RESUMO
OBJECTIVES: Biofilm formation on dental implant surfaces can cause peri-implant mucositis and peri-implantitis. Lectins are involved in interactions between bacteria or between bacteria and their hosts. Disrupting these interactions via specific sugars can result in reduced adhesion and biofilm formation. The purpose of this study was to identify sugars that function as antiadhesion or antibiofilm agents on titanium discs. METHODS: Of the sugars tested, the sugars that did not affect the planktonic growth of Streptococcus oralis, Fusobacterium nucleatum, and Porphyromonas gingivalis were selected. The selected sugars were assessed for their ability to inhibit biofilm formation of bacteria in single and consortium species by crystal violet staining, confocal laser scanning microscopy after live/dead staining, and scanning electron microscopy. The sugars were evaluated for their ability to inhibit activity of the quorum sensing molecule autoinducer 2 (AI-2) by bioluminescence assay. RESULTS: Biofilm formation of single bacteria or consortia of S. oralis, F. nucleatum, and P. gingivalis on titanium discs was significantly inhibited in the presence of d-arabinose. Pretreating titanium discs with d-arabinose for 3 min inhibited biofilm formation at a level comparable to that observed when d-arabinose was present over the entire period, suggesting that d-arabinose had initial anti-adhesive activity. In addition, d-arabinose inhibited the activity of AI-2. CONCLUSIONS: d-Arabinose may be a good candidate for application as an antibiofilm agent and AI-2 inhibitor.
Assuntos
Peri-Implantite , Titânio , Arabinose/farmacologia , Biofilmes , Fusobacterium nucleatum , Humanos , Porphyromonas gingivalis , Titânio/farmacologiaRESUMO
Integrins are cell-surface heterodimeric glycoproteins composed of alpha and beta subunits that mediate cell-cell, cell-extracellular matrix, and cell-pathogen interactions. In this study, we report a specific role of integrin α5ß1 in NLRP3 inflammasome activation in macrophages stimulated by Td92, a surface protein of the periodontopathogen, Treponema denticola. The direct interaction of Td92 with the cell membrane integrin α5ß1 resulted in ATP release and K(+) efflux, which are the main events in NLRP3 activation. This interaction was arginine-glycine-aspartate (RGD)-independent, and Td92 internalization was not required for the activity. An integrin α5ß1 antibody and oxATP, an ATP receptor antagonist, inhibited NLRP3 expression, caspase-1 activation, interleukin-1ß (IL-1ß) secretion, and proIL-1ß synthesis, all of which were regulated by NF-κB activation. Therefore, our data has identified the integrin α5ß1 as a principal cell membrane receptor for both NLRP3 inflammasome activation and IL-1ß transcription by a bacterial protein, which could exaggerate inflammation, a characteristic of periodontitis.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte/metabolismo , Inflamassomos/metabolismo , Integrina alfa5beta1/metabolismo , Trifosfato de Adenosina/metabolismo , Caspase 1/metabolismo , Morte Celular , Linhagem Celular , Humanos , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Potássio/metabolismo , Receptores Purinérgicos P2/metabolismo , Treponema denticola/metabolismo , Regulação para CimaRESUMO
Extracellular fibronectin (Fn) can activate pro-inflammatory pathways and serves as an endogenous danger signalling molecule; thus, it has been suggested as a biomarker for several diseases. In the present study, we found that pathogen-derived activators of the inflammasomes induce the expression and secretion of Fn in macrophages through a mechanism involving adenosine triphosphate and caspase-1 activation. We also found that plasma Fn induces caspase-1 activation and cell death in macrophages, epithelial cells, and fibroblasts. Together, these results indicate that Fn plays a critical role in inflammasome-activated cells by amplifying caspase-1 activation and inducing inflammatory cell death.
