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PubMed1996

Extraction and characterization of the smooth-type lipopolysaccharide from Fusobacterium nucleatum JCM 8532 and its biological activities.

The lipopolysaccharide (LPS) of Fusobacterium nucleatum JCM 8532 was isolated by hot-phenol water extraction. Most of the LPS was extracted in the phenolic phase and shown to be the smooth-type, whereas the aqueous phase contained mainly rough-type LPS. The chemical composition of the LPS was similar to that reported in other studies, but D-quinovosamine, which may be a major component of O-antigenic polysaccharide, and 3-deoxy-D-manno-2-octulosonic acid (Kdoi) were detected for the first time by gas chromatography-mass spectrometry. The biological activities of smooth-type LPS, including limulus activity, lethal toxicity, pyrogenicity, and B lymphocyte mitogenicity, were comparable to those of entero-bacterial LPS. Smooth-type LPS inhibited the cell growth and DNA synthesis of adult and fetal human gingival fibroblasts in a dose-dependent manner, suggesting that LPS may play a role in the occurrence of human gingivitis.

Adult↗

PubMed1990

Production of volatile sulfur compounds by various Fusobacterium species.

In 12 species of Fusobacterium the following characteristics were studied; the desulfhydration of L-cysteine and L-methionine by resting cell suspensions, the formation of alpha-keto-acids from L-cysteine, D-cysteine and L-methionine by cell extracts, and the formation of hydrogen sulfide from L-cysteine, D-cysteine and L-cysteine by cell extracts separated by polyacrylamide gel electrophoresis. Multiple forms of L-cysteine desulfhydrase activity were found in most of the species. In some of them also D-cysteine desulfhydrase activity was demonstrated. Seven of the species had high L-methionine gamma-lyase activity. L-cysteine activity was present in 5 of the species.

Carbon-Sulfur Lyases↗

PubMed1990

Amino acid utilization by Fusobacterium nucleatum grown in a chemically defined medium.

The present investigation evaluated amino acid utilization by 120 strains of Fusobacterium nucleatum in a chemically defined medium and attempted to relate the patterns to 3 proposed subspecies of F. nucleatum. Strains were inoculated into a chemically defined medium, with and without 2 g/l glucose, consisting of 14 inorganic salts, 21 amino acids, 23 vitamins and cofactors, and 7 purines and pyrimidines. After 7 days of anaerobic incubation, the spent culture medium, as well as the uninoculated control medium, were analyzed for amino acid content by ion chromatography. Amino acid utilization was determined by the differences in concentrations of amino acids found in inoculated and uninoculated samples. If greater than 34% of the amino acid was removed from the medium, the amino acid was considered to be utilized. Of the 21 amino acids present in the chemically defined medium, 8 amino acids, lysine, glutamine, asparagine, histidine, threonine, serine, glutamate and cysteine were consistently utilized. Four amino acids, tyrosine, tryptophan, methionine and aspartate were utilized by some strains but not others. Nine amino acids, alanine, leucine, isoleucine, glycine, valine, phenylalanine, proline, ornithine, and arginine were not utilized by any of the strains. The utilization patterns did not relate to subspecies formed on the basis of SDS-PAGE and DNA hybridization.

Amino Acids↗

PubMed1990

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.

DNA, Bacterial↗

PubMed1990

Ingestion of Bacteroides buccae, Bacteroides oris, Porphyromonas gingivalis, and Fusobacterium nucleatum by human polymorphonuclear leukocytes in vitro.

The phagocytic ingestion of clinical isolates and reference strains of Bacteroides buccae, Bacteroides oris, Porphyromonas gingivalis and Fusobacterium nucleatum by polymorphonuclear leukocytes (PMNs) was studied. Special attention was focused on the hydrophobicity of the strains. B. buccae strains, less or equally hydrophobic than PMNs, were poorly ingested without opsonization. Hydrophobic, but not hydrophilic, strains of B. oris and both hydrophilic and hydrophobic P. gingivalis and F. nucleatum strains were readily ingested without opsonization. Hydrophobicity thus contributes to the adherence of bacteria to PMNs in some, but not all, species tested. Normal human serum enhanced the ingestion of B. buccae, but failed to do so after heat-inactivation. Heat-inactivation of the immune serum to B. buccae strain ES57 did not reduce opsonic activity suggesting that specific antibodies enhanced the ingestion of B. buccae.

