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The Protective Effect of Recombinant FomA-expressing Lactobacillus acidophilus Against Periodontal Infection
Ma, Li
Ding, Qinfeng
Feng, Xiping
Li, Fei
A number of studies have shown that the outer membrane protein FomA found in Fusobacterium nucleatum demonstrates great potential as an immune target for combating periodontitis. Lactobacillus acidophilus is a useful antigen delivery vehicle for mucosal immunisation, and previous studies by our group have shown that L. acidophilus acts as a protective factor in periodontal health. In this study, making use of the immunogenicity of FomA and the probiotic properties of L. acidophilus, we constructed a recombinant form of L. acidophilus expressing the FomA protein and detected the FomA-specific IgG in the serum and sIgA in the saliva of mice through oral administration with the recombinant strains. When serum containing FomA-specific antibodies was incubated with the F. nucleatum in vitro, the number of Porphyromonas gingivalis cells that coaggregated with the F. nucleatum cells was significantly reduced. Furthermore, a mouse gum abscess model was successfully generated, and the range of gingival abscesses in the immune mice was relatively limited compared with the control group. The level of IL-1β in the serum and local gum tissues of the immune mice was consistently lower than in the control group. Our findings indicated that oral administration of the recombinant L. acidophilus reduced the risk of periodontal infection with P. gingivalis and F. nucleatum.
FomA; Lactobacillus acidophilus; Fusobacterium nucleatum; Porphyromonas gingivalis; periodontal infection
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The FomA Porin from Fusobacterium nucleatum Is a Toll-Like Receptor 2 Agonist with Immune Adjuvant Activity
Toussi, Deana N.
Liu, Xiuping
Massari, Paola
Clinical and Vaccine Immunology : CVI
Many bacterial components selectively activate immune and nonhematopoietic target cells via Toll-like receptor (TLR) signaling; modulation of such host responses defines the immune adjuvant properties of these bacterial products. For example, the outer membrane protein porins from Neisseria, Salmonella, and Shigella are known TLR2 agonists with established systemic and mucosal immune adjuvanticity. Early work indicated that the FomA porin from Fusobacterium nucleatum has immune adjuvant activity in mice. Using a purified recombinant FomA, we have verified its immune stimulatory properties and have defined a role for TLR2 signaling in its in vitro and in vivo activity. FomA induces interleukin 8 (IL-8) secretion and NF-κB-dependent luciferase activity in HEK cells expressing TLR2, IL-6 secretion, and cell surface upregulation of CD86 and major histocompatibility complex (MHC) II in primary B cells from wild-type mice, but it fails to activate cells from TLR2 knockout mice. Accordingly, the immune adjuvant activity of FomA is also TLR2 dependent. In a mouse model of immunization with ovalbumin (OVA), FomA induces enhanced production of OVA-specific IgM and IgG, including IgG1 and IgG2b antibodies, as well as enhanced secretion of IL-10 and IL-6, consistent with a Th2-type adjuvant effect. We also observe a moderate production of anti-FomA antibodies, suggesting that FomA is also immunogenic, a quality that is also TLR2 dependent. Therefore, modulation of host immune responses by FomA may be effective for targeting general host immunity not only to pathogens (as a novel TLR2 adjuvant) but also to F. nucleatum itself (as an antigen), expanding its use as a self-adjuvanted antigen in an immunization strategy against polymicrobial infections, including those by F. nucleatum.
Related Articles
Fusobacterium nucleatum Envelope Protein FomA Is Immunogenic and Binds to the Salivary Statherin-Derived Peptide▿
Nakagaki, Hidetaka
Sekine, Shinichi
Terao, Yutaka
Toe, Masahiro
Tanaka, Muneo
Ito, Hiro-O
Kawabata, Shigetada
Shizukuishi, Satoshi
Fujihashi, Kohtaro
Kataoka, Kosuke
Infection and Immunity
We have previously shown that one of the minimal active regions of statherin, a human salivary protein, for binding to Fusobacterium nucleatum is a YQPVPE amino acid sequence. In this study, we identified the FomA protein of F. nucleatum, which is responsible for binding to the statherin-derived YQPVPE peptide. Overlay analysis showed that a 40-kDa protein of the F. nucleatum cell envelope (40-kDa CE) specifically bound to the YQPVPE peptide. The equilibrium association constant between the affinity-purified 40-kDa CE and the YQPVPE peptide was 4.30 × 106. Further, the purity and amino acid sequence analyses of the purified 40-kDa CE revealed approximately 98.7% (wt/wt) purity and a high degree of homology with FomA, a major porin protein of F. nucleatum. Thus, a FomA-deficient mutant failed to bind to the YQPVPE peptide. In addition, increased levels of a FomA-specific mucosal IgA antibody (Ab) and plasma IgG and IgA Abs were seen only in mice immunized nasally with cholera toxin (CT) and the purified 40-kDa FomA protein. Interestingly, saliva from mice that received FomA plus CT as a mucosal adjuvant nasally prevented in vitro binding of F. nucleatum to statherin-coated polyvinyl chloride plates. Taken together, these results suggest that induction of specific immunity to the 40-kDa FomA protein of F. nucleatum, which specifically binds to the statherin-derived peptide, may be an effective tool for preventing the formation of F. nucleatum biofilms in the oral cavity.
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A proteomic investigation of Fusobacterium nucleatum alkaline-induced biofilms
Chew, Jactty
Zilm, Peter S
Fuss, Janet M
Gully, Neville J
BMC Microbiology
The Gram negative anaerobe Fusobacterium nucleatum has been implicated in the aetiology of periodontal diseases. Although frequently isolated from healthy dental plaque, its numbers and proportion increase in plaque associated with disease. One of the significant physico-chemical changes in the diseased gingival sulcus is increased environmental pH. When grown under controlled conditions in our laboratory, F. nucleatum subspecies polymorphum formed mono-culture biofilms when cultured at pH 8.2. Biofilm formation is a survival strategy for bacteria, often associated with altered physiology and increased virulence. A proteomic approach was used to understand the phenotypic changes in F. nucleatum cells associated with alkaline induced biofilms. The proteomic based identification of significantly altered proteins was verified where possible using additional methods including quantitative real-time PCR (qRT-PCR), enzyme assay, acidic end-product analysis, intracellular polyglucose assay and Western blotting.
Of 421 proteins detected on two-dimensional electrophoresis gels, spot densities of 54 proteins varied significantly (p < 0.05) in F. nucleatum cultured at pH 8.2 compared to growth at pH 7.4. Proteins that were differentially produced in biofilm cells were associated with the functional classes; metabolic enzymes, transport, stress response and hypothetical proteins. Our results suggest that biofilm cells were more metabolically efficient than planktonic cells as changes to amino acid and glucose metabolism generated additional energy needed for survival in a sub-optimal environment. The intracellular concentration of stress response proteins including heat shock protein GroEL and recombinational protein RecA increased markedly in the alkaline environment. A significant finding was the increased abundance of an adhesin, Fusobacterial outer membrane protein A (FomA). This surface protein is known for its capacity to bind to a vast number of bacterial species and human epithelial cells and its increased abundance was associated with biofilm formation.
This investigation identified a number of proteins that were significantly altered by F. nucleatum in response to alkaline conditions similar to those reported in diseased periodontal pockets. The results provide insight into the adaptive mechanisms used by F. nucleatum biofilms in response to pH increase in the host environment.
Fusobacterium nucleatum; Biofilms; Alkaline pH; Periodontal diseases; Proteomics
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A novel vaccine targeting Fusobacterium nucleatum against abscesses and halitosis
Liu, Pei-Feng
Haake, Susan Kinder
Gallo, Richard L.
Huang, Chun-Ming
An abscess in a gum pocket, resulting from bacterial infection, is a common source of chronic halitosis. Although antibiotics are generally prescribed for abscesses, they require multiple treatments with risks of creating resistant bacterial strains. Here we develop a novel vaccine using ultraviolet-inactivated Fusobacterium nucleatum (F. nucleatum), a representative oral bacterium for halitosis. A gum pocket model, established by continuous inoculation of F. nucleatum, was employed to validate the vaccine potency. Mice immunized with inactivated F. nucleatum effectively minimized the progression of abscesses, measured by swollen tissues of gum pockets. Most notably, the immunized mice were capable of eliciting neutralizing antibodies against the production of volatile sulfur compounds of F. nucleatum. The novel vaccine inducing protective immunity provides an alternative option to conventional antibiotic treatments for chronic halitosis associated with abscesses.
