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.     

Porphyromonas gingivalis gingipain is involved in the detachment and aggregation of Aggregatibacter actinomycetemcomitans biofilm

Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans are major periodontal pathogens that cause several types of periodontal disease. Our previous study suggested that P. gingivalis gingipains secreted in the subgingival environment are related to the detachment of A.actinomycetemcomitans biofilms. However, it remains unclear whether arginine‐specific cysteine proteinase (Rgp) and lysine‐specific proteinase (Kgp) play different roles in the detachment of A. actinomycetemcomitans biofilm. The aim of this study was to investigate possible disruptive roles of Kgp and Rgp in the aggregation and attachment of A. actinomycetemcomitans. While P. gingivalis ATCC33277 culture supernatant has an ability to decrease autoaggregation and coaggregation of A. actinomycetemcomitans cells, neither the boiled culture supernatant of ATCC33277 nor the culture supernatant of KDP136 showed this ability. The addition of KYT‐1 and KYT‐36, specific inhibitors of Rgp and Kgp, respectively, showed no influence on the ability of P. gingivalis culture supernatant. The result of gelatin zymography suggested that other proteases processed by gingipains mediated the decrease of A. actinomycetemcomitans aggregations. We also examined the biofilm‐destructive effect of gingipains by assessing the detachment of A. actinomycetemcomitans from polystyrene surfaces. Scanning electron microscope analysis indicated that A. actinomycetemcomitans cells were detached by P. gingivalis Kgp. The quantity of A. actinomycetemcomitans in biofilm was decreased in co‐culture with P. gingivalis. However, this was not found after the addition of KYT‐36. These findings suggest that Kgp is a critical component for the detachment and decrease of A. actinomycetemcomitans biofilms.     

Potential role for Streptococcus gordonii‐derived hydrogen peroxide in heme acquisition by Porphyromonas gingivalis

Streptococcus gordonii, an accessory pathogen and early colonizer of plaque, co‐aggregates with many oral species including Porphyromonas gingivalis. It causes α‐hemolysis on blood agar, a process mediated by H₂O₂ and thought to involve concomitant oxidation of hemoglobin (Hb). Porphyromonas gingivalis has a growth requirement for heme, which is acquired mainly from Hb. The paradigm for Hb heme acquisition involves the initial oxidation of oxyhemoglobin (oxyHb) to methemoglobin (metHb), followed by heme release and extraction through the actions of K‐gingipain protease and/or the HmuY hemophore‐like protein. The ability of S. gordonii to mediate Hb oxidation may potentially aid heme capture during co‐aggregation with P. gingivalis. Hemoglobin derived from zones of S. gordonii α‐hemolysis was found to be metHb. Generation of metHb from oxyHb by S. gordonii cells was inhibited by catalase, and correlated with levels of cellular H₂O₂ production. Generation of metHb by S. gordonii occurred through the higher Hb oxidation state of ferrylhemoglobin. Heme complexation by the P. gingivalis HmuY was employed as a measure of the ease of heme capture from metHb. HmuY was able to extract iron(III)protoporphyrin IX from metHb derived from zones of S. gordonii α‐hemolysis and from metHb generated by the action of S. gordonii cells on isolated oxyHb. The rate of HmuY‐Fe(III)heme complex formation from S. gordonii‐mediated metHb was greater than from an equivalent concentration of auto‐oxidized metHb. It is concluded that S. gordonii may potentially aid heme acquisition by P. gingivalis by facilitating metHb formation in the presence of oxyHb.     

Effects of N‐acyl homoserine lactone analogues on Porphyromonas gingivalis biofilm formation

