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PubMed1990

Proposal of three subspecies of Fusobacterium nucleatum Knorr 1922: Fusobacterium nucleatum subsp. nucleatum subsp. nov., comb. nov.; Fusobacterium nucleatum subsp. polymorphum subsp. nov., nom. rev., comb. nov.; and Fusobacterium nucleatum subsp. vincentii subsp. nov., nom. rev., comb. nov.

Heterogeneity among isolates of Fusobacterium nucleatum has been recognized for many years. The phenotypic properties of 340 strains considered to be F. nucleatum were examined. While these strains were phenotypically similar and fit the description of F. nucleatum, they could be differentiated into three groups on the basis of electrophoretic patterns of whole-cell proteins and DNA homology. Strains in groups I and II showed greater than 80% DNA homology within groups and less than 75% similarity between groups. Strains of group III demonstrated greater than 85% DNA homology to each other and less than 65% similarity to strains in groups I and II. We propose that Fusobacterium nucleatum be divided into the following three subspecies: Fusobacterium nucleatum subsp. nucleatum, with type strain ATCC 25586; Fusobacterium nucleatum subsp. polymorphum, with type strain ATCC 10953; and Fusobacterium nucleatum subsp. vincentii, with type strain ATCC 49256.

Bacterial Proteins↗

PubMed2024

Development of SacB-based counterselection for efficient allelic exchange in Fusobacterium nucleatum.

Fusobacterium nucleatum, prevalent in the oral cavity, is significantly linked to overall human health. Our molecular comprehension of its role in oral biofilm formation and its interactions with the host under various pathological circumstances has seen considerable advancements in recent years, primarily due to the development of various genetic tools for DNA manipulation in this bacterium. Of these, counterselection-based unmarked in-frame mutation methods have proved notably effective. Under suitable growth conditions, cells carrying a counterselectable gene die, enabling efficient selection of rare, defined allelic exchange mutants. The sacB gene from Bacillus subtilis, encoding levansucrase, is a widely used counterselective marker partly due to the easy availability of sucrose. Yet, its potential application in F. nucleatum genetic study remains untested. We demonstrated that F. nucleatum cells expressing sacB in either a shuttle or suicide plasmid exhibit a lethal sensitivity to supplemental sucrose. Utilizing sucrose counterselection, we created an in-frame deletion of the F. nucleatum tonB gene, a critical gene for energy-dependent transport processes in Gram-negative bacteria, and a precise knock-in of the luciferase gene immediately following the stop codon of the hslO gene, the last gene of a five-gene operon possibly related to the natural competence of F. nucleatum. Post-counterselection with 5% sucrose, chromosomal plasmid loss occurred in all colonies, leading to gene alternations in half of the screened isolates. This sacB-based counterselection technique provides a reliable method for isolating unmarked gene mutations in wild-type F. nucleatum, enriching the toolkit for fusobacterial research.IMPORTANCEInvestigations into Fusobacterium nucleatum's role in related diseases significantly benefit from the strategies of creating unmarked gene mutations, which hinge on using a counterselective marker. Previously, the galk-based allelic exchange method, although effective, faced an inherent limitation-the need for a modified host. This study aims to surmount this limitation by substituting galK with sacB for gene modification in F. nucleatum. Our application of the sacB-based methodology successfully yielded a tonB in-frame deletion mutant and a luciferase gene knock-in at the precise chromosomal location in the wild-type background. The new method augments the existing toolkit for F. nucleatum research and has far-reaching implications due to the easy accessibility to the counterselection compound sucrose. We anticipate its broader adoption in further exploration, thereby reinforcing its critical role in propelling our understanding of F. nucleatum.

Fusobacterium nucleatum↗

PubMed2005

Efficient gene transfer and targeted mutagenesis in Fusobacterium nucleatum.

