Curcumin inhibits growth of Porphyromonas gingivalis by arrest of bacterial dipeptidyl peptidase activity.

Background: Curcumin is a multi-functional polyphenol with anti-bacterial and anti-inflammatory effects and may have potential for treatment of periodontal diseases. The present study was conducted to examine the molecular basis of the anti-bacterial effect of curcumin against Porphyromonas gingivalis using metabolome analysis. Materials and Methods: P. gingivalis were incubated with 10 µg/mL curcumin, and then metabolites were analyzed with CE-TOF/MS. Expression levels of sigma factors were also evaluated using RT-PCR assays. The activities of dipeptidyl peptidases (DPPs) were assessed by examining the degradation reactions of MCA-labeled peptides. Results: The relative amounts of various glycogenic amino acids were significantly decreased when P. gingivalis was incubated with curcumin. Furthermore, the metabolites on the amino acid degradation pathway, including high-energy compounds such as ATP, various intermediate metabolites of RNA/DNA synthesis, nucleoside sugars and amino sugars were also decreased. Additionally, the expression levels of sigma-54 and sigma-70 were significantly decreased, and the same results as noted following nutrient starvation. Curcumin also significantly suppressed the activities of some DPPs, while the human DPP-4 inhibitors markedly inhibited the growth of P. gingivalis and activities of the DPPs. Conclusions: Curcumin suppresses the growth of P. gingivalis by inhibiting DPPs and also interferes with nucleic acid synthesis and central metabolic pathways, beginning with amino acid metabolism.

The polymicrobial pathogenicity of Porphyromonas gingivalis.

Accumulating microbiome data and mechanistic studies in vitro and in vivo have refined our understanding of the oral microbiota as a functionally integrated polymicrobial community. Emergent properties of these communities are driven to a large extent by interspecies communication which can be based on physical association, secreted small molecules or nutritional exchange. Porphyromonas gingivalis is a consensus periodontal pathogen; however, virulence is only expressed in the context of a polymicrobial community. Multivalent fimbriae mediate attachment to other oral species which can initiate a distinct transcriptional program in both constituents of the binding pair. P. gingivalis also responds to small molecules and nutritional cues produced by partner organisms. Physiological interdependence forms the basis of complex networks of cooperating organisms which begin to resemble an organismal entity exhibiting a spectrum of pathogenic potential.

Interspecies metabolite transfer fuels the methionine metabolism of Fusobacterium nucleatum to stimulate volatile methyl mercaptan production.

The major oral odor compound methyl mercaptan (CH3SH) is strongly associated with halitosis and periodontitis. CH3SH production stems from the metabolism of polymicrobial communities in periodontal pockets and on the tongue dorsum. However, understanding of CH3SH-producing oral bacteria and their interactions is limited. This study aimed to investigate CH3SH production by major oral bacteria and the impact of interspecies interactions on its generation. Using a newly constructed large-volume anaerobic noncontact coculture system, Fusobacterium nucleatum was found to be a potent producer of CH3SH, with that production stimulated by metabolic interactions with Streptococcus gordonii, an early dental plaque colonizer. Furthermore, analysis of extracellular amino acids using an S. gordonii arginine-ornithine antiporter (ArcD) mutant demonstrated that ornithine excreted from S. gordonii is a key contributor to increased CH3SH production by F. nucleatum. Further study with 13C, 15N-methionine, as well as gene expression analysis, revealed that ornithine secreted by S. gordonii increased the demand for methionine through accelerated polyamine synthesis by F. nucleatum, leading to elevated methionine pathway activity and CH3SH production. Collectively, these findings suggest that interaction between S. gordonii and F. nucleatum plays a key role in CH3SH production, providing a new insight into the mechanism of CH3SH generation in oral microbial communities. A better understanding of the underlying interactions among oral bacteria involved in CH3SH generation can lead to the development of more appropriate prophylactic approaches to treat halitosis and periodontitis. An intervention approach like selectively disrupting this interspecies network could also offer a powerful therapeutic strategy.IMPORTANCEHalitosis can have a significant impact on the social life of affected individuals. Among oral odor compounds, CH3SH has a low olfactory threshold and halitosis is a result of its production. Recently, there has been a growing interest in the collective properties of oral polymicrobial communities, regarded as important for the development of oral diseases, which are shaped by physical and metabolic interactions among community participants. However, it has yet to be investigated whether interspecies interactions have an impact on the production of volatile compounds, leading to the development of halitosis. The present findings provide mechanistic insights indicating that ornithine, a metabolite excreted by Streptococcus gordonii, promotes polyamine synthesis by Fusobacterium nucleatum, resulting in a compensatory increase in demand for methionine, which results in elevated methionine pathway activity and CH3SH production. Elucidation of the mechanisms related to CH3SH production is expected to lead to the development of new strategies for managing halitosis.

