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PubMed2002

Cloning and characterization of the L-cysteine desulfhydrase gene of Fusobacterium nucleatum.

Hydrogen sulfide and methyl mercaptan are the two major compounds associated with oral malodor. These compounds are highly toxic, and are thought to play an important role in periodontal disease. Fusobacterium nucleatum, an oral bacterium, produces large amounts of hydrogen sulfide from L-cysteine by the enzymatic action of L-cysteine desulfhydrase. We cloned and sequenced the cdl gene encoding L-cysteine desulfhydrase from F. nucleatum ATCC 10953, and revealed that the structural cdl gene consists of 921 bp and encodes a 33.4-kDa protein. The cloned gene was inserted into an expression vector, pDEST17, and expressed in Escherichia coli as a fused protein. The purified enzyme was tested for substrate specificity using various SH-containing compounds. Only L-cysteine served as a substrate for L-cysteine desulfhydrase to produce hydrogen sulfide.

Amino Acid Sequence↗

PubMed2002

Growth pH and transient increases in amino acid availability influence polyglucose synthesis by Fusobacterium nucleatum grown in continuous culture.

Fusobacterium nucleatum is a Gram-negative anaerobe that has been implicated in the aetiology of several diseases including periodontal diseases. Like other fusobacteria, it derives energy from the fermentation of amino acids and, in resting (non-growing) cells, this enables the organism to transport glucose and synthesise intracellular polyglucose (IP). The continued availability and fermentation of amino acids inhibits IP breakdown. We have grown F. nucleatum in continuous culture in a chemically defined medium under amino acid limitation and determined the fate of glucose during growth at steady state and during transient increases in the concentration (pulses) of serine and glutamate. When grown under steady state conditions, IP synthesis dramatically increased at culture pH values of 6.1 and 7.8 and appeared to be a result of cell stress. IP synthesis also increased when the culture was pulsed with serine or glutamate but was rapidly metabolised as the added amino acids were depleted. These results may help to explain the role of IP synthesis in response to environmental stress.

Amino Acids↗

PubMed2006

Coaggregation of Porphyromonas gingivalis and Fusobacterium nucleatum PK 1594 is mediated by capsular polysaccharide and lipopolysaccharide.

Previous reports have shown that coaggregation between Porphyromonas gingivalis and Fusobacterium nucleatum, two important periodontopathogens, is mediated by a galactoside on the surface of P. gingivalis and a lectin on F. nucleatum. In the present study, purified capsular polysaccharide (CPS) and lipopolysaccharide (LPS) of P. gingivalis PK 1924 (serotype K5) were found to be able to bind to F. nucleatum cells and to inhibit binding of F. nucleatum to P. gingivalis serotype K5. Sugar binding studies showed that the requirements for binding of P. gingivalis serotype K5 CPS and LPS to the F. nucleatum lectin are: the presence of a metal divalent ion, an axial free hydroxyl group at position 4 and free equatorial hydroxyl groups at position 3 and 6 of d-galactose. These data suggest that P. gingivalis serotype K5- CPS and LPS act as receptors mediating coaggregation between P. gingivalis and fusobacteria.

Adhesins, Bacterial↗

PubMed1997

Release and activation of human neutrophil matrix metallo- and serine proteinases during phagocytosis of Fusobacterium nucleatum, Porphyromonas gingivalis and Treponema denticola.

The phagocytic ingestion of reference strains and clinical isolates of Fusobacterium nucleatum, Porphyromonas gingivalis, and Treponema denticola by polymorphonuclear leukocytes (PMNs) and the concomitant release of PMN granule proteinases were studied by specific functional and immunological assays. PMNs were incubated with the microorganisms anaerobically at 37 degrees C for indicated time periods. The suspensions and pellets were used for phagocytic ingestion assay and electron microscopic study, respectively. The supernatants were used for the measurements of the amounts and activities of the released PMN enzymes including PMN gelatinase (MMP-9), collagenase (MMP-8), serine proteases (elastase and cathepsin G), and lactate dehydrogenase (LDH). Both fluorescence microscopy and transmission electron microscopy showed that F. nucleatum, P. gingivalis and T. denticola were ingested by the PMNs in comparable numbers. However, measurements of the enzymes released from the triggered PMNs revealed major differences among the three species. High amount of elastase was released from the PMNs triggered by F. nucleatum, but not by P. gingivalis or T. denticola. The treatment of PMNs with P. gingivalis whole cells resulted in the release of gelatinase partly in the 82 kD active form, suggesting proteolytic activation of the degranulated 92 kD proMMP-9. The 82 kD active form of gelatinase was not detected upon triggering the PMNs with F. nucleatum and T. denticola. The PMN-bacteria interaction did not result in release of LDH from triggered PMNs indicating the proteinase release was not due to the PMN cell death. The results show that the susceptibilities of the 3 potentially periodontopathogenic microorganisms, F. nucleatum, P. gingivalis and T. denticola to phagocytic ingestion are not directly related to the amounts and activities of PMN enzymes released during the bacteria-PMN interactions. As PMN degranulation is considered as one of the major pathogenic mechanisms in periodontitis, the observed differences among the microorganisms may be important virulence characteristics of these species.