Vaccine; F. nucleatum; Abscesses; Halitosis
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Direct Recognition of Fusobacterium nucleatum by the NK Cell Natural Cytotoxicity Receptor NKp46 Aggravates Periodontal Disease
Chaushu, Stella
Wilensky, Asaf
Gur, Chamutal
Shapira, Lior
Elboim, Moran
Halftek, Gili
Polak, David
Achdout, Hagit
Bachrach, Gilad
Mandelboim, Ofer
PLoS Pathogens
Periodontitis is a common human chronic inflammatory disease that results in the destruction of the tooth attachment apparatus and tooth loss. Although infections with periopathogenic bacteria such as Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum) are essential for inducing periodontitis, the nature and magnitude of the disease is determined by the host's immune response. Here, we investigate the role played by the NK killer receptor NKp46 (NCR1 in mice), in the pathogenesis of periodontitis. Using an oral infection periodontitis model we demonstrate that following F. nucleatum infection no alveolar bone loss is observed in mice deficient for NCR1 expression, whereas around 20% bone loss is observed in wild type mice and in mice infected with P. gingivalis. By using subcutaneous chambers inoculated with F. nucleatum we demonstrate that immune cells, including NK cells, rapidly accumulate in the chambers and that this leads to a fast and transient, NCR1-dependant TNF-α secretion. We further show that both the mouse NCR1 and the human NKp46 bind directly to F. nucleatum and we demonstrate that this binding is sensitive to heat, to proteinase K and to pronase treatments. Finally, we show in vitro that the interaction of NK cells with F. nucleatum leads to an NCR1-dependent secretion of TNF-α. Thus, the present study provides the first evidence that NCR1 and NKp46 directly recognize a periodontal pathogen and that this interaction influences the outcome of F. nucleatum-mediated periodontitis.
Author Summary
Periodontal disease is a common bacterial-induced inflammatory process in which F. nucleatum and P. gingivalis infections lead to the destruction of the teeth supporting attachment apparatus. Previous reports demonstrated that immune cells aggravate the severity of the disease. However, whether NK cells in general and NKp46 (a major killer receptor expressed by NK cells) in particular, play a protective or destructive role in this disease is unknown. Using mice deficient in NCR1 (the mouse orthlogue of NKp46), we demonstrate that oral infection of mice with F. nucleatum, but not with P. gingivalis results in an NCR1-dependent alveolar bone loss. In addition, we show that F. nucleatum is recognized by NCR1 and NKp46 directly and that this recognition leads to the secretion of TNF-α, a central cytokine critically involved in the pathogenesis of periodontal destruction. Collectively, we show that NCR1 and NKp46 play a critical role in the pathogenesis of F. nucleatum-mediated periodontitis.
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Serum Antibodies to Porphyromonas gingivalis Chaperone HtpG Predict Health in Periodontitis Susceptible Patients
Shelburne, Charles E.
Shelburne, P. Sandra
Dhople, Vishnu M.
Sweier, Domenica G.
Giannobile, William V.
Kinney, Janet S.
Coulter, Wilson A.
Mullally, Brian H.
Lopatin, Dennis E.
PLoS ONE
Chaperones are ubiquitous conserved proteins critical in stabilization of new proteins, repair/removal of defective proteins and immunodominant antigens in innate and adaptive immunity. Periodontal disease is a chronic inflammatory infection associated with infection by Porphyromonas gingivalis that culminates in the destruction of the supporting structures of the teeth. We previously reported studies of serum antibodies reactive with the human chaperone Hsp90 in gingivitis, a reversible form of gingival disease confined to the oral soft tissues. In those studies, antibodies were at their highest levels in subjects with the best oral health. We hypothesized that antibodies to the HSP90 homologue of P. gingivalis (HtpG) might be associated with protection/resistance against destructive periodontitis.
Methodology/Principal Findings
ELISA assays using cloned HtpG and peptide antigens confirmed gingivitis subjects colonized with P. gingivalis had higher serum levels of anti-HtpG and, concomitantly, lower levels of attachment loss. Additionally, serum antibody levels to P. gingivalis HtpG protein were higher in healthy subjects compared to patients with either chronic or aggressive periodontitis. We found a negative association between tooth attachment loss and anti-P. gingivalis HtpG (p = 0.043) but not anti-Fusobacterium nucleatum (an oral opportunistic commensal) HtpG levels. Furthermore, response to periodontal therapy was more successful in subjects having higher levels of anti-P. gingivalis HtpG before treatment (p = 0.018). There was no similar relationship to anti-F. nucleatum HtpG levels. Similar results were obtained when these experiments were repeated with a synthetic peptide of a region of P. gingivalis HtpG.
Our results suggest: 1) anti-P. gingivalis HtpG antibodies are protective and therefore predict health periodontitis-susceptable patients; 2) may augment the host defence to periodontitis and 3) a unique peptide of P. gingivalis HtpG offers significant potential as an effective diagnostic target and vaccine candidate. These results are compatible with a novel immune control mechanism unrelated to direct binding of bacteria.
Related Articles
Mixed infection with Porphyromonas gingivalis and Fusobacterium nucleatum in a murine lesion model: potential synergistic effects on virulence.
Feuille, F
Ebersole, J L
Kesavalu, L
Stepfen, M J
Holt, S C
Infection and Immunity
These studies determined the characteristics of tissue destruction in a murine abscess model elicited by mixed infection with the periodontopathogens Fusobacterium nucleatum and Porphyromonas gingivalis. The interbacterial effects of this synergism, the kinetics of the relationship of the bacterial interaction, and the characteristics of the bacteria required for the tissue destruction were studied. Infection of mice with P. gingivalis and F. nucleatum strains elicited lesions of various sizes as a function of infective dose. Primary infection with F. nucleatum plus P. gingivalis at various ratios (i.e., <1:1) resulted in a significantly greater lesion size (P < 0.001) compared with that resulting from primary infection with P. gingivalis alone. At F. nucleatum/P. gingivalis ratios of > or = 1:1, spreading lesion formation and progression were significantly (P < 0.001) decreased, suggesting that bacterial interaction (i.e., coaggregation) may have inhibited the spread of the P. gingivalis infection to a site distant from the initial injection. Infection with F. nucleatum and P. gingivalis simultaneously (at different sites) or F. nucleatum administered within 4 h prior to or 1 h following P. gingivalis infection significantly enhanced the ability of P. gingivalis to form large phlegmonous lesions. Chemical inhibition of the P. gingivalis trypsin-like protease activity or the use of a trypsin-negative P. gingivalis strain abrogated tissue destruction either alone or in combination with F. nucleatum. Therefore, it was possible to examine aspects of virulence of these pathogens in a murine lesion model by either altering bacterial ratios, manipulating the time of infection, or targeting vital bacterial virulence factors.
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Porphyromonas gingivalis entry into gingival epithelial cells modulated by Fusobacterium nucleatum is dependent on lipid rafts
Saito, Atsushi
Kokubu, Eitoyo
Inagaki, Satoru
Imamura, Kentaro
Kita, Daichi
Lamont, Richard J.
Ishihara, Kazuyuki
Microbial pathogenesis
Host cell invasion by a major periodontal pathogen, Porphyromonas gingivalis, has been proposed as an important mechanism involved in host–pathogen interactions in periodontal and cardiovascular diseases. The present study sought to gain insight into the underlying mechanism(s) involved in previously demonstrated fusobacterial modulation of host cell invasion by P. gingivalis. An immortalized human gingival cell line Ca9-22 was dually infected with P. gingivalis ATCC 33277 and Fusobacterium nucleatum TDC 100, and intracellular invasion was assessed by scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM). SEM observation showed that P. gingivalis and F. nucleatum formed consortia and were in the process of penetrating into Ca9-22 by 30–60 min after infection. In CSLM, Ca9-22 cells that contained both P. gingivalis and F. nucleatum were frequently observed after 2 h, although cells that contained exclusively P. gingivalis were also found. Infection by P. gingivalis and/or F. nucleatum revealed evident colocalization with a lipid raft marker, GM1-containing membrane microdomains. In an antibiotic protection assay, depletion of epithelial plasma membrane cholesterol resulted in a significant reduction of recovered P. gingivalis or F. nucleatum (~33% of untreated control; p < 0.001). This inhibition was also confirmed by CSLM. Sequential infection experiments showed that timing of infection by each species could critically influence the invasion profile. Co-infection with F. nucleatum significantly enhanced host cell invasion by P. gingivalis 33277, its serine phophatase SerB mutant and complemented strains, suggesting that the SerB does not play a major role in this fusobacterial enhancement of P. gingivalis invasion. Thus, the interaction between F. nucleatum and host cells may be important in the fusobacterial enhancement of P. gingivalis invasion. Collectively, these results suggest that lipid raft-mediated process is at least one of the potential mechanisms involved in fusobacterium-modulated host cell invasion by P. gingivalis.
Polymicrobial infection; Host invasion; Porphyromonas gingivalis; Gingival epithelial cells; Periodontitis; Fusobacterium nucleatum
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Mutualistic Biofilm Communities Develop with Porphyromonas gingivalis and Initial, Early, and Late Colonizers of Enamel▿
Periasamy, Saravanan
Kolenbrander, Paul E.