Asahi Y, Noiri Y, Igarashi J, Asai H, Suga H, Ebisu S. Effects of N‐acyl homoserine lactone analogues on Porphyromonas gingivalis biofilm formation. J Periodont Res 2009; doi: 10.1111/j.1600‐0765.2009.01228.x © 2009 John Wiley & Sons A/S Background and Objective: The gram‐negative anaerobic rod Porphyromonas gingivalis in oral biofilms is a primary etiological agent of periodontal disease. Biofilm formation of various gram‐negative bacteria is regulated by a quorum‐sensing circuit that relies on N‐acyl homoserine lactones (HSLs). Some synthetic N‐acyl HSL analogues act as quorum‐sensing inhibitors and suppress biofilm formation in Pseudomonas aeruginosa. Development of chemical control agents against oral biofilms is necessary, because until now, biofilms have been removed only by mechanical debridement. The present study investigated the effect of N‐acyl HSL analogues on P. gingivalis biofilm formation, with the aim of developing new drugs that inhibit oral biofilm formation. Material and Methods: A flow‐cell model was used for P. gingivalis biofilm formation. Seventeen synthetic N‐acyl HSL analogues were quantitatively assessed by spectrophotometry. The effects of three antagonistic compounds against P. gingivalis biofilm formation were further examined by confocal laser scanning microscopy, and investigated for primary attachment using spectrophotometry and phase contrast microscopy. Results: Ten out of 17 analogues affected P. gingivalis biofilm formation. Three out of 10 analogues significantly decreased biofilm‐forming cells (p < 0.05), and these biofilm structures were less well formed three‐dimensionally. There were no quantitative or qualitative differences in cell attachment between the control and the three analogue‐treated groups. Conclusion: Three synthetic N‐acyl HSL analogues inhibited biofilm formation in P. gingivalis. We suggest that these analogues influence the development stage of P. gingivalis biofilm formation.     

Disruption of heterotypic community development by Porphyromonas gingivalis with small molecule inhibitors

Porphyromonas gingivalis is one of the main etiological organisms in periodontal disease. On oral surfaces P. gingivalis is a component of multispecies biofilm communities and can modify the pathogenic potential of the community as a whole. Accumulation of P. gingivalis in communities is facilitated by interspecies binding and communication with the antecedent colonizer Streptococcus gordonii. In this study we screened a library of small molecules to identify structures that could serve as lead compounds for the development of inhibitors of P. gingivalis community development. Three small molecules were identified that effectively inhibited accumulation of P. gingivalis on a substratum of S. gordonii. The structures of the small molecules are derived from the marine alkaloids oroidin and bromoageliferin and contain a 2‐aminoimidazole or 2‐aminobenzimidazole moiety. The most active compounds reduced expression of mfa1 and fimA in P. gingivalis, genes encoding the minor and major fimbrial subunits, respectively. These fimbrial adhesins are necessary for P. gingivalis co‐adhesion with S. gordonii. These results demonstrate the potential for a small molecular inhibitor‐based approach to the prevention of diseases associated with P. gingivalis.     

Treponema denticola improves adhesive capacities of Porphyromonas gingivalis

Porphyromonas gingivalis, an important etiological agent of periodontal disease, is frequently found associated with Treponema denticola, an anaerobic spirochete, in pathogenic biofilms. However, interactions between these two bacteria are not well understood at the molecular level. In this study, we seek to link the influence of T. denticola on the expression of P. gingivalis proteases with its capacities to adhere and to form biofilms. The P. gingivalis genes encoding Arg‐gingipain A (RgpA), Lys‐gingipain (Kgp), and hemagglutinin A (HagA) were more strongly expressed after incubation with T. denticola compared with P. gingivalis alone. The amounts of the three resulting proteins, all of which contain hemagglutinin adhesion domains, were increased in culture supernatants. Moreover, incubation of P. gingivalis with T. denticola promoted static and dynamic biofilm formation, primarily via a time‐dependent enhancement of P. gingivalis adhesion capacities on bacterial partners such as Streptococcus gordonii. Adhesion of P. gingivalis to human cells was also increased. These results showed that interactions of P. gingivalis with other bacterial species, such as T. denticola, induce increased adhesive capacities on various substrata by hemagglutinin adhesion domain‐containing proteins.     

Behavior of two Tannerella forsythia strains and their cell surface mutants in multispecies oral biofilms