Assuntos
Fibronectinas/metabolismo , Inflamassomos/fisiologia , Macrófagos/metabolismo , Ativação Transcricional/imunologia , Trifosfato de Adenosina/fisiologia , Caspase 1/metabolismo , Morte Celular , Linhagem Celular , Ativação Enzimática , Fibronectinas/genética , Flagelina/imunologia , Humanos , Lipopolissacarídeos/farmacologia , Macrófagos/imunologia , Receptores Purinérgicos P2/metabolismo , Salmonella typhimurium/imunologia , Transdução de SinaisRESUMO
This study aimed to verify, in in vivo settings, whether quorum-sensing inhibition molecules could attenuate alveolar bone loss induced by Porphyromonas gingivalis/Fusobacterium nucleatum co-infection and reduce the bacterial colonization of periodontal tissues. In BALB/c mice, periodontitis was induced through oral inoculation with P. gingivalis and F. nucleatum six times during a 42-d period. Quorum sensing inhibitors (a furanone compound and D-ribose) were administered simultaneously with bacterial infection. Linear and volumetric modifications of interproximal alveolar bone levels were compared between groups using micro-computed tomography. Total bacteria, and P. gingivalis and F. nucleatum DNA in periodontal tissues, were quantified using real-time PCR. Radiographic linear measurements demonstrated a significant reduction of alveolar bone loss, of approximately 40%, in mice treated with quorum sensing inhibitors when compared with the co-infection group. This was confirmed by a significant increase of residual bone volume in the test group. While total bacterial genes in the treatment group significantly decreased by 93% in periodontal tissue samples when quorum sensing inhibitors were administered, no significant differences of P. gingivalis DNA were found. Quorum sensing inhibitors reduced periodontal breakdown and bacterial infection in periodontal tissues after co-infection with P. gingivalis and F. nucleatum.
Assuntos
Coinfecção , Periodontite , Percepção de Quorum/efeitos dos fármacos , Perda do Osso Alveolar , Animais , DNA Bacteriano/análise , Modelos Animais de Doenças , Furanos/administração & dosagem , Furanos/antagonistas & inibidores , Fusobacterium nucleatum/genética , Fusobacterium nucleatum/patogenicidade , Expressão Gênica , Genes Bacterianos , Interações Hospedeiro-Patógeno , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Periodontite/diagnóstico por imagem , Periodontite/microbiologia , Periodontite/patologia , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/patogenicidade , Ribose/administração & dosagem , Ribose/antagonistas & inibidores , Microtomografia por Raio-XRESUMO
Synaptotagmin-1 (Syt1) is a major Ca(2+) sensor for synchronous neurotransmitter release, which requires vesicle fusion mediated by SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). Syt1 utilizes its diverse interactions with target membrane (t-) SNARE, SNAREpin, and phospholipids, to regulate vesicle fusion. To dissect the functions of Syt1, we apply a single-molecule technique, alternating-laser excitation (ALEX), which is capable of sorting out subpopulations of fusion intermediates and measuring their kinetics in solution. The results show that Syt1 undergoes at least three distinct steps prior to lipid mixing. First, without Ca(2+), Syt1 mediates vesicle docking by directly binding to t-SNARE/phosphatidylinositol 4,5-biphosphate (PIP(2)) complex and increases the docking rate by 10(3) times. Second, synaptobrevin-2 binding to t-SNARE displaces Syt1 from SNAREpin. Third, with Ca(2+), Syt1 rebinds to SNAREpin, which again requires PIP(2). Thus without Ca(2+), Syt1 may bring vesicles to the plasma membrane in proximity via binding to t-SNARE/PIP(2) to help SNAREpin formation and then, upon Ca(2+) influx, it may rebind to SNAREpin, which may trigger synchronous fusion. The results show that ALEX is a powerful method to dissect multiple kinetic steps in the vesicle fusion pathway.