Journal of Bacteriology
Porphyromonas gingivalis is present in dental plaque as early as 4 h after tooth cleaning, but it is also associated with periodontal disease, a late-developing event in the microbial successions that characterize daily plaque development. We report here that P. gingivalis ATCC 33277 is remarkable in its ability to interact with a variety of initial, early, middle, and late colonizers growing solely on saliva. Integration of P. gingivalis into multispecies communities was investigated by using two in vitro biofilm models. In flow cells, bacterial growth was quantified using fluorescently conjugated antibodies against each species, and static biofilm growth on saliva-submerged polystyrene pegs was analyzed by quantitative real-time PCR using species-specific primers. P. gingivalis could not grow as a single species or together with initial colonizer Streptococcus oralis but showed mutualistic growth when paired with two other initial colonizers, Streptococcus gordonii and Actinomyces oris, as well as with Veillonella sp. (early colonizer), Fusobacterium nucleatum (middle colonizer), and Aggregatibacter actinomycetemcomitans (late colonizer). In three-species flow cells, P. gingivalis grew with Veillonella sp. and A. actinomycetemcomitans but not with S. oralis and A. actinomycetemcomitans. Also, it grew with Veillonella sp. and F. nucleatum but not with S. oralis and F. nucleatum, indicating that P. gingivalis and S. oralis are not compatible. However, P. gingivalis grew in combination with S. gordonii and S. oralis, demonstrating its ability to overcome the incompatibility when cultured with a second initially colonizing species. Collectively, these data help explain the observed presence of P. gingivalis at all stages of dental plaque development.
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Acquisition of Plasmin Activity by Fusobacterium nucleatum subsp. nucleatum and Potential Contribution to Tissue Destruction during Periodontitis
Darenfed, H.
Grenier, D.
Mayrand, D.
Infection and Immunity
Fusobacterium nucleatum subsp. nucleatum has been associated with a variety of oral and nonoral infections such as periodontitis, pericarditis, bone infections, and brain abscesses. Several studies have shown the role of plasmin, a plasma serine protease, in increasing the invasive capacity of microorganisms. In this study, we investigated the binding of human plasminogen to F. nucleatum subsp. nucleatum, and its subsequent activation into plasmin. Plasminogen-binding activity of bacterial cells was demonstrated by a solid-phase dot blot assay using an anti-plasminogen antibody. The binding activity was heat resistant and involved cell-surface lysine residues since it was abolished in the presence of the lysine analog ɛ-aminocaproic acid. Activation of plasminogen-coated bacteria occurred following incubation with either streptokinase, urokinase-type plasminogen activator (u-PA), or a Porphyromonas gingivalis culture supernatant. In the case of the P. gingivalis culture supernatant, a cysteine protease was likely involved in the activation. The plasmin activity generated on the cell surface of F. nucleatum subsp. nucleatum could be inhibited by aprotinin. Activation of plasminogen by u-PA was greatly enhanced when plasminogen was bound to bacteria rather than in a free soluble form. u-PA-activated plasminogen-coated F. nucleatum subsp. nucleatum was found to degrade fibronectin, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tissue inhibitor of metalloproteinase-1 was also degraded by the plasmin activity generated on the bacterial cells. This study suggests a possible role for plasminogen, which is present in affected periodontal sites, in promoting tissue destruction and invasion by nonproteolytic bacteria such as F. nucleatum subsp. nucleatum.
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The Streptomyces-produced antibiotic fosfomycin is a promiscuous substrate for Archaeal isopentenyl phosphate kinase
Mabanglo, Mark F.
Serohijos, Adrian W. R.
Poulter, C. Dale
Isopentenyl phosphate kinase (IPK) catalyzes the phosphorylation of isopentenyl phosphate to form the isoprenoid precursor isopentenyl diphosphate (IPP) in the archaeal mevalonate pathway. This enzyme is highly homologous to fosfomycin kinase (FomA), an antibiotic resistance enzyme found in a few strains of Streptomyces and Pseudomonas whose mode of action is inactivation by phosphorylation. Superposition of Thermoplasma acidophilum (THA) IPK and FomA structures aligns their respective substrates and catalytic residues, including H50 and K14 in THA IPK, and H58 and K18 in S. wedmorensis FomA. These residues are conserved only in the IPK and FomA members of the phosphate subdivision of the amino acid kinase superfamily. We measured the fosfomycin kinase activity of THA IPK, Km = 15.1 ± 1.0 mM and kcat = (4.0 ± 0.1) × 10−2 s−1, resulting in a catalytic efficiency, kcat/Km = 2.6 M−1s−1, that is five orders of magnitude less than the native reaction. Fosfomycin is a competitive inhibitor of IPK, Ki = 3.6 ± 0.2 mM. Molecular dynamics simulation of the IPK•fosfomycin•MgATP complex identified two binding poses for fosfomycin in the IP binding site, one of which results in a complex analogous to the native IPK•IP•ATP complex that it engages H50 and the lysine triangle formed by K5, K14, and K205. The other binding pose leads to a dead-end complex that engages K204 near the IP binding site to bind fosfomycin. Our findings suggest a mechanism for acquisition of FomA-based antibiotic resistance in fosfomycin producing organisms.
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Characterization of extracellular polymeric matrix, and treatment of Fusobacterium nucleatum and Porphyromonas gingivalis biofilms with DNase I and proteinase K
Ali Mohammed, Marwan Mansoor
Nerland, Audun H.
Al-Haroni, Mohammed
Bakken, Vidar
Journal of Oral Microbiology
Biofilms are organized communities of microorganisms embedded in a self-produced extracellular polymeric matrix (EPM), often with great phylogenetic variety. Bacteria in the subgingival biofilm are key factors that cause periodontal diseases; among these are the Gram-negative bacteria Fusobacterium nucleatum and Porphyromonas gingivalis. The objectives of this study were to characterize the major components of the EPM and to test the effect of deoxyribonuclease I (DNase I) and proteinase K.
F. nucleatum and P. gingivalis bacterial cells were grown in dynamic and static biofilm models. The effects of DNase I and proteinase K enzymes on the major components of the EPM were tested during biofilm formation and on mature biofilm. Confocal laser scanning microscopy was used in observing biofilm structure.
Proteins and carbohydrates were the major components of the biofilm matrix, and extracellular DNA (eDNA) was also present. DNase I and proteinase K enzymes had little effect on biofilms in the conditions used. In the flow cell, F. nucleatum was able to grow in partially oxygenated conditions while P. gingivalis failed to form biofilm alone in similar conditions. F. nucleatum supported the growth of P. gingivalis when they were grown together as dual species biofilm.
DNase I and proteinase K had little effect on the biofilm matrix in the conditions used. F. nucleatum formed biofilm easily and supported the growth of P. gingivalis, which preferred anaerobic conditions.
Subgingival biofilm; extracellular polymeric matrix; Fusobacterium nucleatum; Porphyromonas gingivalis; static and dynamic biofilm models; confocal laser scanning microscopy
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Interleukin-8 and Intercellular Adhesion Molecule 1 Regulation in Oral Epithelial Cells by Selected Periodontal Bacteria: Multiple Effects of Porphyromonas gingivalis via Antagonistic Mechanisms
Huang, George T.-J.
Kim, Daniel
Lee, Jonathan K.-H.
Kuramitsu, Howard K.
Haake, Susan Kinder
Infection and Immunity
Interaction of bacteria with mucosal surfaces can modulate the production of proinflammatory cytokines and adhesion molecules produced by epithelial cells. Previously, we showed that expression of interleukin-8 (IL-8) and intercellular adhesion molecule 1 (ICAM-1) by gingival epithelial cells increases following interaction with several putative periodontal pathogens. In contrast, expression of IL-8 and ICAM-1 is reduced after Porphyromonas gingivalis ATCC 33277 challenge. In the present study, we investigated the mechanisms that govern the regulation of these two molecules in bacterially infected gingival epithelial cells. Experimental approaches included bacterial stimulation of gingival epithelial cells by either a brief challenge (1.5 to 2 h) or a continuous coculture throughout the incubation period. The kinetics of IL-8 and ICAM-1 expression following brief challenge were such that (i) secretion of IL-8 by gingival epithelial cells reached its peak 2 h following Fusobacterium nucleatum infection whereas it rapidly decreased within 2 h after P. gingivalis infection and remained decreased up to 30 h and (ii) IL-8 and ICAM-1 mRNA levels were up-regulated rapidly 2 to 4 h postinfection and then decreased to basal levels 8 to 20 h after infection with either Actinobacillus actinomycetemcomitans, F. nucleatum, or P. gingivalis. Attenuation of IL-8 secretion was facilitated by adherent P. gingivalis strains. The IL-8 secreted from epithelial cells after F. nucleatum stimulation could be down-regulated by subsequent infection with P. gingivalis or its culture supernatant. Although these results suggested that IL-8 attenuation at the protein level might be associated with P. gingivalis proteases, the Arg- and Lys-gingipain proteases did not appear to be solely responsible for IL-8 attenuation. In addition, while P. gingivalis up-regulated IL-8 mRNA expression, this effect was overridden when the bacteria were continuously cocultured with the epithelial cells. The IL-8 mRNA levels in epithelial cells following sequential challenge with P. gingivalis and F. nucleatum and vice versa were approximately identical and were lower than those following F. nucleatum challenge alone and higher than control levels or those following P. gingivalis challenge alone. Thus, together with the protease effect, P. gingivalis possesses a powerful strategy to ensure the down-regulation of IL-8 and ICAM-1.
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Proteomics of Streptococcus gordonii within a model developing oral microbial community
Hendrickson, Erik L
Wang, Tiansong
Dickinson, Brittany C
Whitmore, Sarah E
Wright, Christopher J
Lamont, Richard J
Hackett, Murray
BMC Microbiology
Streptococcus gordonii is one of several species that can initiate the formation of oral biofilms that develop into the complex multispecies microbial communities referred to as dental plaque. It is in the context of dental plaque that periodontal pathogens such as Porphyromonas gingivalis cause disease. We have previously reported a whole cell quantitative proteomics investigation of P. gingivalis in a model dental plaque community of S. gordonii, P. gingivalis, and Fusobacterium nucleatum. Here we report the adaptation of S. gordonii to the same model.