As a member of subgingival multispecies biofilms, Tannerella forsythia is commonly associated with periodontitis. The bacterium has a characteristic cell surface (S‐) layer modified with a unique O‐glycan. Both the S‐layer and the O‐glycan were analyzed in this study for their role in biofilm formation by employing an in vitro multispecies biofilm model mimicking the situation in the oral cavity. Different T. forsythia strains and mutants with characterized defects in cell surface composition were incorporated into the model, together with nine species of select oral bacteria. The influence of the T. forsythia S‐layer and attached glycan on the bacterial composition of the biofilms was analyzed quantitatively using colony‐forming unit counts and quantitative real‐time polymerase chain reaction, as well as qualitatively by fluorescence in situ hybridization and confocal laser scanning microscopy. This revealed that changes in the T. forsythia cell surface did not affect the quantitative composition of the multispecies consortium, with the exception of Campylobacter rectus cell numbers. The localization of T. forsythia within the bacterial agglomeration varied depending on changes in the S‐layer glycan, and this also affected its aggregation with Porphyromonas gingivalis. This suggests a selective role for the glycosylated T. forsythia S‐layer in the positioning of this species within the biofilm, its co‐localization with P. gingivalis, and the prevalence of C. rectus. These findings might translate into a potential role of T. forsythia cell surface structures in the virulence of this species when interacting with host tissues and the immune system, from within or beyond the biofilm.     

Bifidobacteria inhibit the growth of Porphyromonas gingivalis but not of Streptococcus mutans in an in vitro biofilm model

There is growing interest in the use of probiotic bifidobacteria for enhancement of the therapy, and in the prevention, of oral microbial diseases. However, the results of clinical studies assessing the effects of bifidobacteria on the oral microbiota are controversial, and the mechanisms of actions of probiotics in the oral cavity remain largely unknown. In addition, very little is known about the role of commensal bifidobacteria in oral health. Our aim was to study the integration of the probiotic Bifidobacterium animalis subsp. lactis Bb12 and of oral Bifidobacterium dentium and Bifidobacterium longum isolates in supragingival and subgingival biofilm models and their effects on other bacteria in biofilms in vitro using two different in vitro biofilms and agar‐overlay assays. All bifidobacteria integrated well into the subgingival biofilms composed of Porphyromonas gingivalis, Actinomyces naeslundii, and Fusobacterium nucleatum and decreased significantly only the number of P. gingivalis in the biofilms. The integration of bifidobacteria into the supragingival biofilms containing Streptococcus mutans and A. naeslundii was less efficient, and bifidobacteria did not affect the number of S. mutans in biofilms. Therefore, our results suggest that bifidobacteria may have a positive effect on subgingival biofilm and thereby potential in enhancing gingival health; however, their effect on supragingival biofilm may be limited.     

A bacterial‐biofilm‐induced oral osteolytic infection can be successfully treated by immuno‐targeting an extracellular nucleoid‐associated protein

Periodontal disease exemplifies a chronic and recurrent infection with a necessary biofilm component. Mucosal inflammation is a hallmark response of the host seen in chronic diseases, such as colitis, gingivitis, and periodontitis (and the related disorder peri‐implantitis). We have taken advantage of our recently developed rat model of human peri‐implantitis that recapitulates osteolysis, the requirement of biofilm formation, and the perpetuation of the bona fide disease state, to test a new therapeutic modality with two novel components. First we used hyperimmune antiserum directed against the DNABII family of proteins, now known to be a critical component of the extracellular matrix of bacterial biofilms. Second we delivered the antiserum as cargo in biodegradable microspheres to the site of the biofilm infection. We demonstrated that delivery of a single dose of anti‐DNABII in poly(lactic‐co‐glycolic acid) (PLGA) microspheres induced significant resolution of experimental peri‐implantitis, including marked reduction of inflammation. These data support the continued development of a DNABII protein‐targeted therapeutic for peri‐implantitis and other chronic inflammatory pathologies of the oral cavity in animals and humans.     

Biofilm formation by Porphyromonas gingivalis and Streptococcus gordonii

Confocal scanning laser microscopy (CSLM) was used to visualize and quantify biofilm formation by the oral bacteria Streptococcus gordonii and Porphyromonas gingivalis. A saliva-coated glass coverslip under continuous bacterial challenge and conditions of low shear force was used to investigate attachment to the salivary pellicle and also the effect of cell-cell interactions on the extent of colonization and biofilm development. S. gordonii bound to the salivary pellicle and outcompeted P. gingivalis for attachment sites. Both P. gingivalis and S. gordonii failed to establish substantial biofilm formation independently. However, biofilm formation did occur subsequent to initial adherence of P. gingivalis to S. gordonii cells deposited on the salivary pellicle. The commensal species S. gordonii may, therefore, provide an attachment substrate for colonization and biofilm accretion by the potential pathogen, P. gingivalis.     