Bacterial Adhesion↗

PubMed1991

DNA probe detection of Eikenella corrodens, Wolinella recta and Fusobacterium nucleatum in subgingival plaque.

This cross-sectional study used species-specific DNA probes to examine subgingival plaque specimens for the presence of Eikenella corrodens, Wolinella recta, and Fusobacterium nucleatum in adults with untreated periodontitis or gingivitis and in healthy controls. W. recta and F. nucleatum were more prevalent in diseased sites from the periodontitis group when compared with the controls (81% vs 22% and 83% vs 20% respectively). E. corrodens was detected in 62% of the control sites and 81% of the periodontitis sites. Because the control sites commonly contained this organism, E. corrodens may not be useful in differentiating between health and disease. In addition, the relationship between the prevalence of W. recta and F. nucleatum and the prevalence of the established periodontal pathogens, Actinobacillus actinomycetemcomitans, Bacteroides intermedius and Bacteroides gingivalis, was examined. Positive detection of W. recta and F. nucleatum correlated closely with the presence of A. actinomycetemcomitans, B. intermedius and B. gingivalis. Therefore, W. recta and F. nucleatum do not appear to be unique indicators of periodontal disease.

Adult↗

PubMed1991

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.

Amino Acids↗

PubMed1991

Comparison of the amino acid uptake profile of reference and clinical isolates of Fusobacterium nucleatum subspecies.

Human isolates of Fusobacterium nucleatum subspecies appear to colonize different niches in the oral cavity, which may be reflected in their nutritional properties. Consequently the utilization of nitrogenous substrates, their sources of energy (supplied here as amino acids) were compared between the 3 subspecies using the reference strain and fresh clinical isolates of each subspecies. All strains incorporated mainly acidic and basic amino acids but significant differences occurred between subspecies. Both reference and clinical isolates of F. nucleatum subspecies polymorphum utilized all amino acids in the medium but the levels of glutamate, arginine and cysteine were noticeably higher in the reference strain. By contrast, F. nucleatum subspecies fusiforme used a very restricted range of amino acids, of which only glutamate, arginine, histidine and cysteine were taken up at greater than 0.5 mM. F. nucleatum subspecies nucleatum utilized fewer amino acids than F. nucleatum subspecies polymorphum but higher concentrations were taken up by the former. Clinical isolates of F. nucleatum subspecies nucleatum incorporated polar and nonpolar neutral amino acids poorly but their levels increased steadily as a clinical isolate was subcultured over a period of 4 months, and was eventually similar to the reference strain. The effect of adding the key catabolic substrate, glutamate (10 mM), on the amino acid uptake profile of F. nucleatum subspecies nucleatum resulted in the complete suppression of the dibasic amino acids arginine, ornithine and histidine. Strains of this subspecies could grow on glutamate as a major source of carbon and energy but, morphologically, the cells appeared somewhat distended and had a tendency to clump.

Amino Acids↗

PubMed1992

A non-lectin-like mechanism by which Fusobacterium nucleatum 10953 adheres to and activates human lymphocytes.

Most (approximately 80%) strains of Fusobacterium nucleatum adhere to human erythrocytes in a lectin-like manner that is strongly inhibited by N-acetyl-D-galactosamine (GalNAc). In this study, we investigated the capacity of F. nucleatum 10953, a strain that is weakly inhibited by GalNAc, to adhere to and activate human lymphocytes in vitro. Experiments using [3H]-labeled bacteria and scanning electron microscopy clearly showed that 10953 adhered to lymphocytes and that adherence was blocked by L-arginine+GalNAc greater than L-arginine much greater than GalNAc. Adherence was Ca(2+)-dependent, inhibited by pretreatment of the bacteria with proteases or heat, and unaffected by paraformaldehyde fixation of the bacteria. Strain 10953 induced lymphocyte mitogenesis that was blocked by L-arginine but not by GalNAc. These results suggest that certain strains of F. nucleatum, such as 10953, express a distinct, non-lectin-like mechanism by which they adhere to and activate lymphocytes. Activation of lymphocytes may be an important mechanism in the pathogenesis of periodontal diseases associated with these bacteria.

