Sequence conservation and distribution of the fusobacterial immunosuppressive protein gene,fipA

Fusobacterium nucleatum is a gram-negative anaerobe involved in various diseases, including periodontitis. Recently, other investigators isolated the F. nucleatum FDC 364 fusobacterial immunosuppressive protein (FIP). One subunit, FipA, impairs T-cell activation in vitro and shows homology with beta-ketothiolases. However, its distribution and variability among fusobacteria was not reported. Cloned fipA gene sequences from F. nucleatum ssp. polymorphum (ATCC 10953) and F. nucleatum ssp. nucleatum (ATCC 23726) shared 89 and 92% identity, respectively, with FDC 364 fipA, and 90 and 94% identity, respectively, with the FDC 364 FipA predicted amino acid sequence. Southern blot analyses of chromosomal DNA from fusobacterial strains, including F. nucleatum and other Fusobacterium species, were performed using partial fipA sequences as probes. The results indicate that fipA is highly conserved among the F. nucleatum strains examined and that fipA homologues are widely distributed among fusobacteria. A clear relationship between immune suppression, metabolism and the FipA protein remains to be determined.     

Fusobacterium nucleatum GroEL induces risk factors of atherosclerosis in human microvascular endothelial cells and ApoE(-/-) mice

Infection and inflammation are risk factors in the initiation and progression of atherosclerosis. Periodontitis is one of the most prevalent chronic inflammations of the oral cavity, and has been reported to be associated with systemic disease. In this study, we evaluated whether the heat-shock protein GroEL of Fusobacterium nucleatum, one of the most prevalent bacteria in periodontitis, induces factors that predispose to atherosclerosis in human microvascular endothelial cells (HMEC-1) and apolipoprotein E-deficient (ApoE(-/-)) mice. GroEL induced the expression of chemokines such as monocyte chemoattractant protein-1 and interleukin-8 as well as cell adhesion molecules, such as intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and E-selectin. GroEL induced the activity of tissue factor and reduced the activity of the tissue factor pathway inhibitor. Foam cell formation was induced by GroEL. GroEL-injected ApoE(-/-) mice showed significant atherosclerotic lesion progression compared with control mice. Serum levels of risk factors for atherosclerosis such as interleukin-6, C-reactive protein, and low-density lipoprotein were increased in GroEL-injected ApoE(-/-) mice compared with control mice, whereas serum levels of high-density lipoprotein were decreased. We could detect significantly higher levels of anti-F. nucleatum GroEL antibody in serum and F. nucleatum DNA in gingival crevicular fluid from patients with periodontitis than in that from healthy subjects. Our results indicate that the host response to the GroEL of periodontal pathogens like F. nucleatum may be a mechanism involved in atherosclerosis, supporting the association of periodontitis and systemic infection.     

具核梭杆菌与大肠癌关系的研究进展

牙周炎与全身健康和疾病关系密切。近年来,牙周炎与大肠癌（colorectal cancer,CRC）发生发展的关系已引起越来越多学者的关注。具核梭杆菌是牙周炎重要的致病菌之一,其感染可能参与了CRC的发生发展,本文拟就其研究进展作一综述。     

The distribution and frequency of Fusobacterium nucleatum subspecies in the human oral cavity

Three reference strains of Fusobacterium nucleatum and 32 human oral isolates were compared by a variety of physiological tests, enzyme electrophoretic profiles, SDS-PAGE patterns, DNA base composition and hybridization to test their possible site specificity and frequency of the recently described subspecies of F. nucleatum. Nine of the 11 isolates assigned to F. nucleatum subspecies nucleatum were from diseased sites, whereas isolates from healthy sites were all identified as F. nucleatum subspecies polymorphum or F. nucleatum subspecies fusiforme. Strains of the latter subspecies were the least frequently isolated (2 of 32). These results, although still inconclusive because of the relatively small sample size, nevertheless confirmed the heterogeneity of F. nucleatum and indicate that most human oral isolates from subgingival sites probably belong to F. nucleatum subspecies nucleatum.     