Extracellular matrix influence in Streptococcus mutans gene expression in a cariogenic biofilm

Caries etiology is biofilm–diet‐dependent. Biofilms are highly dynamic and structured microbial communities enmeshed in a three‐dimensional extracellular matrix. The study evaluated the expression dynamics of Streptococcus mutans genes associated with exopolysaccharides (EPS) (gtfBCD, gbpB, dexA), lipoteichoic acids (LTA) (dltABCD, SMU_775c) and extracellular DNA (eDNA) (lytST, lrgAB, ccpA) during matrix development within a mixed‐species biofilm of S. mutans, Actinomyces naeslundii and Streptococcus gordonii. Mixed‐species biofilms using S. mutans strains UA159 or ΔgtfB formed on saliva‐coated hydroxyapatite discs were submitted to a nutritional challenge (providing an abundance of sucrose and starch). Biofilms were removed at eight developmental stages for gene expression analysis by quantitative polymerase chain reaction. The pH of spent culture media remained acidic throughout the experimental periods, being lower after sucrose and starch exposure. All genes were expressed at all biofilm developmental phases. EPS‐ and LTA‐associated genes had a similar expression profile for both biofilms, presenting lower levels of expression at 67, 91 and 115 hours and a peak of expression at 55 hours, but having distinct expression magnitudes, with lower values for ΔgtfB (eg, fold‐difference of ~382 for gtfC and ~16 for dltB at 43 hours). The eDNA‐associated genes presented different dynamics of expression between both strains. In UA159 biofilms lrgA and lrgB genes were highly expressed at 29 hours (which were ~13 and ~5.4 times vs ΔgtfB, respectively), whereas in ΔgtfB biofilms an inverse relationship between lytS and lrgA and lrgB expression was detected. Therefore, the deletion of gtfB influences dynamics and magnitude of expression of genes associated with matrix main components.     

Candida–streptococcal mucosal biofilms display distinct structural and virulence characteristics depending on growth conditions and hyphal morphotypes

Candida albicans and streptococci of the mitis group form communities in multiple oral sites, where moisture and nutrient availability can change spatially or temporally. This study evaluated structural and virulence characteristics of Candida–streptococcal biofilms formed on moist or semidry mucosal surfaces, and tested the effects of nutrient availability and hyphal morphotype on dual‐species biofilms. Three‐dimensional models of the oral mucosa formed by immortalized keratinocytes on a fibroblast‐embedded collagenous matrix were used. Infections were carried out using Streptococcus oralis strain 34, in combination with a C. albicans wild‐type strain, or pseudohyphal‐forming mutant strains. Increased moisture promoted a homogeneous surface biofilm by C. albicans. Dual biofilms had a stratified structure, with streptococci growing in close contact with the mucosa and fungi growing on the bacterial surface. Under semidry conditions, Candida formed localized foci of dense growth, which promoted focal growth of streptococci in mixed biofilms. Candida biofilm biovolume was greater under moist conditions, albeit with minimal tissue invasion, compared with semidry conditions. Supplementing the infection medium with nutrients under semidry conditions intensified growth, biofilm biovolume and tissue invasion/damage, without changing biofilm structure. Under these conditions, the pseudohyphal mutants and S. oralis formed defective superficial biofilms, with most bacteria in contact with the epithelial surface, below a pseudohyphal mass, resembling biofilms growing in a moist environment. The presence of S. oralis promoted fungal invasion and tissue damage under all conditions. We conclude that moisture, nutrient availability, hyphal morphotype and the presence of commensal bacteria influence the architecture and virulence characteristics of mucosal fungal biofilms.     