Fusobacterium nucleatum is a Gram-negative anaerobe important in dental biofilm ecology and infectious diseases with significant societal impact. The lack of efficient genetic systems has hampered molecular analyses in this microorganism. We previously reported construction of a shuttle plasmid, pHS17, using the native fusobacterial plasmid pFN1 and an erythromycin resistance cassette. However, the host range of pHS17 was restricted to F. nucleatum, ATCC 10953, and the transformation efficiency was limited. This study was undertaken to improve genetic systems for molecular analysis in F. nucleatum. We identified a second F. nucleatum strain, ATCC 23726, which is transformed with improved efficiency compared to ATCC 10953. Two novel second generation pFN1-based shuttle plasmids, pHS23 and pHS30, were developed and enable transformation of ATCC 23726 at 6.2 x 10(4) and 1.5 x 10(6) transformants/mug plasmid DNA, respectively. The transformation efficiency of pHS30, which harbors a catP gene conferring resistance to chloramphenicol, was more than 1000-fold greater than that of pHS17. The improved transformation efficiency facilitated disruption of the chromosomal rnr gene using a suicide plasmid pHS19, the first demonstration of targeted mutagenesis in F. nucleatum. These results provide significant advances in the development of systems for molecular analysis in F. nucleatum.

Chloramphenicol Resistance↗

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↗

PubMed1997

Increase of heat-shock protein and induction of gamma/delta T cells in peritoneal exudate of mice after injection of live Fusobacterium nucleatum.

Fusobacterium nucleatum and Actinobacillus actinomycetemcomitans are Gram-negative rod periodontal pathogens. The peritoneal cavity of Institute of Cancer Research (ICR) mice was used as the local infection model. In vivo production of heat-shock proteins (hsp) was studied by injection of 1/10 minimum lethal dose (MLD) of each live bacteria into mice. Heat-shock proteins 70 and 60 were examined in the extract of peritoneal exudate cells (PEC) from mice injected intraperitoneally with either F. nucleatum or A. actinomycetemcomitans by using sodium dodecylsulphate-polyacrylamide gel electrophoresis and immunoblotting analysis. Although hsp are present in PEC without injection of the bacteria, both hsp increased and reached a peak on day 3 after F. nucleatum injection but not after A. actinomycetemcomitans. Kinetic study of gamma/delta cells in PEC after injection of bacteria showed that the increase of gamma/delta T cells was observed only in the PEC from mice injected with F. nucleatum but not A. actinomycetemcomitans. The gamma/delta T cells in PEC were either CD3+ and CD4+ or CD3+ and CD8+. The differential cell count of PEC suggested that gamma/delta T-cell induction is related to the expansion of the macrophage population. The phagocytic and chemiluminescence responses of macrophages against the same bacteria were compared after intensive immunization with live F. nucleatum and A. actinomycetemcomitans. Elevations of chemiluminescence response and phagocytic function by immunization were observed in the macrophages of mice immunized with F. nucleatum. These results suggest the sequential appearance of hsp, gamma/delta T cells and macrophage activation after fusobacterial infection.

Actinobacillus Infections↗

PubMed2000

Influence of antimicrobial subinhibitory concentrations on hemolytic activity and bacteriocin-like substances in oral Fusobacterium nucleatum.

Fusobacterium nucleatum is considered for its role in colonization of initial and late microorganisms in dental plaque and for its coaggregation with other bacterial species. It is known that action of different antimicrobial substances may interfere with either virulence factors or with host-bacteria interaction. The goal of this study was to examine the influence of subinhibitory concentrations of chlorhexidine, triclosan, penicillin G and metronidazole on hemolytic activity and bacteriocin-like substance production of oral F. nucleatum. A high resistance to penicillin G was observed and 63% of the isolates were beta-lactamase positive. All the tested isolates were susceptible to metronidazole. F. nucleatum isolates grown with or without antimicrobials were alpha-hemolytics. Bacteriocin-like substance production was increased in isolates grown with penicillin G. Impaired production of hemolytic or antagonic substances can suggest a role in the regulation of oral microbiota.

Adult↗

PubMed2000

Isolation and characterization of a human neutrophil aggregation defective mutant of Fusobacterium nucleatum.