Bacteriological Techniques↗

PubMed1981

Complement-dependent histamine release from rat peritoneal mast cells, induced by lipopolysaccharides from Bacteroides oralis, Fusobacterium nucleatum and Veillonella parvula.

The capacity of phenol/water extracted lipopolysaccharides (LPS) from Bacteroides, Fusobacterium and Veillonella to induce histamine release from rat peritoneal mast cells has been evaluated by means of a bioassay using atropinized guinea pig ileum and a fluorescence assay. Histamine release was found to be completely complement dependent. It was also found that LPS of Veillonella have the greatest capacity to induce histamine release and Bacteroides have the weakest.

Animals↗

PubMed1985

Modulation of HLA-DR antigens in the gingival epithelium in vitro by heat-killed Fusobacterium nucleatum and E. coli lipopolysaccharide.

The in vitro influence of the periodontopathic organism Fusobacterium nucleatum (FN) on gingival tissue was examined using an organ culture system. Treatment of gingival explants obtained from periodontally diseased sites with suspensions of FN, stimulated the expression of HLA-DR antigens by Langerhans cells (LC) in a dose-dependent fashion, and produced a maintenance of the LC markers T6 and ATPase. Similar effects were seen when E. coli lipopolysaccharide (LPS) was substituted for suspensions of FN. With both FN and LPS the expression of HLA-DR by gingival keratinocytes was maintained throughout the 72-h culture period, despite the cytotoxic effects of these agents. Using a variety of immunohistological techniques and a monoclonal antibody specific for the strain of FN used, it was possible to demonstrate the uptake of FN antigens by LC within the gingival epithelium.

Cell Count↗

PubMed1984

Antibacterial properties of human lysozyme toward Fusobacterium nucleatum Fev1.

Human lysozyme in physiologic concentrations (17-50 micrograms/ml) had apparently no effects on growth rate and viability of exponentially growing Fusobacterium nucleatum Fev1 cells, but cells in the stationary phase were affected. When grown in the presence of active lysozyme about 70% of the cells in late exponential phase (24-h culture) were able to form colonies, compared to about 100% in the control culture. About 24 h later the colony forming abilities were about 5% and 20%, respectively. Addition of lysozyme to suspensions of cells taken from any growth phase did not lead to any significant decrease in turbidity, that is, no more than 10% decrease at 650 nm. Control cells treated with acridine orange fluoresced with a uniform bright orange color, while the lysozyme treated stationary phase cells fluoresced more faintly. Intracellular granules were more preponderant in the latter cells. When incubated with the hydrophobic probe 8-anilinonaphthalene-1-sulfonic acid (ANS), lysozyme exposed cells gave approximately 20% higher fluorescence intensity than the control cells. Changes in the ultrastructure of the lysozyme treated cells were best studied in the transmission electron microscope using ultrathin sectioned preparations. The peptidoglycan layer became disorganized and apparently dissolved and the ordered structure of the cell wall had disappeared in zones. The cells, however, still retained their form, and only a few per cent had lost their cellular content. This explains why the turbidity of the solution did not change significantly.

Cell Wall↗

PubMed1989

Utilization of amino acids and peptides by Fusobacterium nucleatum.

Fusobacterium nucleatum strains ATCC 10953, Fevl, F1, F3, and F6 utilized amino acids, in particular glutamate, histidine, and aspartate were common to all strains. Strain differences were observed in the utilization of threonine, serine, lysine, tyrosine, and methionine, and only strain ATCC 10953 utilized all these amino acids. The glutamate and histidine pools were in all cases fully depleted before the other amino acids were attacked and at the same time all strains except 10953 started to utilize peptides at a noticeable rate. For strain Fevl, glutamyl- and aspartyl-containing peptides seemed to be of considerable nutritional importance, and this strain did not grow on a medium based on amino acids alone. On the other hand, strain 10953 did not utilize any peptides to a noticeable extent, and it could grow on an amino acid based medium.

Amino Acids↗

PubMed1990

Growth conditions and outer membrane proteins of Fusobacterium nucleatum.

Fusobacterium nucleatum strains ATCC 10953, ATCC 25586, F1 F3, F6, and Fev1 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.