1122 S. gordonii proteins were detected in S. gordonii control samples, 915 in communities with F. nucleatum, 849 with P. gingivalis, and 649 with all three organisms. Quantitative comparisons showed extensive proteome changes in association with F. nucleatum or P. gingivalis individually or both P. gingivalis and F. nucleatum together. The changes were species specific, though the P. gingivalis interaction may be dominant, indicated by large differences between the proteomes with F. nucleatum or P. gingivalis but limited changes between communities with P. gingivalis or both P. gingivalis and F. nucleatum. The results were inspected manually and an ontology analysis conducted using DAVID. Extensive changes were seen in nutrition pathways with increases in energy metabolism and changes in the resulting byproducts, while the acid and sugar repressed PTS (phosphoenolpyruvate dependent phosphotransferase system) sugar transport systems showed decreases. These results were seen across all the multispecies samples, though with different profiles according to the partner species. F. nucleatum association decreased proteins for the metabolic end products acetate and ethanol but increased lactate, the primary source of acidity from streptococcal cultures. P. gingivalis containing samples had a reduction in levels of proteins for ethanol and formate but increased proteins for both acetate and lactate production. The communities also showed increases in exopolysaccharide synthesis, amino acid biosynthesis, and oxidative stress protection and decreases in adhesion and transporter proteins.
This study showed that S. gordonii demonstrates species specific responses during interactions with F. nucleatum or P. gingivalis. Extensive changes were seen in energy metabolism and byproduct production implicating nutrient transfer as an important community interaction.
Streptococcus gordonii; Oral biofilm; Proteomics; Model community; Porphyromonas gingivalis; Fusobacterium nucleatum
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Setup of an In Vitro Test System for Basic Studies on Biofilm Behavior of Mixed-Species Cultures with Dental and Periodontal Pathogens
Standar, Kerstin
Kreikemeyer, Bernd
Redanz, Sylvio
Münter, Wanja L.
Laue, Michael
Podbielski, Andreas
PLoS ONE
Caries and periodontitis are important human diseases associated with formation of multi-species biofilms. The involved bacteria are intensively studied to understand the molecular basis of the interactions in such biofilms. This study established a basic in vitro single and mixed-species culture model for oral bacteria combining three complimentary methods. The setup allows a rapid screening for effects in the mutual species interaction. Furthermore, it is easy to handle, inexpensive, and reproducible.
Streptococcus mitis, S. salivarius and S. sanguinis, typical inhabitants of the healthy oral cavity, S. mutans as main carriogenic species, and Porphyromonas gingivalis, Fusobacterium nucleatum, Parvimonas micra, S. intermedius and Aggregatibacter actinomycetemcomitans as periodontitis-associated bacteria, were investigated for their biofilm forming ability. Different liquid growth media were evaluated. Safranin-staining allowed monitoring of biofilm formation under the chosen conditions. Viable counts and microscopy permitted investigation of biofilm behavior in mixed-species and transwell setups.
S. mitis, F. nucleatum, P. gingivalis and P. micra failed to form biofilm structures. S. mutans, S. sanguinis, S. intermedius and S. salivarius established abundant biofilm masses in CDM/sucrose. A. actinomycetemcomitans formed patchy monolayers. For in depth analysis S. mitis, S. mutans and A. actinomycetemcomitans were chosen, because i) they are representatives of the physiological-, cariogenic and periodontitis-associated bacterial flora, respectively and ii) their difference in their biofilm forming ability. Microscopic analysis confirmed the results of safranin staining. Investigation of two species combinations of S. mitis with either S. mutans or A. actinomycetemcomitans revealed bacterial interactions influencing biofilm mass, biofilm structure and cell viability.
This setup shows safranin staining, microscopic analysis and viable counts together are crucial for basic examination and evaluation of biofilms. Our experiment generated meaningful results, exemplified by the noted S. mitis influence, and allows a fast decision about the most important bacterial interactions which should be investigated in depth.
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N-acetylcysteine and the human serum components that inhibit bacterial invasion of gingival epithelial cells prevent experimental periodontitis in mice
Alam, Jehan
Baek, Keum Jin
Choi, Yun Sik
Kim, Yong Cheol
Choi, Youngnim
Journal of Periodontal & Implant Science
We previously reported that human serum significantly reduces the invasion of various oral bacterial species into gingival epithelial cells in vitro. The aims of the present study were to characterize the serum component(s) responsible for the inhibition of bacterial invasion of epithelial cells and to examine their effect on periodontitis induced in mice.
Immortalized human gingival epithelial (HOK-16B) cells were infected with various 5- (and 6-) carboxy-fluorescein diacetate succinimidyl ester-labeled oral bacteria, including Fusobacterium nucleatum, Provetella intermedia, Porphyromonas gingivalis, and Treponiema denticola, in the absence or presence of three major serum components (human serum albumin [HSA], pooled human IgG [phIgG] and α1-antitrypsin). Bacterial adhesion and invasion were determined by flow cytometry. The levels of intracellular reactive oxygen species (ROS) and activation of small GTPases were examined. Experimental periodontitis was induced by oral inoculation of P. gingivalis and T. denticola in Balb/c mice.
HSA and phIgG, but not α1-antitrypsin, efficiently inhibited the invasion of various oral bacterial species into HOK-16B cells. HSA but not phIgG decreased the adhesion of F. nucleatum onto host cells and the levels of intracellular ROS in HOK-16B cells. N-acetylcysteine (NAC), a ROS scavenger, decreased both the levels of intracellular ROS and invasion of F. nucleatum into HOK-16B cells, confirming the role of ROS in bacterial invasion. Infection with F. nucleatum activated Rac1, a regulator of actin cytoskeleton dynamics. Not only HSA and NAC but also phIgG decreased the F. nucleatum-induced activation of Rac1. Furthermore, both HSA plus phIgG and NAC significantly reduced the alveolar bone loss in the experimental periodontitis induced by P. gingivalis and T. denticola in mice.
NAC and the serum components HSA and phIgG, which inhibit bacterial invasion of oral epithelial cells in vitro, can successfully prevent experimental periodontitis.
Graphical Abstract
Albumins; Bacteria; Epithelial cells; Immunoglobulin G; Periodontitis
Related Articles
Interactions between Periodontal Bacteria and Human Oral Epithelial Cells: Fusobacterium nucleatum Adheres to and Invades Epithelial Cells
Han, Yiping W.
Shi, Wenyuan
Huang, George T.-J.
Kinder Haake, Susan
Park, No-Hee
Kuramitsu, Howard
Genco, Robert J.
Infection and Immunity
Bacteria are causative agents of periodontal diseases. Interactions between oral bacteria and gingival epithelial cells are essential aspects of periodontal infections. Using an in vitro tissue culture model, a selected group of gram-negative anaerobic bacteria frequently associated with periodontal diseases, including Bacteroides forsythus, Campylobacter curvus, Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedia, were examined for their ability to adhere to and invade primary cultures of human gingival epithelial cells (HGEC). The effects of these bacteria on the production of interleukin-8 (IL-8), a proinflammatory chemokine, were also measured. These studies provided an initial demonstration that F. nucleatum adhered to and invaded HGEC and that this was accompanied by high levels of IL-8 secretion from the epithelial cells. The attachment and invasion characteristics of F. nucleatum were also tested using KB cells, an oral epithelial cell line. The invasion was verified by transmission electron microscopy and with metabolic inhibitors. Invasion appeared to occur via a "zipping" mechanism and required the involvement of actins, microtubules, signal transduction, protein synthesis, and energy metabolism of the epithelial cell, as well as protein synthesis by F. nucleatum. A spontaneous mutant, lam, of F. nucleatum, isolated as defective in autoagglutination, was unable to attach to or invade HGEC or KB cells, further indicating the requirement of bacterial components in these processes. Sugar inhibition assays indicated that lectin-like interactions were involved in the attachment of F. nucleatum to KB cells. Investigation of these new virulence phenotypes should improve our understanding of the role of F. nucleatum in periodontal infections.
Related Articles
Strain-Specific Colonization Patterns and Serum Modulation of Multi-Species Oral Biofilm Development
Biyikoğlu, Basak
Ricker, Austin
Diaz, Patricia I.
