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PubMed1994

Purification and quantification of Fusobacterium necrophorum leukotoxin by using monoclonal antibodies.

Monoclonal antibodies (Mabs) were produced to the leukotoxin of Fusobacterium necrophorum. Two mAbs (F7B10 and E12E9) partially neutralized leukotoxin activity, as determined by a tetrazolium (MTT)-dye reduction assay with bovine polymorphonuclear neutrophils as target cells. Immunoblot analysis showed that both clones reacted with antigens of 110 and 131 kilodaltons. Epitope analysis showed that the two mAbs recognized the same epitope. An affinity column containing immobilized mAb F7B10 was used to purify leukotoxin from crude toxin. Affinity chromatography of 1 ml of culture supernatant resulted in 0.67 microgram or 1350 units of leukotoxin. Leukotoxin was quantitated by a sandwich enzyme-linked immunosorbent assay using mAb F7B10 as the capture antibody and as the biotinylated indicator. The minimal detectable level was approximately 1 ng, corresponding to 2 leukotoxin units in the sample.

Animals↗

PubMed1994

Studies on the factors affecting the hemolytic activity of Fusobacterium necrophorum.

The in vitro activity of the hemolysin of Fusobacterium necrophorum was determined using the hemolysis of horse erythrocytes as an assay. The effects of medium composition and pH on hemolysin production were investigated. Calf serum and casitone stimulated a comparatively higher hemolytic activity in F. necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme, respectively. However, sugars, such as glucose, galactose and fructose were inhibitors of hemolytic activity. The spectrum of erythrocyte sensitivity to the hemolysin indicated that horse and quail erythrocytes were more sensitive to the hemolysin of both F. necrophorum subsp. necrophorum and subsp. funduliforme, than were cat, dog, rabbit, pigeon and human erythrocytes. Cat erythrocytes were however insensitive to the hemolysin of subsp. funduliforme. Cattle, sheep and chicken erythrocytes were insensitive to the hemolysin of the two subspecies. Medium pH near neutral were more effective in enhancing hemolytic activity, and hemolytic activity was positively correlated with growth. In general, F. necrophorum subsp. necrophorum was more hemolytic than subsp. funduliforme.

Animals↗

PubMed1996

Evidence for phospholipase B activity in Fusobacterium necrophorum cultures and its association with hemolysin/leucocidin activities.

Phospholipase B (PLB) activity was present in Fusobacterium necrophorum cultures and it correlated closely with virulence and co-purified with the hemolysin/leucocidin activities. All three activities were associated with a large molecule or molecular complex (6 x 10(2)-2 x 10(3) kDa) exhibiting varying degrees of aggregation. These were present mainly in the culture medium and to a lesser extent in cell sonicates. The PLB and toxin activities were sensitive to heat, dissociating agents, proteolytic enzymes, prolonged purification regimes, freeze-drying and repeated freeze-thawing. The toxin(s) were stable over a broad range of pH, did not require divalent ions or reducing agents and could be kept for several months as an ammonium sulfate precipitate at 4 degrees C, or stored as a concentrated liquid in the presence of proteolytic inhibitors at - 20 degrees C.

Animals↗

PubMed1996

The effects of physical and chemical agents on the secretion and stability of a Fusobacterium necrophorum hemolysin.

The effect of various agents as enhancers or inhibitors of hemolysin secretion by Fusobacterium necrophorum was investigated. The hemolysin secreted in phosphate buffered saline (PBS) alone was inactivated shortly after secretion. Tween-80 or albumin preserved the hemolytic activity in PBS in which cultures of F. necrophorum had been suspended. Hemoglobin was found to enhance hemolysin secretion. However, higher concentrations diminished secretion. Chloramphenicol, an inhibitor of protein synthesis, exhibited no effect on the hemolytic activity. However, the addition of sodium azide, an energy metabolic inhibitor, significantly reduced the hemolytic activity. Lower temperatures and pH above 9 and below 6 all yielded a low hemolytic activity. Cells suspended in Tween-80 prior to sonication yielded a substantial amount of extracellular hemolytic activity with low intracellular activity detected. However, cells suspended in PBS alone yielded a low extracellular activity but rather a high intracellular activity. The same spectrum of red blood cells of different species were found to be sensitive to both the extracellular and intracellular hemolysins.