Autoradiography↗

PubMed1991

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

A 65-kilodalton protein was identified in the outer membrane of Fusobacterium nucleatum Fev1 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 extractable with 2% sodium dodecylsulfate (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.

Amino Acids↗

PubMed1995

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- and 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.

Antibodies, Bacterial↗

PubMed1990

Interaction of Fusobacterium nucleatum 191 with human peripheral blood lymphocytes.

Fusobacterium nucleatum is frequently isolated in high numbers from the subgingival plaque of patients with periodontal diseases. Adherence of these bacteria to several host tissues is lectin-like in that it is inhibited by galactose, lactose, and N-acetyl-galactosamine. Since F. nucleatum has the capacity to activate human peripheral blood lymphocytes, the purpose of this study was to determine whether F. nucleatum adheres to lymphocytes and, if so, to determine whether lectin-like interactions facilitate lymphocyte activation. Adherence of F. nucleatum strain 191 to lymphocytes was confirmed by agglutination assays, scanning electron microscopy, and by using fluorescein- or [3H]-labeled bacteria. Saturation of adherence was reached at about 35 bacteria per lymphocyte. F. nucleatum adhered to greater than 90% of the lymphocytes, and adhered in higher numbers to B cell-enriched subpopulations than to T cell-enriched subpopulations. Adherence was inhibited by N-acetyl-galactosamine, lactose greater than galactose, alpha- and beta-methyl galactoside, but not by 100 mM glucose, mannose, N-acetyl-glucosamine, or other sugars tested. Lymphocytes underwent mitogenesis when preincubated with bacteria and washed to remove weakly adherent and non-adherent bacteria. Mitogenesis induced by low concentrations of F. nucleatum was reduced, but not completely eliminated, by N-acetyl-galactosamine. However, N-acetyl-galactosamine had no effect on mitogenesis induced by preincubation with high concentrations of bacteria. These results suggest that adherence of F. nucleatum facilitates activation of lymphocytes, particularly at low concentrations of bacteria.

Acetylgalactosamine↗

PubMed1990

Binding specificity of Fusobacterium nucleatum to human erythrocytes, polymorphonuclear leukocytes, fibroblasts, and HeLa cells.

Eighteen strains of Fusobacterium nucleatum were studied with respect to their adherence to human erythrocytes, polymorphonuclear leukocytes, fibroblasts, and HeLa cells. Four types of adherence were found: 1) sugar-sensitive adherence 2) amino acid-sensitive adherence 3) unknown adherence 4) weak adherence. There was no obvious correlation between the patterns of hemagglutination and adherence to other cells. The results indicate not only a complex network of bacterial receptors on human cells, but also a variety of types of bacterial adherence as expressed by the selectivity of the bacterial binding to specific cell surfaces.

Acetylgalactosamine↗

PubMed1991

Identification of components in Fusobacterium nucleatum chemostat-culture supernatants that are potent inhibitors of human gingival fibroblast proliferation.

The present investigation concerned the effect of chemostat-culture cell-free supernatants of Fusobacterium nucleatum on the growth and synthetic activity of human gingival fibroblasts in vitro. Human gingival fibroblasts were cultured in fetal calf serum supplemented Dulbecco-Vogt medium containing various dilutions of conditioned or unconditioned bacterial culture medium. Cell proliferation was monitored by assessing cell growth over 5 days or incorporation of [3H]-thymidine into DNA. Protein and proteoglycan synthesis were monitored by the incorporation of [3H]-proline and [35S]-sulfate, respectively, into macromolecules. While the conditioned culture medium caused a complete inhibition of cell growth and incorporation of [3H]-thymidine DNA, there was no discernible effect on protein or proteoglycan synthesis. This indicated that the cells remained viable yet unable to divide. Such a view was supported by the observation that the inhibitory effect was reversible upon removal of the conditioned medium. This activity had a molecular size less than 30,000, was heat-stable and nonvolatile. Chemical analysis of the conditioned bacterial culture supernatants indicated that high proportions of butyrate, ammonium, and acetate were present. When these components were added to unconditioned medium and tested, most of the inhibitory activity could be attributed to ammonium and butyrate. Since many bacteria which constitute the subgingival microflora release ammonium and butyrate, a very high concentration of these metabolites may well accumulate. Clearly, the potential for inhibition of fibroblast proliferation has ramifications related to diminished tissue repair following bacterially-induced periodontal destruction.

Ammonia↗

PubMed1993

Purification of arginine-sensitive hemagglutinin from Fusobacterium nucleatum and its role in coaggregation.