A YadA‐like autotransporter,Hag1 in Veillonella atypica is a multivalent hemagglutinin involved in adherence to oral streptococci,Porphyromonas gingivalis,and human oral buccal cells

Dental biofilm development is a sequential process, and adherence between microbes and the salivary pellicle (adhesion) as well as among different microbes (co‐adhesion or coaggregation) plays a critical role in building a biofilm community. The Veillonella species are among the most predominant species in the oral cavity and coaggregate with many initial, early, middle, and late colonizers. Similar to oral fusobacteria, they are also considered bridging species in biofilm development. However, the mechanism of this ability has yet to be reported, due to the previous lack of a genetic transformation system in the entire genus. In this study, we used our recently discovered transformable Veillonella strain, Veillonella atypica OK5, to probe the mechanism of coaggregation between Veillonella species and other oral bacteria. By insertional inactivation of all eight putative hemagglutinin genes, we identified one gene, hag1, which is involved in V. atypica coaggregation with the initial colonizers Streptococcus gordonii, Streptococcus oralis and Streptococcus cristatus, and the periodontal pathogen Porphyromonas gingivalis. The hag1 mutant also abolished adherence to human buccal cells. Inhibition assays using various chemical or physiological treatments suggest different mechanisms being involved in coaggregation with different partners. The entire hag1 gene was sequenced and shown to be the largest known bacterial hemagglutinin gene.     

Resveratrol attenuates the pathogenic and inflammatory properties of Porphyromonas gingivalis

Porphyromonas gingivalis has been strongly associated with chronic periodontitis, which affects tooth‐supporting tissues. This Gram‐negative anaerobic bacterium produces a repertoire of virulence factors that modulate tissue destruction directly or indirectly by the induction of inflammatory processes. The aim of this study was to investigate the effects of resveratrol, a major polyphenol found in grapes and wine, on the growth and virulence properties of P. gingivalis as well as on gingival keratinocyte tight junction integrity and the host inflammatory response. Resveratrol exhibited antibacterial activity that may result from damage to the bacterial cell membrane. Resveratrol also killed a pre‐formed P. gingivalis biofilm and reduced bacterial adherence to matrix proteins. In addition, resveratrol had a protective effect on the integrity of the keratinocyte tight junctions by inhibiting its breakdown by P. gingivalis. This may be related to the ability of resveratrol to inhibit the protease activities of P. gingivalis. Lastly, resveratrol reduced P. gingivalis‐mediated activation of the NF‐κB signaling pathway and attenuated TREM‐1 gene expression as well as soluble TREM‐1 secretion in monocytes. The effect on NF‐κB activation likely results from the ability of resveratrol to act as a PPAR‐γ agonist. In summary, the antibacterial, anti‐adherence, and antiprotease properties of resveratrol, as well as its ability to protect the gingival keratinocyte barrier and attenuate the inflammatory response in monocytes suggest that it may be a promising novel therapeutic agent for treating periodontal disease.     

Gingipain‐dependent degradation of mammalian target of rapamycin pathway proteins by the periodontal pathogen Porphyromonas gingivalis during invasion

Porphyromonas gingivalis and Tannerella forsythia are gram‐negative pathogens strongly associated with periodontitis. Their abilities to interact, invade and persist within host cells are considered crucial to their pathogenicity, but the mechanisms by which they subvert host defences are not well understood. In this study, we set out to investigate whether P. gingivalis and T. forsythia directly target key signalling molecules that may modulate the host cell phenotype to favour invasion and persistence. Our data identify, for the first time, that P. gingivalis, but not T. forsythia, reduces levels of intracellular mammalian target of rapamycin (mTOR) in oral epithelial cells following invasion over a 4‐h time course, via the action of gingipains. The ability of cytochalasin D to abrogate P. gingivalis‐mediated mTOR degradation suggests that this effect is dependent upon cellular invasion. We also show that levels of several other proteins in the mTOR signalling pathway are modulated by gingipains, either directly or as a consequence of mTOR degradation including p‐4E‐BP1. Taken together, our data suggest that P. gingivalis manipulates the mTOR pathway, providing evidence for a potentially novel mechanism by which P. gingivalis mediates its effects on host cell responses to infection.     

The role of Porphyromonas gingivalis gingipains in platelet activation and innate immune modulation