Fusobacterium nucleatum is known to adhere to human polymorphonuclear neutrophils (PMNs) and cause them to aggregate. In this study, we isolated a spontaneously occurring aggregation defective (AGG(-)) mutant and this mutant will be used for future study of the interactions between this bacterium and human PMN. Genomic DNA fingerprinting by random-primed polymerase chain reaction method revealed a difference between the parent strain and the AGG(-) mutant. This mutant also showed an altered phenotype in both microbicidal and phagocytic assays, suggesting that the bacterial factor involved in the aggregation may also be very important for the phagocytosis and, subsequently, the killing by human PMNs. Further study of this mutant may help to clarify the molecular mechanisms of the interaction between this pathogen and human PMNs.

Bacterial Adhesion↗

PubMed2006

Crystallization and preliminary X-ray data of the FadA adhesin from Fusobacterium nucleatum.

Fusobacterium nucleatum is a gram-negative anaerobe prevalent in the oral cavity that is associated with periodontal disease, preterm birth and infections in other parts of the human body. The bacteria attach to and invade epithelial and endothelial cells in the gum tissue and elsewhere via a 13.7 kDa adhesin protein FadA (Fusobacterium adhesin A). FadA exists in two forms: the intact form (pre-FadA), consisting of 129 amino acids, and the mature form (mFadA), which lacks an 18-residue signal sequence. Both forms have been expressed in Escherichia coli and purified. mFadA has been crystallized. The crystals belong to the hexagonal space group P6(1) or P6(5), with unit-cell parameters a = b = 59.3, c = 125.7 A and one molecule per asymmetric unit. The crystals exhibit an unusually high solvent content of 74%. Synchrotron X-ray data have been collected to 1.9 A. The crystals are suitable for X-ray structure determination. The crystal structure of FadA may provide a basis for the development of therapeutic agents to combat periodontal disease and other infections associated with F. nucleatum.

Adhesins, Bacterial↗

PubMed1984

Suppression of human peripheral blood lymphocytes by Fusobacterium nucleatum.

Fusobacterium nucleatum has been implicated in the pathogenesis of several diseases, including urinary tract infections, bacteremia, pericarditis, otitis media, and disorders of the oral cavity such as pulpal infections, alveolar bone abscesses, and periodontal disease. In this study, we examined sonic extracts of F. nucleatum strain FDC 364 for its ability to alter human lymphocyte function. We found that the soluble cytoplasmic fraction (CF) of the sonic extract was able to cause a dose-dependent inhibition of human lymphocyte responsiveness to Con A, PHA, PWM, and the recall antigen SKSD. Suppression involved altered DNA, RNA, and protein synthesis; there was no effect on cell viability. The suppressive activity is nondialyzable and heat labile. To achieve maximal suppression in 96-hr cell cultures, the CF had to be added to cells during the first 24 hr of incubation. Inhibition was reduced when the CF was added at 48 hr, and no suppression was observed when addition was at 72 or 96 hr (along with [3H]TdR). Furthermore, cells could be protected from the suppressive effects of the CF by washing within 24 hr of exposure. Suppression did not involve nonspecific effects on thymidine utilization. Although the mechanism of action of the F. nucleatum immunosuppressive activity has not yet been determined, we can rule out a requirement for monocytes/macrophages and activation of T suppressor cells. It has been proposed that impaired host defense may play a pivotal role in the pathogenesis of many diseases. The data presented in this paper suggest that local and/or systemic immunosuppression could be initiated by F. nucleatum. This immunosuppression may alter the nature and consequences of host-parasite interactions, thereby enhancing the pathogenicity of F. nucleatum itself or that of some other opportunistic organism.

Bacterial Proteins↗

PubMed1986

Desulfuration of cysteine and methionine by Fusobacterium nucleatum.

Fusobacterium nucleatum is a Gram-negative anaerobic rod-shaped bacterium frequently isolated from human dental plaque. It is capable of the desulfuration of cysteine and methionine, resulting in the formation of sulfide and thiol volatiles, respectively. Intact cells, as well as cell-free extracts produced by French pressure cell lysis of F. nucleatum, hydrolyzed radiolabeled cysteine to produce sulfide, pyruvic acid, and ammonia. The hydrolysis products of radiolabeled methionine were a volatile thiol, ketobutyrate, and ammonia. Both activities were associated with the cytoplasmic component, not the membrane. The desulfuration mechanisms are heat-labile, inhibited by the presence of excess substrate, and rates are dependent upon substrate concentration. These dissimilar pathways by F. nucleatum can account in part for the presence of sulfur-containing volatile products that occur in the mouth.