Cigarette smoke extract impairs gingival epithelial barrier function.

We previously showed that junctional adhesion molecule 1 (JAM1) and coxsackievirus and adenovirus receptor (CXADR), tight junction-associated proteins, have important roles to maintain epithelial barrier function in gingival tissues. Smoking is considered to be a significant risk factor for periodontal disease. The present study was conducted to examine the effects of cigarette smoke extract (CSE) on JAM1 and CXADR in human gingival epithelial cells. CSE was found to cause translocation of JAM1 from the cellular surface to EGFR-positive endosomes, whereas CXADR did not. Using a three-dimensional multilayered gingival epithelial tissue model, CSE administration was found to increase permeability to lipopolysaccharide and peptidoglycan, whereas overexpression of JAM1 in the tissue model prevented penetration by those substrates. Furthermore, vitamin C increased JAM1 expression, and inhibited penetration of LPS and PGN induced by CSE. These findings strongly suggest that CSE disrupts gingival barrier function via dislocation of JAM1, thus allowing bacterial virulence factors to penetrate into subepithelial tissues. Furthermore, they indicate that vitamin C increases JAM1 expression and prevents disruption of gingival barrier function by CSE.

Surface pre-reacted glass-ionomer eluate protects gingival epithelium from penetration by lipopolysaccharides and peptidoglycans via transcription factor EB pathway.

Surface pre-reacted glass-ionomer (S-PRG) filler, produced by PRG technology for use with various dental materials, is bioactive and known to release ions from a glass-ionomer phase. We previously reported that coxsackievirus and adenovirus receptor (CXADR), a tight junction associated protein, was located in the epithelial barrier of gingival epithelium. In the present study, the tissue protective effects of an S-PRG eluate prepared with S-PRG filler were investigated using a three-dimensional human gingival epithelial tissue model. The results showed that the S-PRG eluate specifically induced CXADR expression at the transcriptional level of messenger RNA as well as the protein level, and also nuclear translocation of transcription factor EB (TFEB) in gingival epithelial cells. Furthermore, shigyakusan, a TFEB inhibitor, canceled induction of the CXADR protein by the S-PRG eluate. Additionally, gingival epithelial permeation by 40-kDa dextran, lipopolysaccharide, and peptidoglycan in the 3D-tissue models was prevented by the eluate, with those effects abrogated by knockdown of CXADR. These findings suggest that S-PRG eluate increases CXADR expression via the TFEB pathway, thus inhibiting penetration of bacterial virulence factors into subepithelial tissues.

Fusobacterium nucleatum Metabolically Integrates Commensals and Pathogens in Oral Biofilms.