Albumins↗

PubMedDate not supplied

Adherence of Fusobacterium necrophorum biovar A and B strains to erythrocytes and tissue culture cells.

Fusobacterium necrophorum biovar A and biovar B were examined for haemagglutinability and ability to adhere to cells. Six biovar A strains agglutinated tannic-acid-treated chicken erythrocytes (1/32-1/64), as well as untreated chicken erythrocytes (1/16-1/64). They adhered well to MDBK and FL cells. Haemagglutination by them was not inhibited with D-mannose. In six biovar B strains, the haemagglutination titre was low for chicken erythrocytes and showed no increase for tannic-acid-treated chicken erythrocytes. These strains exhibited a weak ability to adhere to MDBK and FL cells.

Bacterial Adhesion↗

PubMed2006

Antimicrobial actions of benzimidazoles against the oral anaerobes Fusobacterium nucleatum and Prevotella intermedia.

BACKGROUND/OBJECTIVE: Benzimidazoles are widely used as proton-pump inhibitors to control stomach hyperacidity and have been found also to have antimicrobial actions against Helicobacter pylori and oral streptococci. Our primary aim was to determine if they are active also against oral anaerobes associated with gingivitis. Our major focus was on catabolism because it leads to production of inflammatory metabolites such as butyrate and ammonia. The benzimidazoles are effective in the protonated form at acid pH values and cause irreversible inhibition of enzymes associated with formation of drug-target disulfide bonds. METHODS: Fusobacterium nucleatum ATCC 25586 and Prevotella intermedia ATCC 25611 were grown anaerobically in suspension cultures, harvested, washed and exposed to the benzimidazole lansoprazole at pH values of 4 or 5 before being washed and used for standard assays to detect inhibition of catabolic functions, uptake of the agent and lethality. RESULTS: Lansoprazole was found to be a bacteriostatic, multi-target antimicrobial against F. nucleatum under anaerobic conditions inhibitory for amino acid fermentation and also for glycolysis of glucose or fructose. ID(50) values for fermentation of amino acids and dipeptides by F. nucleatum ranged from 0.05 mM for lysine to 0.25 mM for serine. Fructose catabolism was highly sensitive with an ID(50) value of 0.03 mM apparently related to high sensitivity of the phosphoenolpyruvate:fructose phosphotransferase system, while the ID(50) for glucose catabolism by intact cells was some 0.07 mM. Fermentation of aspartate or aspartylaspartate by P. intermedia was found to be lansoprazole-sensitive with ID(50) values of about 0.18 and 0.20 mM, respectively. CONCLUSION: Catabolism of amino acids, dipeptides and sugars by oral anaerobes associated with gingivitis are sensitive to the inhibitory actions of lansoprazole. Thus, catabolic pathways are potential targets for use of benzimidazoles against bacteria involved in gingivitis.

2-Pyridinylmethylsulfinylbenzimidazoles↗

PubMed2005

Fluorescence based measurements of Fusobacterium nucleatum coaggregation and of fusobacterial attachment to mammalian cells.

Fusobacterium nucleatum is a common oral anaerobe associated with gingivitis, periodontal disease and preterm deliveries. Coaggregation among oral bacteria is considered to be a significant factor in dental plaque development. Adhesion to host cells was suggested to be important for the F. nucleatum virulence associated with oral inflammation and with preterm births. An uncharacterized fusobacterial galactose inhibitible adhesin mediates coaggregation of F. nucleatum 12230 and F. nucleatum PK1594 with the periodontal pathogen Porphyromonas gingivalis. This adhesin is also involved with the attachment of both fusobacterial strains to host cells. However, it has been suggested that additional unidentified fusobacterial adhesins are involved in F. nucleatum virulence associated with preterm births. In this study, a fluorescence-based high throughput sensitive and reproducible method was developed for measuring bacterial coaggregation and bacterial attachment to mammalian cells. Using this method we found that coaggregation of F. nucleatum 4H with P. gingivalis and its attachment to murine macrophages is less inhibitible by galactose than that of F. nucleatum PK1594. These findings suggest that F. nucleatum 4H can serve as a model organism for identifying nongalactose inhibitible F. nucleatum adhesins considered to be involved in fusobacterial attachment to mammalian cells.