Cysteine↗

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↗

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↗

PubMed1992

Characterization of shared antigens of Fusobacterium nucleatum and Fusobacterium necrophorum.

Fusobacterium nucleatum and Fusobacterium necrophorum are gram-negative, non-spore-forming anaerobic rods, frequently isolated from the normal flora and diseased lesions of the human oral cavity, gastrointestinal and genitourinary tracts. F. necrophorum is also known to be an animal pathogen. Studies were undertaken with rabbit anti-F. nucleatum sera and with human adult periodontitis (AP) sera that demonstrated the sharing of antigens between the two species. Immunodiffusion and immunoelectrophoresis studies of Fusobacterium species with rabbit anti-F. nucleatum sera demonstrated the presence of shared antigen(s) between F. nucleatum and F. necrophorum. Adsorption studies of AP sera in an enzyme-linked immunosorbent assay demonstrated the presence of antibodies reacting with the shared antigens of the two species. Immunoblot (IB) analysis of a soluble protein preparation of the two species of Fusobacterium, when allowed to react with rabbit anti-F. nucleatum 10197 serum, demonstrated 53 kDa and 30 kDa bands present in members of the two species. Further, IB analysis of protein preparations of the two species with AP sera indicated the presence of antibodies reacting with the shared 53 kDa band and in some cases the 30 kDa band. During serological testing with antisera or host immune studies with human sera to these species, the presence of shared antigens must be considered.

Adult↗

PubMed1992

Fusobacterium nucleatum subsp. fusiforme subsp. nov. and Fusobacterium nucleatum subsp. animalis subsp. nov. as additional subspecies within Fusobacterium nucleatum.

Using a variety of physiological, biochemical, and molecular systematic analyses, we have shown previously that there are four groups within the species Fusobacterium nucleatum. Two of these groups of strains correspond to the recently proposed taxa F. nucleatum subsp. nucleatum and F. nucleatum subsp. polymorphum. In this paper we show that the two remaining groups are distinct and formally propose that they should be recognized as F. nucleatum subsp. fusiforme (type strain, NCTC 11326) and F. nucleatum subsp. animalis (type strain, NCTC 12276). The tests which we used did not allow a full assessment of the status of F. nucleatum subsp. vincentii compared with F. nucleatum subsp. nucleatum.

Electrophoresis↗

PubMed1999

Utilization of D-amino acids by Fusobacterium nucleatum and Fusobacterium varium.

The utilization of D- and L-amino acids with acidic, basic or polar side chains was demonstrated by HPLC. Two species of the anaerobe Fusobacterium utilized D-lysine and the L isomers of glutamate, glutamine, histidine, lysine and serine. Only F. varium used L-arginine, D-glutamate and D-serine as substrates, whereas F. nucleatum specifically utilized D-histidine and D-glutamine. D-Glutamate accumulated in F. nucleatum cultures supplemented with D-glutamine, and ornithine was detected when either DL- or L-arginine was included in F. varium cultures. Based on literature precedents, D-glutamate and D-histidine are isomerized to their L isomers prior to degradation, but separate catabolic pathways are possible for each enantiomer of lysine and serine.

Amino Acids↗

PubMed1994

Similarities between Fusobacterium nucleatum and bacteroides fragilis studied by two DNA probes derived from Fusobacterium nucleatum.

A polymerase chain reaction (PCR)-amplified oligonucleotide DNA probe corresponding to a Fusobacterium nucleatum Fevl DNA region coding for a 40-kDa major outer-membrane protein (OMP) and a randomly cloned 2.1 kb DNA probe were found to recognize DNA from the Gram-negative bacteria Fusobacterium nucleatum and Bacteroides fragilis on Southern blots and slot blots. The results indicate sequence similarity within the DNA fragments studied. Immunoblots tested with polyclonal antibodies against whole cells of F. nucleatum revealed only weak antigen similarity between these species.