Periodontitis results from an ecological shift in the composition of subgingival biofilms. Subgingival community maturation is modulated by inter-organismal interactions and the relationship of communities with the host. In an effort to better understand this process, we evaluated biofilm formation, with oral commensal species, by three strains of the subgingivally prevalent microorganism Fusobacterium nucleatum and four strains of the periodontopathogen Porphyromonas gingivalis. We also tested the effect of serum, which resembles gingival exudates, on subgingival biofilms. Biofilms were allowed to develop in flow cells using salivary medium. We found that although not all strains of F. nucleatum were able to grow in mono-species biofilms, forming a community with health-associated partners Actinomyces oris and Veillonella parvula promoted biofilm growth of all F. nucleatum strains. Strains of P. gingivalis also showed variable ability to form mono-species biofilms. P. gingivalis W50 and W83 did not form biofilms, while ATCC 33277 and 381 formed biofilm structures, but only strain ATCC 33277 grew over time. Unlike the enhanced growth of F. nucleatum with the two health-associated species, no strain of P. gingivalis grew in three-species communities with A. oris and V. parvula. However, addition of F. nucleatum facilitated growth of P. gingivalis ATCC 33277 with health-associated partners. Importantly, serum negatively affected the adhesion of F. nucleatum, while it favored biofilm growth by P. gingivalis. This work highlights strain specificity in subgingival biofilm formation. Environmental factors such as serum alter the colonization patterns of oral microorganisms and could impact subgingival biofilms by selectively promoting pathogenic species.
oral multi-species communities; subgingival; Fusobacterium nucleatum; Porphyromonas gingivalis; strain variability; serum; biofilms
Related Articles
A periodontitis-associated multispecies model of an oral biofilm
Park, Jong Hwa
Lee, Jae-Kwan
Um, Heung-Sik
Chang, Beom-Seok
Lee, Si-Young
Journal of Periodontal & Implant Science
While single-species biofilms have been studied extensively, we know notably little regarding multispecies biofilms and their interactions. The purpose of this study was to develop and evaluate an in vitro multispecies dental biofilm model that aimed to mimic the environment of chronic periodontitis.
Streptococcus gordonii KN1, Fusobacterium nucleatum ATCC23726, Aggregatibacter actinomycetemcomitans ATCC33384, and Porphyromonas gingivalis ATCC33277 were used for this experiment. The biofilms were grown on 12-well plates with a round glass slip (12 mm in diameter) with a supply of fresh medium. Four different single-species biofilms and multispecies biofilms with the four bacterial strains listed above were prepared. The biofilms were examined with a confocal laser scanning microscope (CLSM) and scanning electron microscopy (SEM). The minimum inhibitory concentrations (MIC) for four different planktonic single-species and multispecies bacteria were determined. The MICs of doxycycline and chlorhexidine for four different single-species biofilms and a multispecies biofilm were also determined.
The CLSM and SEM examination revealed that the growth pattern of the multispecies biofilm was similar to those of single-species biofilms. However, the multispecies biofilm became thicker than the single-species biofilms, and networks between bacteria were formed. The MICs of doxycycline and chlorhexidine were higher in the biofilm state than in the planktonic bacteria. The MIC of doxycycline for the multispecies biofilm was higher than were those for the single-species biofilms of P. gingivalis, F. nucleatum, or A. actinomycetemcomitans. The MIC of chlorhexidine for the multispecies biofilm was higher than were those for the single-species biofilms of P. gingivalis or F. nucleatum.
To mimic the natural dental biofilm, a multispecies biofilm composed of four bacterial species was grown. The 24-hour multispecies biofilm may be useful as a laboratory dental biofilm model system.
Graphical Abstract
Biofilms; Microbial sensitivity tests; Microbiology; Periodontitis
Related Articles
Localization of the Fusobacterium nucleatum T18 adhesin activity mediating coaggregation with Porphyromonas gingivalis T22.
Kinder, S A
Holt, S C
Journal of Bacteriology
Adherence of pathogenic bacteria is often an essential first step in the infectious process. The ability of bacteria to adhere to one another, or to coaggregate, may be an important factor in their ability to colonize and function as pathogens in the periodontal pocket. Previously, a strong and specific coaggregation was demonstrated between two putative periodontal pathogens, Fusobacterium nucleatum and Porphyromonas gingivalis. The interaction appeared to be mediated by a protein adhesin on the F. nucleatum cells and a carbohydrate receptor on the P. gingivalis cells. In this investigation, we have localized the adhesin activity of F. nucleatum T18 to the outer membrane on the basis of the ability of F. nucleatum T18 vesicles to coaggregate with whole cells of P. gingivalis T22 and the ability of the outer membrane fraction of F. nucleatum T18 to inhibit coaggregation between whole cells of F. nucleatum T18 and P. gingivalis T22. Proteolytic pretreatment of the F. nucleatum T18 outer membrane fraction resulted in a loss of coaggregation inhibition, confirming the proteinaceous nature of the adhesin. The F. nucleatum T18 outer membrane fraction was found to be enriched for several proteins, including a 42-kDa major outer membrane protein which appeared to be exposed on the bacterial cell surface. Fab fragments prepared from antiserum raised to the 42-kDa outer membrane protein were found to partially but specifically block coaggregation. These data support the conclusion that the 42-kDa major outer membrane protein of F. nucleatum T18 plays a role in mediating coaggregation with P. gingivalis T22.
Related Articles
Identification and Localization of Extraradicular Biofilm-Forming Bacteria Associated with Refractory Endodontic Pathogens
Noguchi, Nobuo
Noiri, Yuichiro
Narimatsu, Masahiro
Ebisu, Shigeyuki
Applied and Environmental Microbiology
Bacterial biofilms have been found to develop on root surfaces outside the apical foramen and be associated with refractory periapical periodontitis. However, it is unknown which bacterial species form extraradicular biofilms. The present study aimed to investigate the identity and localization of bacteria in human extraradicular biofilms. Twenty extraradicular biofilms, used to identify bacteria using a PCR-based 16S rRNA gene assay, and seven root-tips, used to observe immunohistochemical localization of three selected bacterial species, were taken from 27 patients with refractory periapical periodontitis. Bacterial DNA was detected from 14 of the 20 samples, and 113 bacterial species were isolated. Fusobacterium nucleatum (14 of 14), Porphyromonas gingivalis (12 of 14), and Tannellera forsythensis (8 of 14) were frequently detected. Unidentified and uncultured bacterial DNA was also detected in 11 of the 14 samples in which DNA was detected. In the biofilms, P. gingivalis was immunohistochemically detected in all parts of the extraradicular biofilms. Positive reactions to anti-F. nucleatum and anti-T. forsythensis sera were found at specific portions of the biofilm. These findings suggested that P. gingivalis, T. forsythensis, and F. nucleatum were associated with extraradicular biofilm formation and refractory periapical periodontitis.
Related Articles
Immunomodulating effect of fosfomycin on gut-derived sepsis caused by Pseudomonas aeruginosa in mice.
Matsumoto, T
Tateda, K
Miyazaki, S
Furuya, N
Ohno, A
Ishii, Y
Hirakata, Y
Yamaguchi, K
Antimicrobial Agents and Chemotherapy
We evaluated the protective effect of fosfomycin (FOM) and an enantiomer of fosfomycin [FOM (+); an isomer of FOM with no bactericidal activity] on murine gut-derived sepsis caused by Pseudomonas aeruginosa. Endogenous bacteremia was induced by administering cyclophosphamide (CY) and ampicillin to specific-pathogen-free mice fed P. aeruginosa. Treatment of mice with FOM at 250 mg/kg of body weight per day twice a day after the second CY administration significantly increased the survival rate compared to that for control mice treated with saline. Treatment with FOM (+) at 20 and 100 mg/kg also significantly increased the survival rate (from 30% for control mice to 80% for treated mice). The bacterial counts in the liver and blood were both significantly lower in FOM(+)-treated mice in comparison with those in liver and blood of saline-treated control mice. FOM(+) administration affected neither the bacterial colonization in the intestinal tract nor the leukocyte counts in the peripheral blood of the mice. After intravascular inoculation of P. aeruginosa, treatment of mice with FOM (+) did not enhance bacterial clearance from the blood of mice pretreated or not enhance bacterial clearance from the blood of mice pretreated or not pretreated with CY, FOM(+) significantly suppressed tumor necrosis factor alpha, interleukin-1 beta, and interleukin-6 levels in the serum of mice after gut-derived sepsis. These results indicate that both FOM and FOM(+) have protective effects against P. aeruginosa bacteremia, despite a lack of specific activity of FOM(+), and suggest that FOM may possess immunomodulating activity and that it induces a protective effect. The protective mechanism is speculated to be that FOM modulates the vivo production of inflammatory cytokines.
Related Articles
RNA-oligonucleotide quantification technique (ROQT) for the enumeration of uncultivated bacterial species in subgingival biofilms
Teles, F.R.F.
Teles, R.P.
Siegelin, Y.
Paster, B.
Haffajee, A.D.
Socransky, S.S.