�������ھߺ���˾��������Էμ���������о�

目的检测慢性阻塞性肺疾病急性加重期（AE—COPD）患者呼吸道内具核梭杆菌（h．）的定植,探讨n．与AE—COPD间的关系。方法选择2008年10月至2009年4月中国医科大学附属第一医院、盛京医院确诊为AE—COPD的患者53例,采集呼吸道分泌物,提取细菌DNA,应用SYBR。GreenIPremixExTaq＇…模式的实时PCR技术,对n．进行定量检测,并计算其所占总菌的比例。同时记录患者FEVl占预计值比例（％）及口腔牙周指标。结果53例样本中有32例检出Fn．,Fn．检出率为60．38％,占总菌的比例为（44．58±16．47）％。Fn．相对含量与FEVl占预计值比例呈负相关关系（P〈0．05）。F凡．相对含量与简化口腔卫生指数及附着丧失水平呈正相关关系（P〈0．05）;与牙龈出血指数、探诊深度、缺失牙数无相关性（P〉0．05）。结论AE—COPD患者呼吸道内存在Fn,Fn相对含量随着肺功能的减弱而增加;Fn．与AE—COPD密切相关。     

Spontaneous gingival antibody production to Fusobacterium nucleatum outer membrane in patients with adult periodontitis

The local antibody response to Fusobacterium nucleatum outer membrane (FnOM) was analyzed in patients with adult periodontitis (AP) at the single cell level. Furthermore, we analyzed whether periodontal hygienic treatment could alter the antibody response. The number of IgG- and IgM-producing cells were investigated in gingival samples collected from 20 patients with AP. The patients were divided into 2 groups, before (BT, n =9) and after (AT, n =11) periodontal hygienic treatment. Four healthy gingival samples were used as controls. The results obtained showed that local antibody production against FnOM occurred in gingiva of patients with AP, but not in healthy gingiva. The IgG anti-FnOM was the predominant isotype observed. However, there was no statistically significant difference between the BT and AT groups. These results indicate that periodontal hygienic treatment was not sufficient to alter significantly the number of IgG- arid IgM-secreting cells present in gingival tissue of AP patients, but it promoted a reduction of IgG anti-FnOM secreting cells. The presence of anti-FnOM antibodies in AP but not in control patients indicates that this bacteria may play a role in the pathogenesis of periodontal disease.     

牙龈卟啉单胞菌和具核梭杆菌多克隆抗体的制备及应用效果评价

目的    制备高效价高纯度的牙龈卟啉单胞菌（P. gingivalis）和具核梭杆菌（F. nucleatum）多克隆抗体,并评价其应用效果。方法    将新鲜培养的P. gingivalis和F. nucleatum菌液灭活后与佐剂乳化混匀,作为抗原皮下多点注射免疫雄性新西兰大白兔,定期免疫并在抗体效价达到预期后采用颈动脉取血获得抗血清。采用间接ELISA法测定多克隆抗体交叉反应。应用硫酸铵沉淀法纯化多克隆抗体,并通过Western-blot方法鉴定抗体的纯度。荧光显微镜观察多克隆抗体应用于免疫荧光实验的效果。结果    免疫6周后,血清抗体效价达到1∶320 000 ~ 1∶640 000,相互之间未出现交叉反应,且纯度较高。免疫荧光实验中可观察到明显的荧光,并可见P. gingivalis的球杆状形态和F. nucleatum的短杆状形态。结论    成功制备了P. gingivalis和F. nucleatum多克隆抗体,其特异性良好,为后续的免疫学相关实验奠定了基础。     

Effects of the oral spirochete Treponema denticola on interleukin-8 expression from epithelial cells

This communication demonstrates that the interaction of the oral spirochete Treponema denticola 35405 with KB epithelial cells does not lead to the induction of interleukin-8 production as occurs with a variety of other bacteria. Utilizing the dentilisin protease mutant K1 of T. denticola, this property was demonstrated to be primarily a function of the expression of the protease by strain 35405.     