Fusobacterium nucleatum is a common constituent of the oral microbiota in both periodontal health and disease. Previously, we discovered ornithine cross-feeding between F. nucleatum and Streptococcus gordonii, where S. gordonii secretes ornithine via an arginine-ornithine antiporter (ArcD), which in turn supports the growth and biofilm development of F. nucleatum; however, broader metabolic aspects of F. nucleatum within polymicrobial communities and their impact on periodontal pathogenesis have not been addressed. Here, we show that when cocultured with S. gordonii, F. nucleatum increased amino acid availability to enhance the production of butyrate and putrescine, a polyamine produced by ornithine decarboxylation. Coculture with Veillonella parvula, another common inhabitant of the oral microbiota, also increased lysine availability, promoting cadaverine production by F. nucleatum. We confirmed that ArcD-dependent S. gordonii-excreted ornithine induces synergistic putrescine production, and mass spectrometry imaging revealed that this metabolic capability creates a putrescine-rich microenvironment on the surface of F. nucleatum biofilms. We further demonstrated that polyamines caused significant changes in the biofilm phenotype of a periodontal pathogen, Porphyromonas gingivalis, with putrescine accelerating the biofilm life cycle of maturation and dispersal. This phenomenon was also observed with putrescine derived from S. gordonii-F. nucleatum coculture. Lastly, analysis of plaque samples revealed cooccurrence of P. gingivalis with genetic modules for putrescine production by S. gordonii and F. nucleatum. Overall, our results highlight the ability of F. nucleatum to induce synergistic polyamine production within multispecies consortia and provide insight into how the trophic web in oral biofilm ecosystems can eventually shape disease-associated communities. IMPORTANCE Periodontitis is caused by a pathogenic shift in subgingival biofilm ecosystems, which is accompanied by alterations in microbiome composition and function, including changes in the metabolic activity of the biofilm, which comprises multiple commensals and pathogens. While Fusobacterium nucleatum is a common constituent of the supra- and subgingival biofilms, its metabolic integration within polymicrobial communities and the impact on periodontal pathogenesis are poorly understood. Here, we report that amino acids supplied by other commensal bacteria induce polyamine production by F. nucleatum, creating polyamine-rich microenvironments. Polyamines reportedly have diverse functions in bacterial physiology and possible involvement in periodontal pathogenesis. We show that the F. nucleatum-integrated trophic network yielding putrescine from arginine through ornithine accelerates the biofilm life cycle of Porphyromonas gingivalis, a periodontal pathogen, from the planktonic state through biofilm formation to dispersal. This work provides insight into how cooperative metabolism within oral biofilms can tip the balance toward periodontitis.

Profiling volatile compounds from culture supernatants of periodontal bacteria using gas chromatography/mass spectrometry/olfactometry analysis with a monolithic silica gel adsorption device.

Halitosis is formed mainly by the volatile compounds produced by periodontal bacteria. Three volatile sulfur compounds (VSCs), hydrogen sulfide, methanethiol, and dimethyl sulfide, have attracted attention as major components of halitosis. However, these compounds cannot account for all odors. In this study, we profiled volatile compounds from the culture supernatants of periodontal bacteria using gas chromatography/mass spectrometry/olfactometry analysis with a monolithic silica gel adsorption device to investigate the potential odorous compounds. Periodontal bacteria have been found to produce volatile compounds belonging to various classes, such as alcohols, ketones, fatty acids, and aromatic compounds, in addition to VSCs. In addition, VSCs different from hydrogen sulfide and methanethiol, which are considered important causative compounds, may also influence to halitosis.

Salivary metabolic signatures of carotid atherosclerosis in patients with type 2 diabetes hospitalized for treatment.

Atherosclerosis is a life-threatening disease associated with morbidity and mortality in patients with type 2 diabetes (T2D). This study aimed to characterize a salivary signature of atherosclerosis based on evaluation of carotid intima-media thickness (IMT) to develop a non-invasive predictive tool for diagnosis and disease follow-up. Metabolites in saliva and plasma samples collected at admission and after treatment from 25 T2D patients hospitalized for 2 weeks to undergo medical treatment for diabetes were comprehensively profiled using metabolomic profiling with gas chromatography-mass spectrometry. Orthogonal partial least squares analysis, used to explore the relationships of IMT with clinical markers and plasma and salivary metabolites, showed that the top predictors for IMT included salivary allantoin and 1,5-anhydroglucitol (1,5-AG) at both the baseline examination at admission and after treatment. Furthermore, though treatment induced alterations in salivary levels of allantoin and 1,5-AG, it did not modify the association between IMT and these metabolites (p interaction > 0.05), and models with these metabolites combined yielded satisfactory diagnostic accuracy for the high IMT group even after treatment (area under curve = 0.819). Collectively, this salivary metabolite combination may be useful for non-invasive identification of T2D patients with a higher atherosclerotic burden in clinical settings.

Potential of Prebiotic D-Tagatose for Prevention of Oral Disease.