Molecular oral microbiology
Approximately 35% of the species present in subgingival biofilms are as yet uncultivated, so their role in periodontal pathogenesis is unknown. The aim of the present study was to develop a high throughput method to quantify a wide range of cultivated and uncultivated taxa in subgingival biofilm samples associated with periodontal disease or health. Oligonucleotides targeting the 16S ribosomal DNA gene were designed, synthesized and labeled with digoxigenin. These probes were hybridized with the total nucleic acids of pure cultures or subgingival biofilm samples. Target species included cultivated taxa associated with periodontal health and disease, as well as uncultivated species, such as TM7 sp OT 346, Mitsuokella sp. OT 131 and Desulfobulbus sp. OT 041. Sensitivity and specificity of the probes were determined. A Universal probe was used to assess total bacterial load. Sequences complementary to the probes were used as standards for quantification. Chemiluminescent signals were visualized after film exposure or using a CCD camera. In a pilot clinical study, 266 subgingival plaque samples from eight periodontally healthy people and 11 patients with periodontitis were examined. Probes were specific and sensitivity reached 104 cells. Fusobacterium nucleatum ss polymorphum and Actinomyces gerencseriae were the most abundant cultivated taxa in clinical samples. Among uncultivated/unrecognized species, Mitsuokella sp. OT 131 and Prevotella sp. OT 306 were the most numerous. Porphyromonas gingivalis and Desulfobulbus sp. OT 041 were only detected in patients with periodontitis. Direct hybridization of total nucleic acids using oligonucleotide probes permitted the quantification of multiple cultivated and uncultivated taxa in mixed species biofilm samples.
bacteria; biofilms; oral; periodontal; uncultivated
Related Articles
Phototoxic effect of blue light on the planktonic and biofilm state of anaerobic periodontal pathogens
Song, Hyun-Hwa
Lee, Jae-Kwan
Um, Heung-Sik
Chang, Beom-Seok
Lee, Si-Young
Lee, Min-Ku
Journal of Periodontal & Implant Science
The purpose of this study was to compare the phototoxic effects of blue light exposure on periodontal pathogens in both planktonic and biofilm cultures.
Strains of Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, and Porphyromonas gingivalis, in planktonic or biofilm states, were exposed to visible light at wavelengths of 400.520 nm. A quartz-tungsten-halogen lamp at a power density of 500 mW/cm2 was used for the light source. Each sample was exposed to 15, 30, 60, 90, or 120 seconds of each bacterial strain in the planktonic or biofilm state. Confocal scanning laser microscopy (CSLM) was used to observe the distribution of live/dead bacterial cells in biofilms. After light exposure, the bacterial killing rates were calculated from colony forming unit (CFU) counts.
CLSM images that were obtained from biofilms showed a mixture of dead and live bacterial cells extending to a depth of 30-45 µm. Obvious differences in the live-to-dead bacterial cell ratio were found in P. gingivalis biofilm according to light exposure time. In the planktonic state, almost all bacteria were killed with 60 seconds of light exposure to F. nucleatum (99.1%) and with 15 seconds to P. gingivalis (100%). In the biofilm state, however, only the CFU of P. gingivalis demonstrated a decreasing tendency with increasing light exposure time, and there was a lower efficacy of phototoxicity to P. gingivalis as biofilm than in the planktonic state.
Blue light exposure using a dental halogen curing unit is effective in reducing periodontal pathogens in the planktonic state. It is recommended that an adjunctive exogenous photosensitizer be used and that pathogens be exposed to visible light for clinical antimicrobial periodontal therapy.
Biofilms; Dental curing lights
Related Articles
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Posted: 07/01/2013
Stephen thinks it's just unnatural that Beyonce, a young, vital singer at the top of her game, is singing at the Superbowl.
Tags: 5 x five, exclusives, mashups, celebrities, beyonce knowles, football, scandals, barack obama, joe biden, inaugurations, pop culture, the word, united nations, kofi annan, jay z, sports illustrated, super bowl
Posted: 12/14/2012
Eugene Mirman combines his two passions.
Tags: eugene mirman, an evening of comedy in a fake underground laboratory, music, flying, security, animals, sci-fi, balls, kofi annan
Posted: 09/17/2012
Kofi Annan enjoys greater diplomatic flexibility as a special envoy than he did Secretary-General.
Tags: kofi annan, exclusives, interviews, extended interviews, military, wars, international affairs, middle east, diplomacy, religion, diversity, ethnic, united nations, work/office, books, muslim
Posted: 09/17/2012
"Interventions: A Life in War and Peace," author and diplomat Kofi Annan discusses our messy world.
Tags: kofi annan, interviews, international affairs, peace, wars, syria, middle east, diplomacy, government, violence, democracy, russia, united nations, books
Posted: 09/17/2012
Kofi Annan predicts a miscalculation in Syria will have far-reaching global consequences.
Tags: kofi annan, exclusives, interviews, extended interviews, international affairs, peace, wars, syria, middle east, diplomacy, government, violence, democracy, russia, united nations, books
Posted: 09/17/2012
Middle Eastern populations critique YouTube's "The Innocence of Muslims" by burning embassies.
Tags: internet, technology, twitter, middle east, international affairs, youtube, movies, protests, rage, violence, controversies, security, democracy, public indecency, my mistake, monty python, fires, jewish, racism, religion, conspiracies, extremism, tosh.0, photos, kofi annan, muslim, cats
Posted: 12/18/2006
The United Nations swears in the hilariously named Ban Ki-moon.
Tags: united nations, kofi annan, ban ki-moon
Posted: 12/05/2006
Jon promotes the Friar's Club roast of John Bolton.
Tags: john bolton, kofi annan, united nations
Posted: 08/22/2006
The U.N. is supposed to be lame -- that's why we put John Bolton there!
Tags: the word, lebanon, france, wars, charlene, jimmy, kofi annan, united nations, balls, jay z
Posted: 05/09/2006
Author Eric Shawn tells Jon what is needed to reform the U.N.
Tags: interviews, eric shawn, united nations, fox news, saddam hussein, kofi annan, security
Posted: 02/21/2006
Sarah Vowell discusses the soul-crushing sadness of the Bush administration.
Tags: interviews, sarah vowell, new york times, npr, george w. bush, kofi annan, state of the union, george h.w. bush, jeb bush, bill clinton
Posted: 02/21/2006
Iraqi President Jalal Talabani gets many microphones angry.
Tags: abu ghraib, bush administration, donald rumsfeld, scott mcclellan, iraq, lynndie england, guantanamo bay, kofi annan, al jazeera
Posted: 09/15/2005
President Bush toasts the U.N. from the prepared remarks, or possibly, the heart.
Tags: united nations, evolution, kofi annan, george w. bush, condoleezza rice, evolution schmevolution, security
Posted: 08/10/2004
Tom Cruise and idealistic Oregonians on tomorrow's show.
Tags: tom cruise, ed helms, ralph nader, kofi annan, jimmy carter
Posted: 10/20/2003
The pussies at the UN approve sending more troops to Iraq.
Tags: headlines, united nations, donald rumsfeld, colin powell, iraq, kofi annan, security, voting
Posted: 07/15/2003
George W. Bush would never trust danged good intelligence.
Tags: mess o'potamia, iraq war, george w. bush, kofi annan
Posted: 11/14/2002
Kofi Annan talks while Bush spreads his legs.
Tags: george w. bush, kofi annan, united nations, iraq
Posted: 11/14/2002
George W. Bush listens with open legs.
Tags: moment of zen, george w. bush, kofi annan, iraq
Posted: 06/25/2002
It almost seems as if the United Nations has a terrible publicist.
Tags: david scheffer, united nations, george w. bush, middle east, kofi annan, palestine, interviews
Posted: 05/02/2002
The new Arafat looks a lot like the old.
Tags: israel, palestine, yasser arafat, kofi annan, united nations
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Be sure to check out our July 2016 issue. Click on the link to the left for a preview of the issue. Members and susbscribers can peruse the issue from cover to cover by logging in as you would log into www.rses.org. If you are not a Member or paid subscriber, feel free to visit the RSES Journal subscription page or the RSES membership information page. Happy reading!
A t the 2015 Annual Business Meeting in Rosemont, IL
last year, the Nominating Committee asked me to
serve a second year as the International President to
help usher in a new business model for our organization. I
agreed to do so. read more


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Mercure Versailles Parly 2, Yvelines (department)
- Free WiFi and free parking
Main amenities
- 89 guestrooms
- Breakfast available
- Bar/lounge
- 24-hour front desk
- Air conditioning
- Daily housekeeping
- Front desk safe
- Laundry service
- Conference space
- Free newspapers in the lobby
- Luggage storage
For families
- Free cribs/infant beds
- Private bathroom
- Daily housekeeping
- Laundry facilities
- Hair dryer
- Blackout drapes/curtains
What's around
- Palace of Versailles (2.3 mi / 3.7 km)
- Parly 2 Shopping Mall (0.4 mi / 0.6 km)
- Theatre Andre Malraux (5.7 mi / 9.1 km)
- Roland Garros (7.9 mi / 12.8 km)
- Chateau de Monte-Cristo (4.6 mi / 7.4 km)
Mercure Versailles Parly 2, Yvelines (department)
Hotel Info: 866-539-8117from $95
- Superior Double Room
- Standard Room, 1 Double Bed
- Superior Room, 1 Queen Bed
- Standard Room, 1 Double Bed
- Privilege, Room, 1 Double Bed
Also popular with travelers from the USA
At a glance
Key facts
Hotel size
- This hotel has 89 rooms
- This hotel is arranged over 4 floors
- Check-in time starts at noon
- Check-out time is noon
Required at check in
- Credit card or cash deposit required
- Government-issued photo ID required
Travelling with others
- Pets allowed *
- Free WiFi in public areas
- Free WiFi in rooms
- Free self parking
Other information
- Resort fee included
Nearby Four Star Yvelines (department) hotels
In the hotel
- Buffet breakfast daily (surcharge)
- Bar/lounge
- Room service (during limited hours)
- Conference space
- Conference space size (feet) - 430
- Conference space size (meters) - 40
- 24-hour front desk
- Dry cleaning/laundry service
- Laundry facilities
- Free newspapers in lobby
- Luggage storage
- Number of buildings/towers - 1
- Elevator/lift
- Safe-deposit box at front desk
In the room
- In-room climate control (air conditioning)
- Air conditioning
- Minibar
- Iron/ironing board (on request)
- Blackout drapes/curtains
- Private bathroom
- Shower/tub combination
- Hair dryer
- Flat-screen TV
- Satellite TV channels
- Desk
- Free newspaper
- Free WiFi
- Daily housekeeping
- In-room safe
Top Yvelines (department) hotels
Recently reviewed hotels in Yvelines (department)
- Hôtel de France InvalidesFrom
"We enjoyed our stay at this hotel. The staff was very friendly and helpful. The rooms were a bit small but always cleaned well daily. The morning breakfast buffet was excellent…"Jun 18, 2016
- Hilton Paris Charles de Gaulle AirportFrom
"Stayed here overnight because of an early flight out of CDG the following morning. Checked in around noon and a room was ready for us. The RER train station is right next to the…"Jun 18, 2016
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"All of the staff here are friendly, helpful and have excellent English. Location is good for Latin Quarter restaurants and not too far from many attractions like Luxembourg…"Jun 18, 2016
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DiningLe Miroir - This bar serves light fare only.