Acetylgalactosamine↗

PubMed1992

The breakdown and utilization of peptides by strains of Fusobacterium nucleatum.

It has been claimed that most strains of Fusobacterium nucleatum require peptides rather than free amino acids for growth. In contrast, we have shown that, under continuous culture conditions, all strains tested grow in a chemically defined medium (CDM). The purposes of this study were to determine whether resting cells of F. nucleatum could attack unsubstituted peptides and whether growing cells could utilize a peptide fraction prepared from a commercial peptone. Resting cells cleaved all 19 peptides, containing 3-6 residues, and the 4 key energy-yielding amino acids--Glu, His, Ser and Lys--were rapidly taken up. A hydrophilic Casitone fraction, rich in Glu, promoted growth and peptides < 1 kDa were rapidly utilized. The cleaved residues metabolized were those previously shown to limit growth in CDM: Glu, Ser, His and Lys. The endopeptidase activities of Porphyromonas gingivalis would provide the necessary peptides for the growth of F. nucleatum, which may partly explain why these two organisms frequently coexist in periodontally diseased sites.

Amino Acids↗

PubMed1993

Serum immunoglobulin G antibodies to Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum and Streptococcus sanguis during experimental gingivitis in young adults.

Twenty-eight young, healthy adults completed an experimental gingivitis study in which blood and clinical recordings were obtained at baseline; after a 4-week period of thorough oral hygiene; after a subsequent 3-week period of plaque accumulation; and after another 2 weeks of thorough oral hygiene. Serum immunoglobulin G antibodies against whole cells of Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum and Streptococcus sanguis were determined using enzyme-linked immunosorbent assay. Mean serum immunoglobulin G antibody levels to P. intermedia, F. nucleatum and S. sanguis remained essentially constant during the experiment, whereas the immunoglobulin G antibodies to P. gingivalis declined during the initial period of oral hygiene and the subsequent period of plaque accumulation to an average of 84.5% of the baseline value. This reduction could be attributed to the people who developed marked gingival inflammation during the period of plaque accumulation, indicating that the systemic host response may be associated with local tissue responses to variations in oral hygiene. These people were, however, also characterized by higher initial serum immunoglobulin G responses to P. gingivalis than people who developed less pronounced gingival inflammation during the experiment. The variability and individuality noted in the host response to potential pathogens have important implications for attempts to use such measures for establishing a diagnosis or prognosis for the individual patient.

Adult↗

PubMed1994

Utilization of glutathione (L-gamma-glutamyl-L-cysteinylglycine) by Fusobacterium nucleatum subspecies nucleatum.

Although fusobacteria use amino acids and peptides as energy source, it is not known whether they are able to actively transport peptides into the cell. In the present study the tripeptide glutathione was used as a model substance to investigate peptide uptake in Fusobacterium nucleatum subsp. nucleatum. Cells harvested after 2 days of growth on blood agar or in their exponential growth phase in broth were suspended in buffer with glutathione, L-cysteinylglycine and L-cysteine. As a measure of cell uptake, the formation of hydrogen sulfide was followed. Cells from blood agar had a low capacity to form hydrogen sulfide from the tripeptide glutathione and the dipeptide L-cysteinylglycine. However, hydrogen sulfide was formed from L-cysteinylglycine, but not from glutathione or from L-cysteine, by cells grown in broth in such a way that it strongly indicated an active transport of L-cysteinylglycine with a Km of 18 microM. Hydrogen sulfide was efficiently formed from glutathione by cells grown in broth in the presence 1 mM glutathione. In these cells a glycylglycine-dependent L-gamma-glutamyl peptidase activity was induced. It is probable that the efficient utilization of glutathione for hydrogen sulfide formation mirrored the uptake of L-cysteinylglycine after an L-gamma-glutamyl peptidase had split L-glutamate off from glutathione.