Assuntos
Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas SNARE/metabolismo , Vesículas Sinápticas/metabolismo , Sinaptotagmina I/metabolismo , Bioensaio , Cálcio/metabolismo , Cinética , Soluções , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
Porphyromonas gingivalis utilizes its major proteases, Arg gingipains (RgpA and RgpB) and Lys gingipain (Kgp), for dysregulation of host immune systems. The aim of this study was to investigate the roles of gingipains in caspase-1 activation and its sequelae in P. gingivalis-infected macrophages. Infection with P. gingivalis at low multiplicity of infections (MOIs), but not at high MOIs, resulted in low levels of interleukin-1ß and lactate dehydrogenase without detectable active caspase-1 in the culture supernatants. The proteins released from caspase-1-activated cells were rapidly degraded by gingipains. However, P. gingivalis with gingipains induced higher intracellular caspase-1 activity in the infected cells than the gingipain-null mutant, which was associated with ATP release from the infected cells. In addition, growing the gingipain-null mutant with gingipains enhanced caspase-1 activation by the mutant. In contrast, inhibition of the protease activity of Kgp or Rgps increased the caspase-1-activating potential of wild-type P. gingivalis, indicating an inhibitory effect of the collaborative action of Kgp and Rgps. These results illuminate the contradictory roles of gingipains in the manipulation of host defence systems by P. gingivalis, as they act by both stimulating and inhibiting innate immune responses.
Assuntos
Adesinas Bacterianas/metabolismo , Caspase 1/metabolismo , Cisteína Endopeptidases/metabolismo , Macrófagos/microbiologia , Porphyromonas gingivalis/enzimologia , Porphyromonas gingivalis/metabolismo , Células Cultivadas , Ativação Enzimática , Cisteína Endopeptidases Gingipaínas , Interações Hospedeiro-Patógeno , Humanos , Macrófagos/imunologiaRESUMO
Parkinson disease and dementia with Lewy bodies are featured with the formation of Lewy bodies composed mostly of α-synuclein (α-Syn) in the brain. Although evidence indicates that the large oligomeric or protofibril forms of α-Syn are neurotoxic agents, the detailed mechanisms of the toxic functions of the oligomers remain unclear. Here, we show that large α-Syn oligomers efficiently inhibit neuronal SNARE-mediated vesicle lipid mixing. Large α-Syn oligomers preferentially bind to the N-terminal domain of a vesicular SNARE protein, synaptobrevin-2, which blocks SNARE-mediated lipid mixing by preventing SNARE complex formation. In sharp contrast, the α-Syn monomer has a negligible effect on lipid mixing even with a 30-fold excess compared with the case of large α-Syn oligomers. Thus, the results suggest that large α-Syn oligomers function as inhibitors of dopamine release, which thus provides a clue, at the molecular level, to their neurotoxicity.
Assuntos
Neurônios/fisiologia , Proteínas SNARE/fisiologia , alfa-Sinucleína/química , alfa-Sinucleína/fisiologia , Animais , Exocitose/efeitos dos fármacos , Exocitose/fisiologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Modelos Neurológicos , Neurônios/efeitos dos fármacos , Neurotoxinas/química , Neurotoxinas/toxicidade , Células PC12 , Ligação Proteica , Estrutura Quaternária de Proteína , Proteolipídeos/metabolismo , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/toxicidade , Vesículas Secretórias/efeitos dos fármacos , Vesículas Secretórias/fisiologia , Transdução Genética , Proteína 2 Associada à Membrana da Vesícula/fisiologia , alfa-Sinucleína/genética , alfa-Sinucleína/toxicidadeRESUMO
OBJECTIVES: This in vitro study was conducted: (i) to evaluate the effect of using cotton pellets soaked with chlorhexidine (CHX) on titanium surface roughness; (ii) to assess the removal of Porphyromonas gingivalis (P. gingivalis) from resorbable blast material (RBM) titanium surfaces using CHX pellets; and (iii) to evaluate the effects of additional brushing on bacterial removal efficiency. MATERIALS AND METHODS: RBM titanium discs were treated with CHX-soaked cotton pellets, and change in surface roughness was measured using confocal microscopy. After the titanium discs were incubated with P. gingivalis for 2 days, the discs were cleaned with CHX pellets for 40 s. The quantity of remaining adherent bacteria was measured using crystal violet assay. Additional brushing was performed with dentifrice for a total of 40 s, and bacterial removal efficiency with brushing and dentifrice was evaluated using crystal violet assay and scanning electron microscopy. RESULTS: The changes in surface roughness after treatment were observed by confocal microscopy. Statistically significant decrease in surface roughness was seen in CHX 40-s group (p < 0.05). Cleaning with CHX-soaked pellets resulted in significant decrease in remaining adherent bacteria. Brushing the bacteria-incubated discs with dentifrice reduced adhering bacteria. There were fewer bacteria left on the CHX-pre-treated discs compared with the brushing-only group, but there were no significant differences when compared with the brushing-only group (p > 0.05). CONCLUSIONS: This study clearly showed that burnishing with CHX influenced the RBM titanium surface, and burnishing with CHX pellets and brushing with dentifrice were efficient in removing bacteria from the contaminated titanium surface.