Platelets are considered to have important functions in inflammatory processes and as actors in the innate immunity. Several studies have shown associations between cardiovascular disease and periodontitis, where the oral anaerobic pathogen Porphyromonas gingivalis has a prominent role in modulating the immune response. Porphyromonas gingivalis has been found in atherosclerotic plaques, indicating spreading of the pathogen via the circulation, with an ability to interact with and activate platelets via e.g. Toll‐like receptors (TLR) and protease‐activated receptors. We aimed to evaluate how the cysteine proteases, gingipains, of P. gingivalis affect platelets in terms of activation and chemokine secretion, and to further investigate the mechanisms of platelet–bacteria interaction. This study shows that primary features of platelet activation, i.e. changes in intracellular free calcium and aggregation, are affected by P. gingivalis and that arg‐gingipains are of great importance for the ability of the bacterium to activate platelets. The P. gingivalis induced a release of the chemokine RANTES, however, to a much lower extent compared with the TLR2/1‐agonist Pam₃CSK₄, which evoked a time‐dependent release of the chemokine. Interestingly, the TLR2/1‐evoked response was abolished by a following addition of viable P. gingivalis wild‐types and gingipain mutants, showing that both Rgp and Kgp cleave the secreted chemokine. We also demonstrate that Pam₃CSK₄‐stimulated platelets release migration inhibitory factor and plasminogen activator inhibitor‐1, and that also these responses were antagonized by P. gingivalis. These results supports immune‐modulatory activities of P. gingivalis and further clarify platelets as active players in innate immunity and in sensing bacterial infections, and as target cells in inflammatory reactions induced by P. gingivalis infection.     

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.     

Effects of short‐chain fatty acids on Actinomyces naeslundii biofilm formation

Actinomyces naeslundii is an early colonizer and has important roles in the development of the oral biofilm. Short‐chain fatty acids (SCFA) are secreted extracellularly as a product of metabolism by gram‐negative anaerobes, e.g. Porphyromonas gingivalis and Fusobacterium nucleatum; and the SCFA may affect biofilm development with interaction between A. naeslundii and gram‐negative bacteria. Our aim was to investigate the effects of SCFA on biofilm formation by A. naeslundii and to determine the mechanism. We used the biofilm formation assay in 96‐well microtiter plates in tryptic soy broth without dextrose and with 0.25% sucrose using safranin stain of the biofilm monitoring 492 nm absorbance. To determine the mechanism by SCFA, the production of chaperones and stress‐response proteins (GrpE and GroEL) in biofilm formation was examined using Western blot fluorescence activity with GrpE and GroEL antibodies. Adding butyric acid (6.25 mm ) 0, 6 and 10 h after beginning culture significantly increased biofilm formation by A. naeslundii, and upregulation was observed at 16 h. Upregulation was also observed using appropriate concentrations of other SCFA. In the upregulated biofilm, production of GrpE and GroEL was higher where membrane‐damaged or dead cells were also observed. The upregulated biofilm was significantly reduced by addition of anti‐GroEL antibody. The data suggest biofilm formation by A. naeslundii was upregulated dependent on the production of stress proteins, and addition of SCFA increased membrane‐damaged or dead cells. Production of GroEL may physically play an important role in biofilm development.     

Liver X receptors contribute to periodontal pathogen‐elicited inflammation and oral bone loss

Periodontal diseases are chronic oral inflammatory diseases that are polymicrobial in nature. The presence of specific bacteria in subgingival plaque such as Porphyromonas gingivalis is associated with microbial dysbiosis and the modulation of host immune response. Bacterially elicited innate immune activation and inflammation are key elements implicated in the destruction of soft and hard tissues supporting the teeth. Liver X receptors (LXRs) are nuclear hormone receptors with important function in lipid homeostasis, inflammation, and host response to infection; however, their contribution to chronic inflammatory diseases such as periodontal disease is not understood. The aim of this study was to define the contribution of LXRs in the development of immune response to P. gingivalis and to assess the roles that LXRs play in infection‐elicited oral bone loss. Employing macrophages, we observed that P. gingivalis challenge led to reduced LXRα and LXRβ gene expression compared with that observed with unchallenged wild‐type cells. Myeloid differentiation primary response gene 88 (MyD88)‐independent, Toll/interleukin‐1 receptor‐domain‐containing adapter‐inducing interferon‐β (TRIF)‐dependent signaling affected P. gingivalis‐mediated reduction in LXRα expression, whereas neither pathway influenced the P. gingivalis effect on LXRβ expression. Employing LXR agonist and mice deficient in LXRs, we observed functional effects of LXRs in the development of a P. gingivalis‐elicited cytokine response at the level of the macrophage, and participation of LXRs in P. gingivalis‐elicited oral bone loss. These findings identify novel importance for LXRs in the pathogenesis of P. gingivalis infection‐elicited inflammation and oral bone loss.