Recent studies have shown phenotypic and metabolic heterogeneity in related species including Streptococcus oralis, a typical oral commensal bacterium, Streptococcus mutans, a cariogenic bacterium, and Streptococcus gordonii, which functions as an accessory pathogen in periodontopathic biofilm. In this study, metabolites characteristically contained in the saliva of individuals with good oral hygiene were determined, after which the effects of an identified prebiotic candidate, D-tagatose, on phenotype, gene expression, and metabolic profiles of those three key bacterial species were investigated. Examinations of the saliva metabolome of 18 systemically healthy volunteers identified salivary D-tagatose as associated with lower dental biofilm abundance in the oral cavity (Spearman's correlation coefficient; r = -0.603, p = 0.008), then the effects of D-tagatose on oral streptococci were analyzed in vitro. In chemically defined medium (CDM) containing D-tagatose as the sole carbohydrate source, S. mutans and S. gordonii each showed negligible biofilm formation, whereas significant biofilms were formed in cultures of S. oralis. Furthermore, even in the presence of glucose, S. mutans and S. gordonii showed growth suppression and decreases in the final viable cell count in a D-tagatose concentration-dependent manner. In contrast, no inhibitory effects of D-tagatose on the growth of S. oralis were observed. To investigate species-specific inhibition by D-tagatose, the metabolomic profiles of D-tagatose-treated S. mutans, S. gordonii, and S. oralis cells were examined. The intracellular amounts of pyruvate-derived amino acids in S. mutans and S. gordonii, but not in S. oralis, such as branched-chain amino acids and alanine, tended to decrease in the presence of D-tagatose. This phenomenon indicates that D-tagatose inhibits growth of those bacteria by affecting glycolysis and its downstream metabolism. In conclusion, the present study provides evidence that D-tagatose is abundant in saliva of individuals with good oral health. Additionally, experimental results demonstrated that D-tagatose selectively inhibits growth of the oral pathogens S. mutans and S. gordonii. In contrast, the oral commensal S. oralis seemed to be negligibly affected, thus highlighting the potential of administration of D-tagatose as an oral prebiotic for its ability to manipulate the metabolism of those targeted oral streptococci.

Saliva and Plasma Reflect Metabolism Altered by Diabetes and Periodontitis.

Periodontitis is an inflammatory disorder caused by disintegration of the balance between the periodontal microbiome and host response. While growing evidence suggests links between periodontitis and various metabolic disorders including type 2 diabetes (T2D), non-alcoholic liver disease, and cardiovascular disease (CVD), which often coexist in individuals with abdominal obesity, factors linking periodontal inflammation to common metabolic alterations remain to be fully elucidated. More detailed characterization of metabolomic profiles associated with multiple oral and cardiometabolic traits may provide better understanding of the complexity of oral-systemic crosstalk and its underlying mechanism. We performed comprehensive profiling of plasma and salivary metabolomes using untargeted gas chromatography/mass spectrometry to investigate multivariate covariation with clinical markers of oral and systemic health in 31 T2D patients with metabolic comorbidities and 30 control subjects. Orthogonal partial least squares (OPLS) results enabled more accurate characterization of associations among 11 oral and 25 systemic clinical outcomes, and 143 salivary and 78 plasma metabolites. In particular, metabolites that reflect cardiometabolic changes were identified in both plasma and saliva, with plasma and salivary ratios of (mannose + allose):1,5-anhydroglucitol achieving areas under the curve of 0.99 and 0.92, respectively, for T2D diagnosis. Additionally, OPLS analysis of periodontal inflamed surface area (PISA) as the numerical response variable revealed shared and unique responses of metabolomic and clinical markers to PISA between healthy and T2D groups. When combined with linear regression models, we found a significant correlation between PISA and multiple metabolites in both groups, including threonate, cadaverine and hydrocinnamate in saliva, as well as lactate and pentadecanoic acid in plasma, of which plasma lactate showed a predominant trend in the healthy group. Unique metabolites associated with PISA in the T2D group included plasma phosphate and salivary malate, while those in the healthy group included plasma gluconate and salivary adenosine. Remarkably, higher PISA was correlated with altered hepatic lipid metabolism in both groups, including higher levels of triglycerides, aspartate aminotransferase and alanine aminotransferase, leading to increased risk of cardiometabolic disease based on a score summarizing levels of CVD-related biomarkers. These findings revealed the potential utility of saliva for evaluating the risk of metabolic disorders without need for a blood test, and provide evidence that disrupted liver lipid metabolism may underlie the link between periodontitis and cardiometabolic disease.