Assuntos
Bactérias/efeitos dos fármacos , Clorexidina/farmacologia , Dentifrícios/farmacologia , Titânio/química , Escovação Dentária , Bactérias/crescimento & desenvolvimento , Aderência Bacteriana/efeitos dos fármacos , Carga Bacteriana , Materiais Dentários/química , Violeta Genciana , Teste de Materiais , Microscopia Confocal , Microscopia Eletrônica de Varredura , Porphyromonas gingivalis/efeitos dos fármacos , Porphyromonas gingivalis/crescimento & desenvolvimento , Propriedades de Superfície , Fatores de TempoRESUMO
Alzheimer's disease (AD) and Parkinson's disease (PD) are caused by ß-amyloid (Aß) and α-synuclein (αS), respectively. Ample evidence suggests that these two pathogenic proteins are closely linked and have a synergistic effect on eliciting neurodegenerative disorders. However, the pathophysiological consequences of Aß and αS coexistence are still elusive. Here, we show that large-sized αS oligomers, which are normally difficult to form, are readily generated by Aß42-seeding and that these oligomers efficiently hamper neuronal SNARE-mediated vesicle fusion. The direct binding of the Aß-seeded αS oligomers to the N-terminal domain of synaptobrevin-2, a vesicular SNARE protein, is responsible for the inhibition of fusion. In contrast, large-sized Aß42 oligomers (or aggregates) or the products of αS incubated without Aß42 have no effect on vesicle fusion. These results are confirmed by examining PC12 cell exocytosis. Our results suggest that Aß and αS cooperate to escalate the production of toxic oligomers, whose main toxicity is the inhibition of vesicle fusion and consequently prompts synaptic dysfunction.
Assuntos
Peptídeos beta-Amiloides/fisiologia , Vesículas Citoplasmáticas/fisiologia , Fusão de Membrana , Proteínas SNARE/antagonistas & inibidores , alfa-Sinucleína/fisiologia , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Exocitose/genética , Humanos , Fusão de Membrana/genética , Células PC12 , Ligação Proteica/genética , Multimerização Proteica/fisiologia , Ratos , Proteínas SNARE/metabolismo , Sinapses/genética , Sinapses/metabolismo , Transfecção , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMO
Fluorescence-based single-vesicle fusion assays provide a powerful method for studying mechanisms underlying complex biological processes of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-mediated vesicle fusion and neurotransmitter release. A crucial element of these assays is the ability of the fluorescent probe(s) to reliably detect key intermediate events of fusion pore opening and content release/mixing. Here, we report a new, reliable, and efficient single-vesicle content-mixing assay using a high affinity, fluorophore tagged host-guest pair, cucurbit[7]uril-Cy3 and adamantane-Cy5 as a fluorescence resonance energy transfer (FRET) pair. The power of these probes is demonstrated by the first successful observation of flickering dynamics of the fusion pore by in vitro assay using neuronal SNARE-reconstituted vesicles.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/análise , Fusão de Membrana , Proteínas SNARE/metabolismo , Adamantano/análise , Adamantano/metabolismo , Animais , Hidrocarbonetos Aromáticos com Pontes/análise , Hidrocarbonetos Aromáticos com Pontes/metabolismo , Carbocianinas/análise , Carbocianinas/metabolismo , Corantes Fluorescentes/metabolismo , Humanos , Imidazóis/análise , Imidazóis/metabolismo , Neurônios/metabolismo , Proteínas SNARE/análiseRESUMO