Hemagglutinin of Fusobacterium nucleatum was extracted from Triton X-100-pronase P-treated cell envelopes, and was purified by affinity chromatography on L-arginine agarose. The hemagglutinin was inactivated by heating at 70 degrees C for 1 min. The activity was inhibited by L-arginine but was not affected by any sugars or by EDTA. The hemagglutinin aggregated 14 out of 17 strains of oral streptococci tested, and the bacterial aggregating activity was also inhibited by L-arginine. The results indicate the dominant role of this hemagglutinin in the adherence of this bacterium both to host cells and to other bacteria.

Arginine↗

PubMed1993

Interleukin 1, interleukin 6 and transforming growth factor-beta production by human gingival mononuclear cells following stimulation with Porphyromonas gingivalis and Fusobacterium nucleatum.

Gingival mononuclear cell production of interleukin 1 (IL-1), interleukin 6 (IL-6) and transforming growth factor-beta (TGF-beta) after stimulation with the putative periodontopathic bacteria, Porphyromonas gingivalis and Fusobacterium nucleatum was investigated. Using an ELISA method, gingival mononuclear cells extracted from 18 adult periodontitis subjects were found to be producing IL-1. However, IL-1 activity could only be detected in 5 out of these 18 cases when tested using a thymocyte proliferation bio-assay, suggesting the presence of IL-1 inhibitors. Depletion of monocytes from peripheral blood cultures resulted in a significant decrease in IL-1 activity following P. gingivalis stimulation while there was no effect in the level of IL-1 activity following stimulation with F. nucleatum. This suggests that P. gingivalis and F. nucleatum stimulate different cell types to produce IL-1. Like IL-1, IL-6 production by gingival mononuclear cells was significantly greater than that produced by the control peripheral blood mononuclear cells. Following P. gingivalis and F. nucleatum stimulation, higher levels of IL-6 could be detected; however, both organisms stimulated similar levels. Intracytoplasmic immunofluorescence staining demonstrated a lower percent TGF-beta+ cells in bacterial stimulated peripheral blood mononuclear cell cultures compared with cells in medium alone. In the gingival mononuclear cell cultures, the percentage TGF-beta+ cells peaked at day 1 in F. nucleatum-stimulated, whereas in P. gingivalis-stimulated cultures the peak TGF-beta+ cells occurred at day 3, again suggesting stimulation of different cell subsets. These results illustrate that different periodontopathic bacteria may stimulate different cell types to produce cytokines which may have synergistic or antagonistic effects.

B-Lymphocytes↗

PubMed1995

Characteristics of multimodal co-aggregation between Fusobacterium nucleatum and streptococci.

The co-aggregation characteristics between Fusobacterium nucleatum and streptococci were examined to clarify the adherence factors participating in the co-aggregation. Nineteen strains of F. nucleatum were classified into 8 groups according to co-aggregation titer and inhibition by L-arginine, L-lysine and N-acetyl-D-galactosamine (or lactose). The inhibition activity was, however, very different from strain to strain. With two fusobacterial strains, two inhibitors, which were both inhibition negative on their own, completely inhibited the co-aggregation when used together in a mixture. In some co-aggregation pairs, the protease treatment of F. nucleatum inactivated one of the adherence factors, and resulted in the change of inhibition characteristics. These results indicate the multimodal co-aggregation of F. nucleatum with streptococci mediated by L-arginine-sensitive, L-lysine-sensitive, N-acetyl-D-galactosamine-sensitive and in some resistant factors, and that the adherence factor or factors participating the co-aggregation change according to the co-aggregation partners.

Acetylgalactosamine↗

PubMed1998

Clonal analysis by ribotyping of Fusobacterium nucleatum isolates obtained from healthy young adults with optimal plaque control.

Fusobacterium nucleatum is a Gram-negative anaerobic rod implicated in the pathogenesis of periodontal disease. However, this organism has also been frequently identified in high numbers in healthy adults. These observations suggest that the species may comprise different clonal types, some of which may participate in disease. The purpose of the present investigation was to use restriction endonuclease analysis (REA) and ribotyping to characterize F. nucleatum clonal types isolated from healthy young adults with optimal plaque control and investigate the stability of some of these clonal types. A group comprising 11 dental students and 11 dental outpatients with optimal plaque control was sampled. Clonal stability was investigated by sampling the dental student group at baseline and at 16 months. One hundred and thirty-two clinical isolates of F. nucleatum were successfully recovered from 15/22 individuals. For the positive subjects, 29 different clonal types were identified by REA and ribotyping, each subject and site being colonized by 1-4 clonal types. For the dental students, 9 and 15 different clonal types were identified at baseline and 16 months, respectively. None of the students harboured identical clonal types at both sampling times. Our results show that ribotyping is a useful technique for monitoring the distributions of F. nucleatum clonal types and indicate that healthy individuals with optimal plaque control can be colonized by more than one F. nucleatum clonal type and that these clonal types appear to be unstable.

Adult↗