits inflammasome activation to subvert immune responses. Several lines of evidence have suggested that the inflammasome activation is modulated by periodontopathic bacteria other than P. gingivalis. Hence, studies on the roles of inflammasomes in the pathogenesis of periodontal diseases began only 8 years ago and many pathological roles of inflammasomes remain to be clarified.