Plasminogen activator inhibitor-1 (PAI-1) is associated with nonalcoholic fatty liver disease (NAFLD) by lipid accumulation in the liver. In this study, we showed that extracellular vesicles (EVs) from the periodontal pathogens Filifactor alocis and Porphyromonas gingivalis induced steatosis by inducing PAI-1 in the liver and serum of mice fed a low-fat diet. PAI-1 induction was not observed in TLR2-/- mice. When tested using HEK-Blue hTLR2 cells, human TLR2 reporter cells, the TLR2-activating ability of serum from NAFLD patients (n = 100) was significantly higher than that of serum from healthy subjects (n = 100). Correlation analysis confirmed that PAI-1 levels were positively correlated with the TLR2-activating ability of serum from NAFLD patients and healthy subjects. Amphiphilic molecules in EVs were involved in PAI-1 induction. Our data demonstrate that the TLR2/PAI-1 axis is important for hepatic steatosis by EVs of periodontal pathogens.
Assuntos
Vesículas Extracelulares , Hepatopatia Gordurosa não Alcoólica , Inibidor 1 de Ativador de Plasminogênio , Receptor 2 Toll-Like , Animais , Humanos , CamundongosRESUMO
PURPOSE: The dental implant surface will be colonized by bacteria once it is exposed to the oral cavity. It is necessary to keep the titanium surface clean to prevent peri-implant diseases. Mechanical instrumentation is widely used, but this may cause damage to the implant surfaces. There is limited information whether surface change resulting from instrumentation influences the adherence of bacteria to the implant surface or influences the ease of removal of bacteria from the titanium surface by daily brushing. Therefore, this in vitro study was performed (1) to evaluate removal of Porphyromonas gingivalis from sand-blasted and acid-etched (SLA) titanium discs after the discs were instrumented by various ultrasonic scaler tips or brushed with a toothbrush with dentifrice using crystal violet assay and scanning electron microscopy (SEM), and (2) to assess the change of surface roughness after the treated discs were brushed with a toothbrush with dentifrice. MATERIALS AND METHODS: SLA discs were treated with various ultrasonic scaler tips and a toothbrush. The titanium discs were incubated with P. gingivalis for 2 days after treatment (ultrasonic scales tips and brush) and then the disc surfaces were brushed for total of 40 seconds (20 seconds, two cycles) with a toothbrush with dentifrice. Differences in adhering bacteria were evaluated using crystal violet assay and SEM. Surface roughness of the treated discs after brushing with dentifrice was measured using confocal microscopy. RESULTS: The change of surface structure was observed after different treatment modalities. Removal of bacteria was increased with the longer time of brushing, and the ultrasonic metal tip group displayed a significantly lower number of bacteria after brushing when compared to other groups. CONCLUSIONS: Within the limits of this study, it may be suggested that when SLA surface is exposed to the oral cavity, it should firstly be treated with metal tips to smoothen the rough surface and thereby reduce attachment of bacteria and facilitate the removal of bacteria by daily oral hygiene procedures.