Animals↗

PubMed2005

Coaggregation ability of Weissella cibaria isolates with Fusobacterium nucleatum and their adhesiveness to epithelial cells.

In the present study, we evaluated the ability of Weissella cibaria isolated from the oral cavity to coaggregate with Fusobacterium nucleatum, and the adhesiveness of these strains to epithelial cells. W. cibaria efficiently coaggregated with F. nucleatum, and adhered to epithelial cells. We tested the effects of various factors on the coaggregation. The coaggregation and adhesiveness of W. cibaria disappeared upon exposure to pronase or LiCl, suggesting that proteinaceous components on the surface of W. cibaria mediated the coaggregation and adhesiveness. In conclusion, W. cibaria may serve as a potential probiotic with the ability to establish an oral flora protecting against oral pathogens.

Amino Acids↗

PubMed2005

The major outer membrane protein of Fusobacterium nucleatum (FomA) folds and inserts into lipid bilayers via parallel folding pathways.

Membrane protein insertion and folding was studied for the major outer membrane protein of Fusobacterium nucleatum (FomA), which is a voltage-dependent general diffusion porin. The transmembrane domain of FomA forms a beta-barrel that is predicted to consist of 14 beta-strands. Here, unfolded FomA is shown to insert and fold spontaneously and quantitatively into phospholipid bilayers upon dilution of the denaturant urea, which was shown previously only for outer membrane protein A (OmpA) of Escherichia coli. Folding of FomA is demonstrated by circular dichroism and fluorescence spectroscopy, by SDS-polyacrylamide gel electrophoresis, and by single-channel recordings. Refolded FomA had a single-channel conductance of 1.1 nS at 1 M KCl, in agreement with the conductance of FomA isolated from membranes in native form. In contrast to OmpA, which forms a smaller eight-stranded beta-barrel domain, folding kinetics of the larger FomA were slower and provided evidence for parallel folding pathways of FomA into lipid bilayers. Two pathways were observed independent of membrane thickness with two different lipid bilayers, which were either composed of dicapryl phosphatidylcholine or dioleoyl phosphatidylcholine. This is the first observation of parallel membrane insertion and folding pathways of a beta-barrel membrane protein from an unfolded state in urea into lipid bilayers. The kinetics of both folding pathways depended on the chain length of the lipid and on temperature with estimated activation energies of 19 kJ/mol (dicapryl phosphatidylcholine) and 70 kJ/mol (dioleoyl phosphatidylcholine) for the faster pathways.

Bacterial Outer Membrane Proteins↗

PubMed2006

Characterization of proteolytic activities of Fusobacterium nucleatum.

This investigation attempted to detect the proteolytic activity of Fusobacterium nucleatum in living cells, lysate cells, and supernatant of cultures. The reactions were optimized in their pH, temperature, reaction time, enzyme source, and substrate volume. Synthetic substrates beta-naphthylamides (Cys-Na, Ser-Na, Leu-Na, Glu-Na, Lys-Na and BANA), carbobenzoxy L-tirosine p-nitrophenylester (CTN), and natural substrate azoalbumin were used. Reaction occurred with Cys-Na, Ser-Na, and Glu-Na in living cells and with Glu-Na, Leu-Na, and CTN substrates in lysate cells. The supernatant reacted only with Glu-Na. Optimal pH ranged from 6.0 to 7.5, except for CTN (pH 13), and optimal temperature, between 30 and 40 degrees C. Optimal reaction time was 60 min, except for Glu-Na in living cells (40 min), lysate cells (20 min), and CTN substrate (80 min). There was no activity with Lys-Na, BANA, and azoalbumin. Proteolytic activity was assessed by several inhibitors and the presence of metallo, serine, cysteine, and aspartic proteases were detected.

Animals↗

PubMed2004

Differential regulation of cytokine genes in gingival epithelial cells challenged by Fusobacterium nucleatum and Porphyromonas gingivalis.