Aggregatibacter actinomycetemcomitans↗

PubMed2004

Active domains of salivary statherin on apatitic surfaces for binding to Fusobacterium nucleatum cells.

Fusobacterium nucleatum can bind to saliva-coated tooth surfaces. However, the nature of the domains of salivary protein that interact with F. nucleatum remains unclear. The ability of individual proteins in human submandibular-sublingual saliva (HSMSL) to bind F. nucleatum cells was examined by dot blot assay; statherin displayed the strongest binding activity. Statherin binding sites were determined based on binding of (125)I-labelled F. nucleatum to statherin-coated hydroxyapatite (sHAP) beads via inhibition assays using synthetic analogous peptide fragments of whole statherin. Analogous peptides corresponding to residues 19-26 and 32-39 of statherin inhibited binding by 77 % and 68 %, respectively. Synthetic peptides were also prepared by serial deletions of individual residues from N- and C-termini of the peptides GPYQPVPE (aa 19-26) and QPYQPQYQ (aa 32-39). The inhibitory effects of peptides YQPVPE (aa 21-26) and PYQPQYQ (aa 33-39) were very similar to those of GPYQPVPE and QPYQPQYQ, respectively. However, additional deletion of residues resulted in significant reduction of the inhibitory effect. Alanine-scan analysis of YQPVPE revealed that all tested peptides retained inhibitory activity; only YAPVPE exhibited significantly decreased inhibitory activity. These findings suggest that YQPVPE and PYQPQYQ may represent the minimal active segments of statherin for binding to F. nucleatum; moreover, Gln may be a key amino acid in the active segment.

Amino Acid Sequence↗

PubMed1999

Arbitrarily primed-polymerase chain reaction for identification and epidemiologic subtyping of oral isolates of Fusobacterium nucleatum.

BACKGROUND: Fusobacterium nucleatum is the most frequently isolated bacterium in periodontal disease and plays an important role in serious infections in other parts of the body. Arbitrarily primed-polymerase chain reaction (AP-PCR) was used to construct primers for specific identification and subtyping of F. nucleatum. Subtypes may differ in virulence and, hence, are important as periodontal pathogens. Subtypes also may differ in antibiotic susceptibility; therefore, knowing the subtypes may influence choice of treatment. METHODS: We analyzed 70 DNA samples of F. nucleatum isolated from patients with periodontal disease (PD) (N = 32) or AIDS-related PD (N = 8) and from healthy carriers (N = 30). From 90 AP-PCR primers screened, five amplification products were selected, cloned in pCR II vector, and sequenced. These sequences were used to design new pairs of specific primers. Sequences were compared to GenBank entries with BLAST and showed no significant matches. RESULTS: Three primer pairs produced bands of approximately 1 Kb (primer 5059S) or 0.5 Kb (primers FN5047 or M1211) with all F. nucleatum DNAs tested. PCR amplification using primer pair M8171 produced a 1 Kb band with isolates from 7 (22%) PD and 5 (63%) PD-AIDS patients and 9 (30%) healthy controls. Using the same primer pair, 2 other bands of approximately 0.5 Kb and 0.4 Kb were observed with DNA from isolates from 2 (6%) PD and all PD-AIDS patients, but were not observed with DNA samples from healthy controls (P<0.0001). All the primer pairs produced no or different amplicon profiles with DNA samples from bacterial species other than F. nucleatum. CONCLUSIONS: Our results suggest that PCR primer pairs 5059S, FN5047 or M1211 can be used to specifically identify F. nucleatum isolates and distinguish them from other bacteria. The primer pair M8171 could also be used to differentiate F. nucleatum isolated from periodontal patients or healthy individuals. These specific primers can be used in PCR analysis for specific identification of F. nucleatum and to distinguish it from other bacteria associated with human periodontitis. These approaches appear promising in facilitating laboratory identification, molecular subtyping, and taxonomy of putative periodontopathogens.

AIDS-Related Opportunistic Infections↗