Assuntos
Implantes Dentários/microbiologia , Raspagem Dentária/instrumentação , Titânio , Escovação Dentária , Ultrassom , Dentifrícios , Teste de Materiais , Microscopia Eletrônica de Varredura , Porphyromonas gingivalis , Propriedades de SuperfícieRESUMO
Periodontitis is a chronic inflammatory disease caused by periodontal pathogens in subgingival plaque and is associated with systemic inflammatory diseases. Extracellular vesicles (EVs) released from host cells and pathogens carry a variety of biological molecules and are of interest for their role in disease progression and as diagnostic markers. In the present study, we analysed the proteome and inflammatory response of EVs derived from macrophages infected with Tannerella forsythia, a periodontal pathogen. The EVs isolated from the cell conditioned medium of T. forsythia-infected macrophages were divided into two distinct vesicles, macrophage-derived EVs and T. forsythia-derived OMVs, by size exclusion chromatography combined with density gradient ultracentrifugation. Proteome analysis showed that in T. forsythia infection, macrophage-derived EVs were enriched with pro-inflammatory cytokines and inflammatory mediators associated with periodontitis progression. T. forsythia-derived OMVs harboured several known virulence factors, including BspA, sialidase, GroEL and various bacterial lipoproteins. T. forsythia-derived OMVs induced pro-inflammatory responses via TLR2 activation. In addition, we demonstrated that T. forsythia actively released OMVs when T. forsythia encountered macrophage-derived soluble molecules. Taken together, our results provide insight into the characterisation of EVs derived from cells infected with a periodontal pathogen.
Assuntos
Vesículas Extracelulares , Periodontite , Humanos , Tannerella forsythia , Proteoma , Periodontite/microbiologia , Macrófagos , ImunidadeRESUMO
Outer membrane vesicles (OMVs) of bacteria harbor physiologically active molecules, and quorum sensing inhibitors (QSIs) are expected to regulate bacterial virulence. In this study, we analyzed the proinflammatory activity of OMVs of the periodontal pathogen Tannerella forsythia treated with d-arabinose and d-galactose as QSIs, which inhibit the biofilm formation of periodontal pathogens and autoinducer 2 activity. Compared to OMVs of nontreated T. forsythia (TF OMVs), OMVs released from QSI-treated T. forsythia, designated TF ara-OMVs and TF gal-OMVs, showed reduced production of TNF-α, IL-1ß, IL-6, and IL-8 in THP-1 monocytes through decreased activation of NF-κB/MAPKs. Using a human NF-κB reporter cell line and bone marrow-derived macrophages from TLR2-/- mice, TF ara-OMVs and TF gal-OMVs showed less activation of TLR2 than TF OMVs. These results demonstrated that QSIs provide a dual advantage against bacterial infection by inhibiting bacterial biofilm formation and generating OMVs with reduced proinflammatory activity.
Assuntos
NF-kappa B , Tannerella forsythia , Humanos , Animais , Camundongos , NF-kappa B/metabolismo , Receptor 2 Toll-Like/metabolismo , Percepção de Quorum , Macrófagos/metabolismoRESUMO
Outer membrane vesicles (OMVs) released from gram-negative bacteria harbor diverse molecules to communicate with host cells. In this study, we evaluated the OMVs of periodontal pathogens for their effects on the activation of dendritic cells and CD4+ T cell differentiation. OMVs of Porphyromonas gingivalis ATCC 33277, Treponema denticola ATCC 33521, and Tannerella forsythia ATCC 43037 ('red complex' pathogens) were isolated by density gradient ultracentrifugation. Mouse bone marrow-derived dendritic cells (BMDCs) were treated with OMVs, and OMV-primed BMDCs were cocultured with naïve CD4+ T cells to analyze the polarization of effector helper T cells. The OMVs upregulated maturation markers, including MHC class II, CD80, CD86, and CD40, on BMDCs. OMVs of P. gingivalis and T. forsythia induced the expression of the proinflammatory cytokines IL-1ß, IL-6, IL-23, and IL-12p70 in BMDCs. In T. denticola OMV-primed BMDCs, proinflammatory cytokines were poorly detected, which may be attributed to posttranslational degradation due to the highly proteolytic nature of OMVs. In cocultures of naïve CD4+ T cells with OMV-primed BMDCs, OMVs of P. gingivalis and T. denticola induced the differentiation of Th17 cells, whereas T. forsythia OMVs induced Th1 cell differentiation. These results demonstrate that OMVs derived from the 'red complex' periodontal pathogens induce maturation of BMDCs and differentiation of naïve CD4+ T cells to Th1 or Th17 cells.