Biological Transport, Active↗

PubMed1995

The influence of intracellular polyglucose and prior growth rate on the survival of Fusobacterium nucleatum under starvation conditions.

Grown in a chemically defined medium containing glucose at a dilution rate of D = 0.065 h-1, Fusobacterium nucleatum D212B-2 produced large amounts of intracellular polyglucose. Aliquots of this culture were starved by anaerobic incubation at 37 degrees C and at various times, assayed for intracellular polyglucose content and viability. This protocol was repeated using cells grown under the same conditions in a chemically defined medium, a medium lacking carbohydrate and in which the organism produced no intracellular polyglucose. Both cultures had 50% survival time values of about 1.5 h and were not eliminated even after 32 h of starvation. It was, therefore concluded that starvation-survival is not influenced by intracellular polyglucose. Starvation-survival was also determined for cells grown in a chemically defined medium at D = 0.048 h-1 and D = 0.12 h-1. The faster-grown cells had a 50% survival time of 3.8 h but were completely eliminated after 8-16 h of starvation. In contrast, slower-grown cells had a 50% survival time of 1.5 h but were not completely eliminated after 32 h of starvation. This illustrates the importance of cell history and technique standardization in comparing the starvation-survival of different organisms.

Adaptation, Physiological↗

PubMed1995

Effect of sonicated material from Fusobacterium nucleatum on the functional capacity of accessory cells derived from dental pulp.

Fusobacterium nucleatum-derived components were studied for their effect on the ability of accessory pulpal cells to induce T-lymphocyte proliferation. In initial experiments, spleen cells were used to establish concentration ranges at which testing of the effect of the bacterial components would be suitable. At low concentrations, bacterial substances induced a stimulatory effect on the proliferation rate of spleen cells, but not when purified T-lymphocytes were incubated in the presence of concanavalin A and pulpal accessory cells. At increasing concentrations, a dose-dependent reduction was observed. Pretreatment with the bacterial products for 4 h gave a stimulatory effect in the absence of concanavalin A for both spleen cells and T-lymphocytes. Removal of low-molecular weight components (<3.5 kDa) did not influence the inhibitory effect, whereas preheating the bacterial substances to 100 degrees C eliminated the observed inhibition. Findings show that heat-sensitive, high-molecular-weight components of F. nucleatum can evoke a concentration-dependent stimulatory or suppressive effect on the proliferation rate of T-lymphocytes in the presence of accessory cells derived from the dental pulp.

Analysis of Variance↗

PubMed1996

The use of allozyme electrophoresis to assess genetic heterogeneity among previously subspeciated isolates of Fusobacterium nucleatum.

Fusobacterium nucleatum has been implicated in the pathogenesis of periodontal diseases. Five subspecies have previously been proposed. The validity of these subdivisions was investigated using allozyme electrophoresis for 21 enzyme mobilities. The 18 F. nucleatum isolates tested had previously been subspeciated and included type strains as well as isolates from both oral and extraoral sites. The results showed 2 distinct genetic groups with fixed differences at 82.5% of the test loci, indicative of a species complex with a number of subspecies within each of the 2 groups. There was also evidence of a correlation between the 2 major groups of isolates and the site from which they were taken. It was concluded that there is a high degree of genetic heterogeneity within the species F. nucleatum and that its current subspeciation is of questionable validity.

Abscess↗

PubMed1996

Induction of systemic murine B-cell responses by Fusobacterium nucleatum and Porphyromonas gingivalis.