Mercure Versailles Parly 2, Yvelines (department)'s small print
Also known as
- Mercure Parly 2
- Mercure Parly 2 Hotel
- Mercure Parly 2 Hotel Versailles
- Mercure Versailles Parly 2
- Mercure Le Chesnay
- Mercure Versailles Parly 2 Hotel Le Chesnay
- Mercure Versailles Parly 2 Le Chesnay, France
- Mercure Versailles Parly 2 Hotel
- Mercure Versailles Parly 2 Le Chesnay
Mandatory fees
The charges below are included in the total price displayed:
- Resort fee
Optional extras
Buffet breakfast is offered for an extra charge of EUR 17.50 for adults and EUR 8.00 for children (approximately)
Pets are allowed for an extra charge of EUR 10.00 per pet, per night
We have included all charges provided to us by this hotel. However, charges can vary, for example, based on length of stay or the room you book.
Recent reviews
Good 3.6 /5 from 124 reviews
Good hotel, but only for one night. There was a ugly smell, like mold in the carpet. The room we got was smoke free, however the staff smokes right in front so when we open the window all their cigarette smell went into our room. Staff was excellent, friendly and helpful. The breakfast was over priced. The discount at the Del Arte Pizza was nice, we received 20% off our bill.
The hotel was about 5 miles from Versailles and well priced for the area. It had a restaurant onsite and a mall just around the corner. The pizzeria, located at the mall, offered a 20% discount for hotel guests. There was also onsite parking. The rooms were nice and included a fridge. There were a few tv channels in English, as well. The front desk staff was very nice and provided suggestions regarding local restaurants and directions to Paris. The front desk staff also spoke English. I would stay at this hotel again.
Room clean and comfortable. Hotel close to Versailles with onsite parking. Staff was friendly, gave advice regarding where to eat and how to drive to Paris. Walking distance to a mall with restaurants (you receive a 20% off at a local pizzeria).
The staff was very pleasant and helpful . Buffet breakfast in hotel was convenient as well as delicious. Hotel conveniently located near shopping mall. Bus stop 1 block away. Much cheaper than the hotels right by the Versaille.
The office was nice. Allowed us to check in early and was extremely helpful. Bar was closed early.
Is the description of this hotel not correct? Tell us


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Links to the Kentucky Academic Standards (KAS) in Arts and Humanities and other documents of interest are located on this page.
Kentucky Academic Standards
Kentucky Academic StandardsAdministrative regulation 704 KAR 3:303 Required Kentucky Academic Standards (KAS) was adopted into law as of June 2013. Curriculum translates these standards and considers all the steps (curriculum mapping, unit development, materials and resources, unit assessments, etc.) it will take to meet these standards. The Continuous Instructional Improvement Technology System (CIITS) provides electronic access to the Kentucky Academic Standards and a lesson planning tool and scheduler to help teachers manage standards-based instruction in the classroom. CIITS directly links and aligns high quality, multi-media instructional resources. These classroom materials are designed to engage students in learning and reinforce the standards being taught. Teachers may also share instructional resources they design through CIITS.


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Steve Martin 2016
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Groggy frogSports Bars, Karaoke, Bar
129 cross street
(860) 378-0043
Dan C.
Despite its dive appearance upon entering, I was pleasantly surprised with the Frogg. I had heard lots of hype about their wings, so I insisted for a group...read more
Joseph C.
I didn't get the chance to try the food but the bar was excellent! I grew up in Southington in the 70s & 80s when there were a number of great bars serving...read more
Eric D.
Stopped by with a bunch of guys after our union meeting for a few beers and some wings. Sign out front said best wings 2015-2016 from wing fest, so we had...read more
Sarah G.
Karaoke with Joey G. On Thursday nights its the best! Stacey and Brian pouring you drinks and keeping you toasty! Karaoke on Fridays and Sundays with Karaoke Mike!read more
Love the Frog!read more
Half price wings all day on Sunday!! Best wings in town!read more
Best wings in town!!read more
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Don't break glasses!read more
Best wings in town!!! Best burgers too!!!read more
Half price on the award winning wings every Sunday....read more
stay away: Not good at all, very filthy- poor service unless you are a local bar fly.It really is as dirty on the inside as it looks from the outside. One word to sum it up- Gross!read more
: An awesome place, good food and service.My place to go for food and friends.read more
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text:
We stayed here for one night. It's a nice hotel. The staff was friendly and helpful, our room was neat and clean. We really liked the heated indoor pool, we just wish it was bigger as it got a little crowded in the evening. The location is good, there are plenty of restaurants close by. Our only complaint would be that the walls are a little thin. We could easily hear our next door neighbors talking and plenty of door slamming out in the hallway. Luckily we always travel with a white noise generator to drown out excess noise. Breakfast was good in the morning, the sausage gravy was great!
- Reservation Options:
- TripAdvisor is proud to partner with Booking.com, Carlson, Hotels.com, Priceline, Expedia, Travelocity, Odigeo and Hotwire so you can book your Country Inn & Suites By Carlson, Princeton reservations with confidence. We help millions of travelers each month to find the perfect hotel for both vacation and business trips, always with the best discounts and special offers.
- Also Known As:
- Country Inn And Suites Princeton
- Country Inn & Suites Princeton Hotel Princeton


text:
Podcast: We Have Brown, We Have Tea, We Have Bipartisanship (Not)
Republicans now have 41. The President is bipartisan, except when he's not. Illinois voters have spoken; we just don't know what they said. Ted Deutch (D-FL) is in, soon. So is John Boozman (R-AR), soon. And we'll hear from Sarah Palin (Tea Party-Earth), soon.
Sounds like another confusing episode of our "It's All Politics" podcast.
Credits (as it were) —
Nonstop talkers: Ron Elving and Ken Rudin
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26 - Pk. Bucket of Food Supply Depot Breakfast Variety
Item # WX2-293533SOLD OUT
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26 - Pk. Bucket of Food Supply Depot Breakfast Variety keeps your bellies full and minds sharp in case of food shortages, emergency or otherwise.
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text:
Yo Gabba GabbaSat, November 24, 2012 - 2:00 PMHonda Center in Anaheim, CA
We are sorry, the date for this event has already passed. This event took place on 11/24/2012. Click here to view upcoming events for Yo Gabba Gabba.
Still unsure? Check out our user reviews for:
Have a question? Need help? Call us at 800.477.5285


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SOUTH BEND, Ind. -- A Florida man has pleaded guilty to a
misdemeanor charge of leaving a threatening telephone message for
then-Notre Dame football coach Tyrone Willingham, saying he was
drunk when he did it.
Andrew French, 26, of Fort Myers, Fla., pleaded guilty Wednesday
in exchange for the government dismissing a felony charge against
him for the November 2003 message left on Willingham's voice mail
while he was still coach at Notre Dame.
French said in court that he was drunk and under peer pressure
when he made the call. French said he did not use good judgment and
said "terrible things" during the 4 a.m. call, including using
racially charged terms about Willingham, now the coach at
Washington, and threatening to burn a cross in the coach's yard.
French admitted to a charge of interfering with Willingham's
ability to enjoy his employment because of threats of harm related
to his race or color.
French told U.S. District Court Magistrate Judge Christopher
Nuechterlein he did not remember making the call after it occurred
but had listened to the tape twice.
"It makes me sick to think I did it," he said.
French has taken part in a pretrial diversion program for about
18 months. Nuechterlein took the guilty plea under advisement,
giving French until July 19 to withdraw the plea. If he does not
withdraw the plea, French faces a maximum penalty of up to a year
in jail and a fine up to $250,000.
The Associated Press left a message at Washington's sports
information department seeking comment from Willingham.


text:
Crazy Substitute Teachers
Enjoy, NG4.02 / 5.00 2,167 Views
Vacuum Salesman
This dude and his Megazord vacuum cleaner4.00 / 5.00 2,624 Views
Markiplier Animated | Mark Of The Survey Corps
This took far too long to make...3.97 / 5.00 5,605 Views
now my favorite out of this series.