Fusobacterium nucleatum Binding to Complement Regulatory Protein CD46 Modulates the Expression and Secretion of Cytokines and Matrix Metalloproteinases by Oral Epithelial Cells

Background: Periodontitis is a chronic inflammatory disease that results in the destruction of the supporting tissues of the teeth. Gingival epithelial cells are an important mechanical barrier and participate in the host inflammatory response to periodontopathogens. The aim of the present study is to investigate the capacity of Fusobacterium nucleatum to bind to the complement regulatory protein CD46 expressed by oral epithelial cells and to determine the impact of the binding on the gene expression and protein secretion of interleukin (IL)‐6, IL‐8, and matrix metalloproteinase (MMP)‐9 by oral epithelial cells. Methods: Binding of recombinant human CD46 to the surface of F. nucleatum was demonstrated by immunologic assays. After stimulation of oral epithelial cells with F. nucleatum, gene expression was determined by real‐time polymerase chain reaction analysis while protein secretion was monitored by enzyme‐linked immunosorbent assays. Results: Heat and protease treatments of bacterial cells reduced CD46 binding. F. nucleatum–bound CD46 mediated the cleavage of C3b in the presence of factor I. Stimulating oral epithelial cells with F. nucleatum at a multiplicity of infection of 50 resulted in a significant upregulation of the gene expression and protein secretion of IL‐6, IL‐8, and MMP‐9 by oral epithelial cells. However, pretreating the epithelial cells with an anti‐CD46 polyclonal antibody attenuated the production of IL‐6, IL‐8, and MMP‐9 in response to F. nucleatum. Such an inhibitory effect was not observed with non‐specific antibodies. Conclusions: The present study demonstrates that F. nucleatum can bind the complement regulatory protein CD46. The interaction of F. nucleatum with epithelial cell surface CD46 may contribute to increasing the levels of proinflammatory mediators and MMPs in periodontal sites and consequently modulate tissue destruction.     

Purification and characterization of a 65-kilodalton diisopropylfluorophosphate-binding protein in the outer membrane of Fusobacterium nucleatum Fev1

Abstract— A 65-kilodalton protein was identified in the outer membrane of Fusobacterium nucleatum Fevl by SDS-PAGE. The relative amount of the protein varied during growth, being greatest in stationary phase. The protein was radio-labeled by [¹²⁵I]-lactoperoxidase treatment of live cells and was only partially cxtractable with 2% sodium dodecylsulfatc (SDS) at room temperature, and therefore assumed to be both exposed to the cell surface and peptidoglycan associated. In intact cells the protein bound diisopropylfluorophosphate (DFP), indicating that it may be a serine protease. DFP-binding activity depended apparently on proper localization of the proteins in the membrane. Several methods were tried in attempts to purify the 65-kilodalton protein; the method that gave best results was preparative SDS-polyacrylamide gel electrophoresis. The isoelectric point was determined to about pH 5. Amino acid composition analysis showed that the 65-kilodalton protein possesses an excess of hydrophilic over hydrophobic residues, polarity index 52%. The N -terminal end of the protein was apparently blocked.     

Effects of the oral spirochete Treponema denticola on interleukin‐8 expression from epithelial cells

This communication demonstrates that the interaction of the oral spirochete Treponema denticola 35405 with KB epithelial cells does not lead to the induction of interleukin‐8 production as occurs with a variety of other bacteria. Utilizing the dentilisin protease mutant K1 of T. denticola, this property was demonstrated to be primarily a function of the expression of the protease by strain 35405.     

Stimulation of Fusobacterium nucleatum biofilm formation by Porphyromonas gingivalis