RESUMO
Dysbiosis of the oral microbiota plays an important role in the progression of periodontitis, which is characterized by chronic inflammation and alveolar bone loss, and associated with systemic diseases. Bacterial extracellular vesicles (EVs) contain various bioactive molecules and show diverse effects on host environments depending on the bacterial species. Recently, we reported that EVs derived from Filifactor alocis, a Gram-positive periodontal pathogen, had osteoclastogenic activity. In the present study, we analysed the osteoclastogenic potency and immunostimulatory activity of EVs derived from the Gram-negative periodontal pathogens Porphyromonas gingivalis and Tannerella forsythia, the oral commensal bacterium Streptococcus oralis, and the gut probiotic strain Lactobacillus reuteri. Bacterial EVs were purified by density gradient ultracentrifugation using OptiPrep (iodixanol) reagent. EVs from P. gingivalis, T. forsythia, and S. oralis increased osteoclast differentiation and osteoclstogenic cytokine expression in osteoclast precursors, whereas EVs from L. reuteri did not. EVs from P. gingivalis, T. forsythia, and S. oralis preferentially activated Toll-like receptor 2 (TLR2) rather than TLR4 or TLR9, and induced osteoclastogenesis mainly through TLR2. The osteoclastogenic effects of EVs from P. gingivalis and T. forsythia were reduced by both lipoprotein lipase and polymyxin B, an inhibitor of lipopolysaccharide (LPS), while the osteoclastogenic effects of EVs from S. oralis were reduced by lipoprotein lipase alone. These results demonstrate that EVs from periodontal pathogens and oral commensal have osteoclastogenic activity through TLR2 activation by lipoproteins and/or LPS.
Assuntos
Vesículas Extracelulares , Boca , Osteoclastos , Diferenciação Celular , Vesículas Extracelulares/metabolismo , Lipopolissacarídeos , Lipase Lipoproteica , Microbiota , Boca/microbiologia , Osteoclastos/metabolismo , Porphyromonas gingivalis/fisiologia , Receptor 2 Toll-Like , Receptor 4 Toll-LikeRESUMO
Surface molecules of pathogens play an important role in stimulating host immune responses. Elucidation of the signaling pathways activated by critical surface molecules in host cells provides insight into the molecular pathogenesis resulting from bacteria-host interactions. MspTL is the most abundant outer membrane protein of Treponema lecithinolyticum, which is associated with periodontitis, and induces expression of a variety of proinflammatory factors. Although bacteria and bacterial components like LPS and flagellin are known to induce IFN-beta, induction by bacterial surface proteins has not been reported. In the present study, we investigated MspTL-mediated activation of signaling pathways stimulating up-regulation of IFN-beta and IFN-stimulated genes in a human monocytic cell line, THP-1 cells, and primary cultured human gingival fibroblasts. MspTL treatment of the cells induced IFN-beta and the IFN-stimulated genes IFN-gamma-inducible protein-10 (IP-10) and RANTES. A neutralizing anti-IFN-beta Ab significantly reduced the expression of IP-10 and RANTES, as well as STAT-1 activation, which was also induced by MspTL. Experiments using specific small interfering RNA showed that MspTL activated TANK-binding kinase 1 (TBK1), but not inducible IkappaB kinase (IKKi). MspTL also induced dimerization of IFN regulatory factor-3 (IRF-3) and translocation into the nucleus. The lipid rapid-disrupting agents methyl-beta-cyclodextrin, nystatin, and filipin inhibited the MspTL internalization and cellular responses, demonstrating that lipid raft activation was a prerequisite for MspTL cellular signaling. Our results demonstrate that MspTL, the major outer protein of T. lecithinolyticum, induced IFN-beta expression and subsequent up-regulation of IP-10 and RANTES via TBK1/IRF-3/STAT-1 signaling secondary to lipid raft activation.