The purpose of this study was to examine the antigenic abilities of Fusobacterium nucleatum strain ATCC 25586 and Porphyromonas gingivalis strain W50 black inbred BALB/cABom mice immunized subcutaneously. Furthermore, we aimed to analyze whether the outer membranes (OM) and whole cells (WC) of F. nucleatum or P. gingivalis had an effect on the levels of antibody response and whether a combination of both could either enhance or suppress the B-cell response. A single-cell assay, solid-phase enzyme-linked immunospot (ELISPOT), was used to analyze the splenic B-cell response (immunoglobulin A (IgA), IgG and IgM). Enzyme-linked immunosorbent assay (ELISA) and immunoblotting were used to verify the specific antibody response in the sera. A statistically significant lower level of spontaneous antibody production was observed in the group immunized with P. gingivalis OM compared with groups immunized with F. nucleatum and saline. The specific antibody titers measured by ELISA indicated that the bacterial preparations were able to induce IgG and IgM response. The preparations containing P. gingivalis OM induced higher humoral response than the preparations containing P. gingivalis WC, but for F. nucleatum such a difference was not observed. The prominent proteins revealed had apparent molecular masses of 40 kDa for F. nucleatum and 115, 55-56 and 43 kDa for P. gingivalis; whereas the immunoreactive proteins were 70, 65 and 40 kDa for mice immunized with F. nucleatum and 115, 55-56, 43 and 33-34 kDa for mice immunized with P. gingivalis. Quantitative analysis of B-cell response at the single cell level with ELISPOT revealed that some component(s) of P. gingivalis OM may have a suppressive ability on splenocytes incubated for a short time.

Animals↗

PubMed1997

Acid tolerance and acid-neutralizing activity of Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum.

The tolerance to acid and the acid-neutralizing activity of three important periodontopathic bacteria, Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum were studied. P. gingivalis strains grew only at neutral pH and did not utilize glucose, whereas strains of P. intermedia and F. nucleatum could grow under acidic conditions and increased their growth by utilizing glucose. P. gingivalis tended to raise the culture pH during growth. P. intermedia and F. nucleatum raised the culture pH during growth in the absence of glucose, while in the presence of glucose they decreased the pH. Resting cell suspensions of all the bacteria raised the pH in the presence of tryptone and casamino acids. Acid-neutralizing activity was confirmed by measuring base production at a fixed pH with a pH-stat. During neutralization, the cells produced cytotoxic substrates, ammonia and organic acids (butyric, isobutyric and isovaleric acids by P. gingivalis; isovaleric and succinic acids by P. intermedia; propionic and butyric acids by F. nucleatum). These findings suggest that deamination of amino acids into ammonia and organic acids occurs simultaneously with base production, resulting in acid neutralization. These results could partially explain the survival of P. intermedia and F. nucleatum in both supragingival and subgingival plaque and the apparent restriction of P. gingivalis to subgingival plaque. The former bacteria may aid in creation of an environment fostering colonization of subgingival plaque by P. gingivalis.

Culture Media↗

PubMed1998

Characterization of a novel N-acetylneuraminic acid-specific Fusobacterium nucleatum PK1594 adhesin.

Fusobacterium nucleatum has been identified as significantly associated with sites with active periodontal disease and, as a group, the oral fusobacteria coaggregate with members of all oral bacteria genera tested. Monoclonal antibodies were prepared and used in conjunction with other potential inhibitors, such as simple sugars and amino acids, to characterize coaggregation interactions, of F. nucleatum PK1594. Four unique monoclonal antibodies, 5H11, 14C7, 19F2 and 29C12, were obtained by their ability to inhibit coaggregation of F. nucleatum PK1594 with Actinomyces israelii PK16. They were also capable of inhibiting other coaggregations including Streptococcus oralis H1, S. oralis J22, Capnocytophaga ochracea ATCC33596, Prevotella denticola PK1277 and Prevotella intermedia PK1511. All of these interactions were completely inhibited by N-acetylneuraminic acid. Neither N-acetylneuraminic acid nor monoclonal antibody 5H11 had any inhibitory effect on other F. nucleatum PK1594 interactions, including all galactose-inhibitable coaggregations. The results indicate that F. nucleatum PK1594 expresses upon its surface a distinct type of adhesin that mediates coaggregation interactions that are inhibited by N-acetylneuraminic acid.

Adhesins, Bacterial↗