BACKGROUND/AIMS: Bacterial infection is a major cause of periapical periodontitis. Eradication of these microorganisms from apical lesions is essential to the success of endodontic treatment. The aim of this study was to clarify the molecular interaction between Fusobacterium nucleatum, Porphyromonas gingivalis and other microorganisms associated with periapical periodontitis. METHODS: Microorganisms isolated from periapical lesions were inoculated into type-I collagen-coated polystyrene microtiter plates and maintained at 37 degrees C under anaerobic conditions for 2 days, after which, the quantity of organized biofilm on the plates was evaluated by crystal violet staining. Growth enhancement via soluble factor was evaluated by separated coculture using a 0.4-mum membrane filter. RESULTS: F. nucleatum exhibited strong adherence to type-I collagen-coated polystyrene microplates. Biofilm formation by F. nucleatum was significantly enhanced by P. gingivalis. It was complemented by compartmentalized coculture with P. gingivalis. Enhancement of biofilm formation by P. gingivalis was only slightly reduced by inactivation of its autoinducer-2-producing gene luxS. CONCLUSION: The results suggest that P. gingivalis enhances biofilm formation by F. nucleatum by releasing diffusible signaling molecules other than autoinducer-2.     

Growth conditions and outer membrane proteins of Fusobacterium nucleatum

Abstract – Fusobacterium nucleatum strains ATCC 10953, ATCC 25586, F1 F3, F6, and Fevl were grown in different media. The influence of growth conditions on the outer membrane proteins (OMPs) was analyzed by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE). There were no apparent differences in the outer membrane protein profiles when cells in the same phase of growth in various rich media were compared. Differences, however, were observed between early logarithmic phase and stationary phase cells. Thus several proteins were only synthesized during late logarithmic and stationary phase. Synthesis of some of these proteins, in particular a 65K and a 14K protein, seemed to depend on the presence of peptides in the medium. In a complete medium, these proteins were synthesized after depletion of some amino acids, and peptides were then utilized.     

Oral bacterial DNA differ in their ability to induce inflammatory responses in human monocytic cell lines

Background: Deoxyribonucleic acids (DNA) of periodontal pathogens, Porphyromonas gingivalis (Pg) and Tannerella forsythia, stimulate cytokine production in human monocytic cells (THP‐1) through Toll‐like receptor 9 (TLR‐9) and nuclear factor‐κB signaling. Fusobacterium nucleatum (Fn) is one of the most frequently isolated bacteria in periodontally diseased tissues and is reported to synergize with Pg, enhancing the pathogenicity. We investigate inflammatory mediator production in THP‐1 cells challenged with Fn and Streptococcus sanguinis (Ss) DNA, a non‐pathogenic oral bacteria, and further assess whether cytokines triggered by whole pathogens or Pg lipopolysaccharide (LPS) are affected by TLR‐9 signaling inhibitors (chloroquine). Methods: THP‐1 cells were stimulated with Pg‐DNA (100 ng/μL), Fn‐DNA (100 ng/μL), Ss‐DNA (100 ng/μL), Pg‐LPS (10 ng/μL), and heat‐killed whole bacteria (multiplicity of infection, 1:100) for 16 hours with or without chloroquine pretreatment (10 μg/mL). Interleukin (IL)‐1β, IL‐6, IL‐8, and tumor necrosis factor‐α levels were determined using enzyme‐linked immunosorbent assay. Statistical analyses included analysis of variance with multiple comparisons using Dunnett or Tukey methods and paired t test. A value of P <0.05 was significant. Results: Inflammatory mediator levels were increased in response to all the stimuli with the exception of Ss‐DNA (P <0.05). Chloroquine pretreatment significantly decreased cytokine production from THP‐1 cells with the exception of IL‐6 production triggered by whole Fn and Ss (P <0.05). Conclusions: Differences exist among oral bacterial DNA in inducing immune responses. By altering the conditions in cytosolic compartments, we can interfere with cellular responses triggered by extracellular receptor activation. Thus, alternative treatment approaches targeted to intracellular receptors might be of benefit in controlling periodontal inflammation.     