Assuntos
Proteínas de Bactérias/fisiologia , Regulação Bacteriana da Expressão Gênica/imunologia , Fator Regulador 3 de Interferon/fisiologia , Interferon beta/genética , Microdomínios da Membrana/imunologia , Monócitos/imunologia , Porinas/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Treponema/imunologia , Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/imunologia , Linhagem Celular Tumoral , Quimiocina CCL5/biossíntese , Quimiocina CCL5/genética , Quimiocina CXCL10/biossíntese , Quimiocina CXCL10/fisiologia , Dimerização , Fibroblastos/enzimologia , Fibroblastos/imunologia , Fibroblastos/microbiologia , Humanos , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/biossíntese , Interferon beta/fisiologia , Microdomínios da Membrana/enzimologia , Microdomínios da Membrana/microbiologia , Monócitos/enzimologia , Monócitos/microbiologia , Periodontite/enzimologia , Periodontite/imunologia , Periodontite/microbiologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Treponema/patogenicidade , Infecções por Treponema/enzimologia , Infecções por Treponema/imunologia , Infecções por Treponema/microbiologia , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
Periodontitis is an inflammatory disease induced by local infection in tooth-supporting tissue. Periodontitis is associated with systemic bone diseases, but little is known about the mechanism of the causal effect of periodontitis on systemic bone resorption. Bacteria-derived extracellular vesicles (EVs) act as natural carriers of virulence factors that are responsible for systemic inflammation. In this study, we investigated the role of EVs derived from Filifactor alocis, a Gram-positive, anaerobic periodontal pathogen, in systemic bone loss and osteoclast differentiation. F. alocis EVs accumulated in the long bones of mice after intraperitoneal administration. These EVs induced proinflammatory cytokines, osteoclastogenesis, and bone resorption via Toll-like receptor 2 (TLR2). The phase separation of F. alocis EVs showed that amphiphilic molecules were responsible for the induced bone resorption and osteoclastogenesis. The osteoclastogenic effects of F. alocis EVs were reduced by lipoprotein lipase. Proteomic analysis of the amphiphilic molecules identified seven lipoproteins. Our results indicate that lipoprotein-like molecules in F. alocis EVs may contribute to systemic bone loss via TLR2.
Assuntos
Doenças Ósseas/microbiologia , Vesículas Extracelulares/metabolismo , Periodontite/microbiologia , Receptor 2 Toll-Like/metabolismo , Animais , Clostridiales , Humanos , CamundongosRESUMO
Filifactor alocis, a gram-positive, obligate anaerobic rod, is an emerging periodontal pathogen that is frequently isolated from patients with periodontitis, peri-implantitis, and apical periodontitis. Recent studies have shown that extracellular vesicles (EVs) from gram-negative periodontal pathogens, so-called outer membrane vesicles (OMVs), harbor various effector molecules responsible for inducing host inflammatory responses. However, there are no reports of EVs from F. alocis. In this study, we purified and characterized the protein profiles of EVs from F. alocis and investigated their immunostimulatory activity on human monocytic THP-1 and human oral keratinocyte HOK-16B cell lines. Highly pure EVs were obtained from F. alocis using density gradient ultracentrifugation. Nanoparticle tracking analysis and transmission electron microscopy showed that F. alocis EVs were between 50 and 270 nm in diameter. Proteome analysis identified 28 proteins, including lipoproteins, autolysins, F. alocis complement inhibitor (FACIN), transporter-related proteins, metabolism-related proteins, and ribosomal proteins. Human cytokine array analysis showed that F. alocis EVs remarkably induced the expression of CCL1, CCL2, MIP-1, CCL5, CXCL1, CXCL10, ICAM-1, IL-1ß, IL-1ra, IL-6, IL-8, MIF, SerpinE, and TNF-α in THP-1 cells and CXCL1, G-CSF, GM-CSF, IL-6, and IL-8 in HOK-16B cells. The immunostimulatory activity of F. alocis EVs was similar to that of the whole bacterial cells. Our findings provide new insight into the role of EVs from gram-positive oral bacteria in periodontal diseases.