IL-8 mRNA in human gingival epithelial cells (HGECs) is up-regulated by Fusobacterium nucleatum, and up-/down-regulated by Porphyromonas gingivalis in a complex interaction in the early stages (< or = 4 h) after infection. The mechanisms involved in this regulation in response to F. nucleatum and/or P. gingivalis infection, and identification of co-regulated cytokine genes, are the focus of this investigation. Heat, formalin or protease treatment of F. nucleatum cells attenuated the IL-8 mRNA up-regulation. NF-kappaB, mitogen-activated protein kinase (MAPK) p38 and MAPK kinase/extracellular signal-regulated kinase (MEK/ERK) pathways were involved in IL-8 mRNA induction by F. nucleatum. Pretreatment of P. gingivalis with heat, formalin or protease enhanced IL-8 mRNA induction. NF-kappaB, MARK p38, and MEK/ERK pathways were also involved in this induction. In contrast, down-regulation of IL-8 mRNA by P. gingivalis involved MEK/ERK, but not NF-kappaB or MAPK p38 pathways. cDNA arrays analysis revealed that mRNA down-regulation by P. gingivalis is a specific reaction that only a number of genes, e.g. IL-1beta, IL-8, macrophage inflammatory protein-2alpha, and migration inhibitory factor-related protein-14, are affected based on examination of 278 cytokine/receptor genes. These data indicate that F. nucleatum and P. gingivalis trigger specific and differential gene regulation pathways in HGECs.

Cell Line↗

PubMed1998

Preterm labor and bacterial intraamniotic infection: arachidonic acid liberation by phospholipase A2 of Fusobacterium nucleatum.

OBJECTIVE: The studies presented in this report were undertaken to evaluate whether Fusobacterium nucleatum, a common anaerobic isolate in intrauterine infection, stimulates arachidonic acid metabolism, a rate-limiting step for prostaglandin synthesis, in the human uterine endometrium. STUDY DESIGN: Effects of F nucleatum on arachidonic acid liberation from human uterine endometrial cells and of F nucleatum extract on lysophosphatidylcholine production in human uterine endometrial cells were investigated. RESULTS: When human uterine endometrial cells labeled with tritiated arachidonic acid to an isotopically steady state were exposed to an extract of F nucleatum, arachidonic acid liberation was stimulated, accompanied by lysophospholipid formation. Similar stimulatory effects on phospholipid degradation were also observed in the experiment with bacterially conditioned media. CONCLUSIONS: These results suggest that F nucleatum stimulates endometrial phospholipid metabolism, related to activity of phospholipase A2, which might induce the onset of labor associated with intraamniotic infection.

Arachidonic Acid↗

PubMed2000

Effects of Streptococcus crista and human saliva on the viability of Fusobacterium nucleatum ATCC 10953.

Associations with facultative species might help planktonic oral anaerobes survive when they traverse saliva. This study investigated whether co-aggregation with Streptococcus crista ATCC 51110 enhanced the viability of Fusobacterium nucleatum ATCC 10953. Two tubes each of co-aggregates and Fus. nucleatum ATCC 10953 alone were prepared. One of each set was resuspended in buffer, and the other in clarified saliva from each of 20 donors. After 1 h, cells were stained for viability. The median percentage of viable Fus. nucleatum ATCC 10953 in buffer with Strep. crista (86%) was significantly higher (p=0.04) than in buffer alone (77%). A similar trend was observed for saliva with Strep. crista (81%) and saliva alone (74%), although that difference was not significant (p=0.41). The median percentage of Fus. nucleatum co-aggregated in buffer (44%) was significantly reduced after incubation in saliva (16%) (p<0.0002). No such change was seen when saliva was replaced with purified salivary proline-rich glycoprotein, which can bind both species. For co-aggregate suspensions, there was no difference in the viability of fusobacteria that were or were not in direct contact with Strep. crista. In both cases, viability was significantly reduced in saliva relative to buffer. Strep. crista may enhance the viability of planktonic Fus. nucleatum ATCC 10953, but it is not yet clear whether that requires co-aggregation. Transmission of fusobacteria through saliva could depend on the interplay between protective factors, such as the presence of streptococci, and antimicrobial factors, which kill cells or disassociate co-aggregates.

Anti-Bacterial Agents↗

PubMed1997

Cloning and expression of FomA, the major outer-membrane protein gene from Fusobacterium nucleatum T18.