Intra-familial distribution of Fusobacterium nucleatumstrains in healthy families with optimal plaque control

Fusobacterium nucleatum, a Gram-negative anaerobic rod associated with periodontal disease, is also found in healthy individuals and is considered part of the indigenous oral microflora. Although intra-familial transmission of periodontal pathogens has been documented, there are no data relating transmission of F. nucleatum. This study investigated the distribution of F. nucleatum strains in 4 strictly healthy families. 32 F. nucleatum strains were isolated from 19 individuals (8 parents and 11 children aged 1-13 years). DNA was extracted and digested with the restriction endonucleases EcoRI, TaqI and HindIII. The digests were separated by electrophoresis through 0.8% agarose gels at 40 V overnight, in TBE buffer containing 1 microg/ml ethidium bromide, and photographed. The DNA was transferred to nylon filters by Southern blotting and hybridized with a digoxigenin labelled E. coli rRNA probe (Kit Dig DNA Labelling mixture - Boehringer). Probed DNA was visualized colorimetrically (CSPD Luminescent Detection Kit Boehringer) and photographed (Amersham). We found that 10/11 children shared identical ribotypes with at least one of their respective parents. Some of the children also harbored a unique additional ribotype. On the basis of indistinguishable restriction endonuclease and ribotype patterns these results support the hypothesis that intra-familial transmission of F. nucleatum is possible.     

Aspects of the growth and metabolism of Fusobacterium nucleatum ATCC 10953 in continuous culture

Fusobacterium nucleatum ATCC 10953, a type strain of one of the newly proposed subspecies of this group of organisms, was grown anaerobically in continuous culture in a chemically defined medium. Its response to conditions of varying pH, nutritional environment, and imposed growth rate were then examined. The organism failed to grow at pH 7.8 but grew at pH 5.8, although the cell yield was greatly reduced. At pH 6.8 the cell yield was halved and less than 50% of available glucose was consumed. The optimum growth pH was around 7.4 when the culture appeared to be limited for both glucose and the amino acids glutamate, histidine and serine. Some intracellular polyglucose (IP) was produced and acetate, butyrate and ammonia were the major fermentation end-products, as they were under all growth conditions tested. Increasing the available glucose or amino acids did not alter cell numbers but the amount of IP was greatly increased. When glucose was omitted from the medium, the cell yield was halved and the culture then became limited for lysine as well as for glutamate, histidine and serine. Growth rate had little overall effect on the organism's physiology and the maximum growth rate at pH 7.4 was 0.20 h-1, a doubling time of 3.5 h. Glucose was thus channelled into stable IP synthesis only when the growth limitation imposed by lack of fermentable amino acids was relieved. The metabolism of IP and the ability to obtain carbon and energy from a variety of substrates may explain why F. nucleatum is one of the most commonly detected organisms in subgingival dental plaque.     

具核梭杆菌与口腔常见微生物粘附作用的研究进展

具核梭杆菌是口腔中含量最高的梭杆菌,可成为早期和晚期定植的微生物的连接桥梁,以共生菌的形式存在,也可成为致病菌参与口腔疾病的发生发展.具核梭杆菌与口腔微生物的相互作用可影响微生物与宿主细胞的相互作用,进而决定口腔的健康状态.粘附是最直接的相互作用方式,也是微生物进入生物膜和侵袭宿主细胞的第一步.本文就具核梭杆菌与口腔常...     

不同孵育时间具核梭杆菌黏附和侵入上皮细胞的变化

目的：观察在具核梭杆菌（F.nucleatum，Fn）黏附和侵入上皮细胞的早期阶段，不同时间黏附量和侵入量的变化以及砌不同菌株之间黏附和侵入能力的差别。方法：将实验菌Fn ATCC10953以及Fn WCD05-1分别配成菌悬液，加入培养有上皮细胞（KB）的24孔细胞培养板中。孵育0．5、1、2、3、4h进行黏附和侵入测定。初步探索时间与砌黏附和侵入KB细胞的数量之间是否存在相关关系。结果：在黏附和侵入早期，随相互作用时间延长，砌黏附和侵入KB细胞的数量增加，并且临床株的黏附和侵入能力明显优于标准株。在达到最大黏附和侵入量以后，细菌量有一定下降，但是至少在短时间内可以保持一定的黏附和侵入水平。达到黏附和侵入的最大值时，临床株的黏附和侵入量分别约是标准株的2倍和4倍。结论：在黏附和侵入早期，随相互作用时间延长，砌黏附和侵入KB细胞的数量增加，并且临床株WCD05-1显示出比标准株ATCC10953更强的黏附和侵入能力。达到最大值后其黏附和侵入量有一定下降。