Let's just be done with it
, by knocking this beautiful bastard's flash up to a perfect five.
Any objections?
Hahaha Hilarious
That was a great characterization of Morrigan. Amazing voice syncing and the facial experessions on Morrigan was amazing awesome super fun time! I was distracted by the boob jiggling. So I had to watch it 10 more times. lol
Hsien-ko next please!! Show us all the stuff she's got hiding in her sleeves!
While I love Johny Utah's Iron Man one this is by far the best one. THE PUBLIC DEMANDS MORE. LOL Seriously though great job
Nice work in grabbing my attention, it was longer then I thought it would be, but in a good way. Cheers all around, this was more on Morrigan then Dante, rofl quite a twist.


text:
The Effingham County High School chapter of the National Honor Society has had the privilege to serve its community by volunteering for Habitat for Humanity over the last couple of months.
The Effingham County Board of Education expects at its December meeting to approve the calendars for the next two school years.
Not every student gets to design, build and test robots in class.
People in Effingham County, and across the state, can give feedback to education leaders on proposed revisions to the Common Core Georgia Performance Standards.
Honor rolls for the first nine-week grading period for Effingham County's eight elementary schools:
Honor rolls for the first nine-week grading period for Effingham County's three middle schools:
Honor rolls for the first nine-week grading period for Effingham County's two high schools:
One tenet of the Effingham College and Career Academy is that preparing students for careers is not a one-size-fits-all process.
Students from Grace Academy are learning at a young age to help others in their community.
More than 300 high school students in Effingham County were honored last week for completing Georgia-Pacific's STEM Scholars program.
More than a hundred South Effingham High School students took advantage of a recent opportunity to apply to college.
SAVANNAH - Fire science students at Savannah Technical College may expand their first responder training with EMS certifications with a new firefighter/EMS diploma.
Georgia Southern University has announced a $225,000 contribution from Gulfstream Aerospace to support research and education at the university. This gift continues Georgia Southern's long relationship with Gulfstream, manufacturer of the world's most technologically advanced business-jet aircraft, which began with former Gulfstream president and CEO Allen E. Paulson.
Ebenezer Middle School's Eagle Winds honor band has been chosen to perform at the 24th annual University of Georgia Middle School Band Festival in December.
A group of Effingham County Middle School students participated in Georgia Southern University's Global Village Competition, as part of the International Festival hosted by the Center for International Studies.
Page 23 of 201
It was a big night at The Corral for the South Effingham High School class of 2016 and graduates entering the military went to the ...
It was smiles and hugs all around Rebel Field on Friday night as the Effingham County High School class of 2016 received its diplomas.
The Effingham County High School Drama Club brought the house down April 29 and 30, with its musical version of "Beauty and the Beast" by ...
ROME-The following area students were named to the spring 2016 dean's list at Berry College:
Berry College announces that Eric Eaton of Rincon earned a BMUS in Music with Elective Studies in Business.
TIFTON-Students who achieved academic excellence in their course work during the spring term were recently recognized at Abraham Baldwin Agricultural College. ABAC cites its ...
Dr. Bonita C. Jacobs, president of UNG, recognized more than 1,200 students who earned a 4.0 during the spring 2016 semester.
HOUSTON-Roan Cowart, a 2006 South Effingham High School graduate, was honored with a tool grant from 3M and the Collision Repair Education Foundation in ...
Page 4 of 4
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Some Things Are Best Left Unseen
by Mike Stanz
posted Jul 9 2014 5:00AM
Thank you, Justin Bieber, for posting a pelvis-up shot of your apparently naked self to Shots.
No one's quite sure what this shot is saying.
"Hey, I've got tattoos on my arm!"
"Hey, check out my gold chain!"
"Hey, I've got a cell phone with a camera and way too much time on my hands."
Whatever the reason, please, please, please! Don't ever do that again.
Thank you.
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BYU football: Hebron 'Loni' Fangupo finds good fit after sudden transfer
The stark environment change that Fangupo is enjoying isn't just outside of the football facilities, either. Through his time at BYU, he's found a unique environment provided by his coaches and teammates that has been beneficial.
"This team is a lot more close than we were at USC," he explained. "It was amazing after the Ole Miss game and at halftime how no one was complaining about their playing time or anything like that. It's all about the team, and that is what helps us be successful. There is a great attitude here that's an unselfish attitude."
Fangupo will finish up this season and then move on to other things. Given how well he's taken to BYU, one would think that he regrets not having arrived earlier — but he doesn't see it that way.
"All I can do is rely on the Lord," he said. "I prayed about where I should go after junior college, and even though BYU was sort of my dream school, the Lord told me that I needed to go to USC. I met my wife there at USC, the love of my life, so how could I regret that? It was meeting my wife at USC that got me to come here to BYU, along with prayer, and I couldn't be happier with how things have worked out."
Other players seeing their first action against Ole Miss:
- Houston Reynolds, 6-foot-2, 296-pound sophomore OL. Reynolds saw almost every rep at left guard after having won the spot during fall practices. It was assumed that there would be a lot of rotation at the OG positions, but the youngest Reynolds brother saw almost every rep at his position during the game.
- Preston Hadley, 6-foot-0, 200-pound junior CB. Hadley accomplished what any incoming JC player aims to do, which is to earn a starting spot. He won the starting boundary corner position during fall and provided very good coverage and run support against Ole Miss.
- Joe Sampson, 5-foot-10, 213-pound junior CB. Sampson rotated with Hadley at the BCB position and, like Hadley, he proved to be very effective throughout the course of the game.
- Ross Apo, 6-foot-3, 206-pound freshman WR. Although Apo saw limited time on special teams against Washington last year, Ole Miss was his first game playing reps at wide receiver. He responded well in catching the first touchdown and accounting for four receptions for 46 yards on the day.
- Uona Kaveinga, 5-foot-11, 236-pound junior LB. Kaveinga started at middle linebacker and recorded five total tackles against the Rebels.
- Austin Heder, 6-foot-1, 241-pound freshman FB/LB. Heder saw limited reps playing fullback during short-yardage situations.
- Jordan Johnson, 5-foot-10, 175-pound freshman CB/KR. Johnson returned one kickoff for 28 yards.
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Bhopal City Link Ltd Tenders
View all Tender Notifications issued by Bhopal City Link Ltd.Till date we found 1 Tender Notices published from Bhopal City Link Ltd.1 live business opportunities are available in BCLL Tenders section.Bhopal City Link Ltd Tenders further filtered by Bid Submission Date, Tender Value, Project Location & Product Category. Get email alert on Tenders from Bhopal City Link Ltd.
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Location: Madhya Pradesh - INDIA
Tender Value: Ref. Document
Selection Of Agency For Dismantling Of Old Bus Stops At Various Locations Of Bhopal City


text:
Spike Technologies, Inc. Overview
Spike Technologies, Inc. filed as an Articles of Incorporation in the State of California and is no longer active. This corporate entity was filed approximately twenty-two years ago on Monday, November 14, 1994 , according to public records filed with California Secretary of State.
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California Secretary of State
|Filing Type:
|Articles of Incorporation
|State ID:
|Date Filed:
|Monday, November 14, 1994
|Registered Agent
|Nikhil Modi
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Other companies at ths same address
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Spike Technologies, LLC
San Diego, CA
San Diego, CA
Officers: Rhonda S. Meyer , Sanjay Mehta and 2 others Qualcomm Global Trading Pte Ltd , William E. Keitel
California Secretary of State
Data last refreshed on Monday, July 25, 2016
Data last refreshed on Monday, July 25, 2016


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Car crash leaves thousands in La Habra without power
LA HABRA – A car crashed into a pole Monday night, downing a power line and leaving homes and businesses without electricity overnight, officials said.
The outage affected about 6,000 customers around the 500 block of South Cypress Street, according to Southern California Edison. Service was estimated to be restored by 3:30 p.m.
The single-car crash took place around 11 p.m., according to the La Habra Police Department. The driver complained of pain at the scene, but was not taken to a hospital.
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Oh I know. the MM must have thought of the following strategy to dupe the investors: 1 green day on Monday to throw unsuspecting longs who dont know whats coming for rest of the week, and then 4 days of redYou have been warned longs
Under-valued, un-loved, keep bashing, i'm buying. 1.50 near term
1 day green, 4 days red.if the gods in heaven are extra merciful on you longs, then 2 days green, 3 days red. you longs lose either way by end of the week. I would not trust this stock with your moneyEasy short. I know a stinking pile of POS when i see 1


text:
1165 Route 22 West #173, North Plainfield, NJ
(908) 756-1950
(908) 756-1950
Cypress Gardens is collection of 214 garden-style apartments located in the Somerset County Borough of North Plainfield.
These spacious apartments range from 850 to 1,075 square feet of living space and boast a variety of appointments including refrigerators, ovens and hardwood flooring throughout.
1165 US Highway 22 WestNorth Plainfield, NJ(908) 756-1950
"Everyone is very professional and they take the tenants requests and wants into careful consideration. There are not enough words to express thank you for Management and Maintenance."
- Janice M.
* Actual driving mileage may differ.


text:
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Artizan Street, Barbican, Shoe Lane, City Business, Guildhall, eLibrary and home delivery service.
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