The major outer-membrane protein. FomA, of Fusobacterium nucleatum has been associated with porin activity, interbacterial adherence and stimulation of host immune cells. Until now, molecular analysis of FomA has not been possible because previous attempts to clone the fomA gene were not successful. The inability to clone F. nucleatum genes led to speculation that Escherichia coli may not be a suitable host. This report concerns the amplification of the fomA gene of F. nucleatum T18 using oligonucleotide primers containing restriction endonuclease sites that allow cloning of fomA into the E. coli expression vector pMMB67. The resultant plasmid, pXWI, was transformed into E. coli DH5 alpha, providing high-level expression of recombinant FomA (rFomA). Amino acid sequencing of rFomA demonstrated that the FomA signal peptide was correctly processed by E. coli signal peptidase I. rFomA was correctly localized to the outer membrane by the E. coli export pathway. The rFomA protein also displayed the heat-modifiable oligomeric and conformational properties of native FomA (nFomA). This demonstration of rFomA expression, processing, export, and secondary and tertiary structure in E. coli provides support for the feasibility of molecular analysis of the structure and function of FomA and other F. nucleatum proteins using recombinant techniques.

Amino Acid Sequence↗

PubMed2000

[Analysis using ELISA test of antibody response to Fusobacterium nucleatum and Eikenella corrodens in subjects with periodontal disease].

BACKGROUND: To know the synthesis of IgG, IgA, and IgM to Fusobacterium nucleatum and Eikenella corrodens in serum, crevicular liquid and saliva in subjects with periodontal disease, using ELISA test. PATIENTS AND METHODS: We studied 26 patients with high bias to the disease aged less than 35 years and 30 individuals with comparable age and with scarce bias to the disease. RESULTS: No differences were found for IgG and IgM titers between the groups, except for IgM to E. corrodens. For IgA, differences were found when the saliva and crevicular liquid were studied for both bacteria, as occurred in serum IgA compared to E. corrodens. In all the cases antibody levels were lower in the patients. CONCLUSIONS: The lower synthesis of IgM and IgA, fundamentally the latter, to F. nucleatum and E. corrodens in patients with periodontal disease would contribute to the pathogenesis of this illness.

Adult↗

PubMed1989

Cytotoxicity against various cell lines of lipopolysaccharides purified from Bacteroides, Fusobacterium, and Veillonella isolated from infected root canals.

The cytotoxicity against two mesenchymal cell lines, L-929 and WI-38, and two epithelial cell lines, KB and HeLa S-3, of lipopolysaccharide (LPS) purified from strains of Bacteroides gingivalis, Fusobacterium nucleatum, and Veillonella parvula isolated from infected root canals was investigated. The inhibition of cell growth by these LPS's was considerable for mesenchymal cell lines, but mild for epithelial cell lines. The cytotoxic effect of F. nucleatum LPS was the greatest and that of B. gingivalis LPS was the least.

Bacteroides↗

PubMed2001

Electrophoretic mobility anomalies associated with PCR amplification of the intergenic spacer region between 16S and 23S ribosomal RNA genes of Fusobacterium necrophorum.

PCR amplification of the intergenic spacer region (ISR) between 16S and 23S rRNA genes among subspecies of the anaerobic bacterium Fusobacterium necrophorum gave identical patterns, with two forms of ISR identified. However, extra bands resulting from anomalous electrophoretic mobility of amplified DNA fragments with certain primer combinations were encountered. Therefore, PCR assays relying solely on banding patterns may be unreliable, and supporting sequence analysis is essential for correct culture identification.

3' Untranslated Regions↗

PubMed1984

Purification and partial characterization of Fusobacterium necrophorum hemagglutinin.

Hemagglutinin (HAin) of Fusobacterium necrophorum was separated from the bacterial cells by trypsinization-sonication, and purified by the gel filtration on Sephadex G-100 column. The final product obtained from gel filtration gave one precipitin line in the immunodiffusion gel and produced a single band in polyacrylamide gel electrophoresis. The molecular weight of the HAin was estimated to be about 19000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It was heat labile and comparatively rich in alanine, glutamine and histidine. Electron microscopy observation revealed that the HAin was a filamentous rod with 0.5-1.0 nm width or frequently showed a cluster form. The hemagglutinability was inhibited by addition of albumins but not by sugars and lipopolysaccharide. Anti HAin rabbit serum inhibited hemagglutination.

Amino Acids↗