Porphyromonas gingivalis induces penetration of lipopolysaccharide and peptidoglycan through the gingival epithelium via degradation of coxsackievirus and adenovirus receptor.

Porphyromonas gingivalis is a major pathogen of human periodontitis and dysregulates innate immunity at the gingival epithelial surface. We previously reported that the bacterium specifically degrades junctional adhesion molecule 1 (JAM1), causing gingival epithelial barrier breakdown. However, the functions of other JAM family protein(s) in epithelial barrier dysregulation caused by P. gingivalis are not fully understood. The present results show that gingipains, Arg-specific or Lys-specific cysteine proteases produced by P. gingivalis, specifically degrade coxsackievirus and adenovirus receptor (CXADR), a JAM family protein, at R145 and K235 in gingival epithelial cells. In contrast, a gingipain-deficient P. gingivalis strain was found to be impaired in regard to degradation of CXADR. Furthermore, knockdown of CXADR in artificial gingival epithelium increased permeability to dextran 40 kDa, lipopolysaccharide and peptidoglycan, whereas overexpression of CXADR in a gingival epithelial tissue model prevented penetration by those agents following P. gingivalis infection. Together, these results suggest that P. gingivalis gingipains breach the stratified squamous epithelium barrier by degrading CXADR as well as JAM1, which allows for efficient transfer of bacterial virulence factors into subepithelial tissues. TAKEAWAYS: P. gingivalis, a periodontal pathogen, degraded coxsackievirus and adenovirus receptor (CXADR), a JAM family protein, in gingival epithelial tissues. P. gingivalis gingipains, cysteine proteases, degraded CXADR at R145 and K235. CXADR degradation by P. gingivalis caused increased permeability to lipopolysaccharide and peptidoglycan through gingival epithelial tissues.

Porphyromonas gingivalis induces penetration of lipopolysaccharide and peptidoglycan through the gingival epithelium via degradation of junctional adhesion molecule 1.

Porphyromonas gingivalis is a major pathogen in severe and chronic manifestations of periodontal disease, which is one of the most common infections of humans. A central feature of P. gingivalis pathogenicity is dysregulation of innate immunity at the gingival epithelial interface; however, the molecular basis underlying P. gingivalis-dependent abrogation of epithelial barrier function remains unknown. Gingival epithelial cells express junctional adhesion molecule (JAM1), a tight junction-associated protein, and JAM1 homodimers regulate epithelial barrier function. Here we show that Arg-specific or Lys-specific cysteine proteases (gingipains) secreted by P. gingivalis can specifically degrade JAM1 at K134 and R234 in gingival epithelial cells, resulting in permeability of the gingival epithelium to 40 kDa dextran, lipopolysaccharide (LPS), and proteoglycan (PGN). A P. gingivalis strain lacking gingipains was impaired in degradation of JAM1. Knockdown of JAM1 in monolayer cells and a three-dimensional multilayered tissue model also increased permeability to LPS, PGN, and gingipains. Inversely, overexpression of JAM1 in epithelial cells prevented penetration by these agents following P. gingivalis infection. Our findings strongly suggest that P. gingivalis gingipains disrupt barrier function of stratified squamous epithelium via degradation of JAM1, allowing bacterial virulence factors to penetrate into subepithelial tissues.

Metabolic crosstalk regulates Porphyromonas gingivalis colonization and virulence during oral polymicrobial infection.

Many human infections are polymicrobial in origin, and interactions among community inhabitants shape colonization patterns and pathogenic potential 1 . Periodontitis, which is the sixth most prevalent infectious disease worldwide 2 , ensues from the action of dysbiotic polymicrobial communities 3 . The keystone pathogen Porphyromonas gingivalis and the accessory pathogen Streptococcus gordonii interact to form communities in vitro and exhibit increased fitness in vivo 3,4 . The mechanistic basis of this polymicrobial synergy, however, has not been fully elucidated. Here we show that streptococcal 4-aminobenzoate/para-amino benzoic acid (pABA) is required for maximal accumulation of P. gingivalis in dual-species communities. Metabolomic and proteomic data showed that exogenous pABA is used for folate biosynthesis, and leads to decreased stress and elevated expression of fimbrial adhesins. Moreover, pABA increased the colonization and survival of P. gingivalis in a murine oral infection model. However, pABA also caused a reduction in virulence in vivo and suppressed extracellular polysaccharide production by P. gingivalis. Collectively, these data reveal a multidimensional aspect to P. gingivalis-S. gordonii interactions and establish pABA as a critical cue produced by a partner species that enhances the fitness of P. gingivalis while diminishing its virulence.

Distinct signatures of dental plaque metabolic byproducts dictated by periodontal inflammatory status.

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.

Dual lifestyle of Porphyromonas gingivalis in biofilm and gingival cells.

Porphyromonas gingivalis is deeply involved in the pathogenesis of marginal periodontitis, and recent findings have consolidated its role as an important and unique pathogen. This bacterium has a unique dual lifestyle in periodontal sites including subgingival dental plaque (biofilm) and gingival cells, as it has been clearly shown that P. gingivalis is able to exert virulence using completely different tactics in each environment. Inter-bacterial cross-feeding enhances the virulence of periodontal microflora, and such metabolic and adhesive interplay creates a supportive environment for P. gingivalis and other species. Human oral epithelial cells harbor a large intracellular bacterial load, resembling the polymicrobial nature of periodontal biofilm. P. gingivalis can enter gingival epithelial cells and pass through the epithelial barrier into deeper tissues. Subsequently, from its intracellular position, the pathogen exploits cellular recycling pathways to exit invaded cells, by which it is able to control its population in infected tissues, allowing for persistent infection in gingival tissues. Here, we outline the dual lifestyle of P. gingivalis in subgingival areas and its effects on the pathogenesis of periodontitis.

Intracellular periodontal pathogen exploits recycling pathway to exit from infected cells.

Although human gingival epithelium prevents intrusions by periodontal bacteria, Porphyromonas gingivalis, the most well-known periodontal pathogen, is able to invade gingival epithelial cells and pass through the epithelial barrier into deeper tissues. We previously reported that intracellular P. gingivalis exits from gingival epithelial cells via a recycling pathway. However, the underlying molecular process remains unknown. In the present study, we found that the pathogen localized in early endosomes recruits VAMP2 and Rab4A. VAMP2 was found to be specifically localized in early endosomes, although its localization remained unclear in mammalian cells. A single transmembrane domain of VAMP2 was found to be necessary and sufficient for localizing in early endosomes containing P. gingivalis in gingival epithelial cells. VAMP2 forms a complex with EXOC2/Sec5 and EXOC3/Sec6, whereas Rab4A mediates dissociation of the EXOC complex followed by recruitment of RUFY1/Rabip4, Rab4A effector, and Rab14. Depletion of VAMP2 or Rab4A resulted in accumulation of bacteria in early endosomes and disturbed bacterial exit from infected cells. It is suggested that these novel dynamics allow P. gingivalis to exploit fast recycling pathways promoting further bacterial penetration of gingival tissues.

Antibacterial Activity of Curcumin Against Periodontopathic Bacteria.

BACKGROUND: Curcumin is a polyphenol extracted from root of turmeric and known to possess multifunctional properties, including antibacterial activity. Although previous studies have investigated the effects of curcumin on microorganisms, available knowledge on the effects of curcumin on periodontopathic bacteria is still limited. In this study, the antibacterial effect of curcumin on periodontopathic bacteria is investigated, particularly Porphyromonas gingivalis. METHODS: Representative periodontopathic bacteria were cultured in media with and without various curcumin concentrations, and the optical density at 600 nm was measured for 60 hours. The inhibitory effect of curcumin on P. gingivalis Arg- and Lys-specific proteinase (RGP and KGP, respectively) activities were assessed using spectrofluorophotometric assay. Analysis of biofilm formation by P. gingivalis with or without Streptococcus gordonii was conducted using confocal laser-scanning microscopy (CLSM). RESULTS: Curcumin inhibited the growth of P. gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Treponema denticola in a dose-dependent manner. Bacterial growth was suppressed almost completely at very low concentrations of curcumin. Conversely, 100 µg/mL curcumin did not suppress the growth of Aggregatibacter actinomycetemcomitans. It also demonstrated inhibitory effects against RGP and KGP activities in a dose-dependent manner. CLSM revealed that curcumin suppressed P. gingivalis homotypic and P. gingivalis-S. gordonii heterotypic biofilm formation in a dose-dependent manner. A concentration of 20 µg/mL curcumin inhibited these P. gingivalis biofilm formations by >80%. CONCLUSION: Curcumin possesses antibacterial activity against periodontopathic bacteria and may be a potent agent for preventing periodontal diseases.

Arginine-Ornithine Antiporter ArcD Controls Arginine Metabolism and Interspecies Biofilm Development of Streptococcus gordonii.

Arginine is utilized by the oral inhabitant Streptococcus gordonii as a substrate of the arginine deiminase system (ADS), eventually producing ATP and NH3, the latter of which is responsible for microbial resistance to pH stress. S. gordonii expresses a putative arginine-ornithine antiporter (ArcD) whose function has not been investigated despite relevance to the ADS and potential influence on inter-bacterial communication with periodontal pathogens that utilize amino acids as a main energy source. Here, we generated an S. gordonii ΔarcD mutant to explore the role of ArcD in physiological homeostasis and bacterial cross-feeding. First, we confirmed that S. gordonii ArcD plays crucial roles for mediating arginine uptake and promoting bacterial growth, particularly under arginine-limited conditions. Next, metabolomic profiling and transcriptional analysis of the ΔarcD mutant revealed that deletion of this gene caused intracellular accumulation of ornithine leading to malfunction of the ADS and suppression of de novo arginine biosynthesis. The mutant strain also showed increased susceptibility to low pH stress due to reduced production of ammonia. Finally, accumulation of Fusobacterium nucleatum was found to be significantly decreased in biofilm formed by the ΔarcD mutant as compared with the wild-type strain, although ornithine supplementation restored fusobacterium biovolume in dual-species biofilms with the ΔarcD mutant and also enhanced single species biofilm development by F. nucleatum. Our results are the first direct evidence showing that S. gordonii ArcD modulates not only alkali and energy production but also interspecies interaction with F. nucleatum, thus initiating a middle stage of periodontopathic biofilm formation, by metabolic cross-feeding.

Porphyromonas gingivalis promotes invasion of oral squamous cell carcinoma through induction of proMMP9 and its activation.

Recent epidemiological studies have revealed a significant association between periodontitis and oral squamous cell carcinoma (OSCC). Furthermore, matrix metalloproteinase 9 (MMP9) is implicated in the invasion and metastasis of tumour cells. We examined the involvement of Porphyromonas gingivalis, a periodontal pathogen, in OSCC invasion through induced expression of proMMP and its activation. proMMP9 was continuously secreted from carcinoma SAS cells, while P. gingivalis infection increased proenzyme expression and subsequently processed it to active MMP9 in culture supernatant, which enhanced cellular invasion. In contrast, Fusobacterium nucleatum, another periodontal organism, failed to demonstrate such activities. The effects of P. gingivalis were observed with highly invasive cells, but not with the low invasivetype. P. gingivalis also stimulated proteinase-activated receptor 2 (PAR2) and enhanced proMMP9 expression, which promoted cellular invasion. P. gingivalis mutants deficient in gingipain proteases failed to activate MMP9. Infected SAS cells exhibited activation of ERK1/2, p38, and NF-kB, and their inhibitors diminished both proMMP9-overexpression and cellular invasion. Together, our results show that P. gingivalis activates the ERK1/2-Ets1, p38/HSP27, and PAR2/NF-kB pathways to induce proMMP9 expression, after which the proenzyme is activated by gingipains to promote cellular invasion of OSCC cell lines. These findings suggest a novel mechanism of progression and metastasis of OSCC associated with periodontitis.