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1.  The Fusobacterium nucleatum Outer Membrane Protein RadD Is an Arginine-Inhibitable Adhesin Required for Inter-Species Adherence and the Structured Architecture of Multi-Species Biofilm 
Molecular microbiology  2008;71(1):35-47.
Summary
A defining characteristic of the suspected periodontal pathogen Fusobacterium nucleatum is its ability to adhere to a plethora of oral bacteria. This distinguishing feature is suggested to play an important role in oral biofilm formation and pathogenesis, with fusobacteria proposed to serve as central “bridging organisms” in the architecture of the oral biofilm bringing together species which would not interact otherwise. Previous studies indicate that these bacterial interactions are mediated by galactose- or arginine-inhibitable adhesins although genetic evidence for the role and nature of these proposed adhesins remains elusive. To characterize these adhesins at the molecular level, the genetically transformable F. nucleatum strain ATCC 23726 was screened for adherence properties, and arginine inhibitable adhesion was evident, while galactose-inhibitable adhesion was not detected. Six potential arginine binding proteins were isolated from the membrane fraction of F. nucleatum ATCC 23726 and identified via mass spectroscopy as members of the outer membrane family of proteins in F. nucleatum. Inactivation of the genes encoding these six candidates for arginine-inhibitable adhesion and two additional homologues revealed that only a mutant derivative carrying an insertion in Fn1526 (now designated as radD) demonstrated significantly decreased co-aggregation with representatives of the Gram-positive “early oral colonizers”. Lack of the 350 kDa outer membrane protein encoded by radD resulted in the failure to form the extensive structured biofilm observed with the parent strain when grown in the presence of Streptococcus sanguinis ATCC 10556. These findings indicate that radD is responsible for arginine-inhibitable adherence of F. nucleatum and provides definitive molecular evidence that F. nucleatum adhesins play a vital role in inter-species adherence and multispecies biofilm formation.
doi:10.1111/j.1365-2958.2008.06503.x
PMCID: PMC2741168  PMID: 19007407
Fusobacterium nucleatum; RadD. Arginine; Adhesin; Biofilm; Co-aggregation
2.  Analysis of interspecies adherence of oral bacteria using a membrane binding assay coupled with polymerase chain reaction-denaturing gradient gel electrophoresis profiling 
Information on co-adherence of different oral bacterial species is important for understanding interspecies interactions within oral microbial community. Current knowledge on this topic is heavily based on pariwise coaggregation of known, cultivable species. In this study, we employed a membrane binding assay coupled with polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) to systematically analyze the co-adherence profiles of oral bacterial species, and achieved a more profound knowledge beyond pairwise coaggregation. Two oral bacterial species were selected to serve as “bait”: Fusobacterium nucleatum (F. nucleatum) whose ability to adhere to a multitude of oral bacterial species has been extensively studied for pairwise interactions and Streptococcus mutans(S. mutans) whose interacting partners are largely unknown. To enable screening of interacting partner species within bacterial mixtures, cells of the “bait” oral bacterium were immobilized on nitrocellulose membranes which were washed and blocked to prevent unspecific binding. The “prey” bacterial mixtures (including known species or natural saliva samples) were added, unbound cells were washed off after the incubation period and the remaining cells were eluted using 0.2 mol·L−1 glycine. Genomic DNA was extracted, subjected to 16S rRNAPCR amplification and separation of the resulting PCR products by DGGE. Selected bands were recovered from the gel, sequenced and identified via Nucleotide BLAST searches against different databases. While few bacterial species bound to S. mutans, consistent with previous findings F.nucleatum adhered to a variety of bacterial species including uncultivable and uncharacterized ones. This new approach can more effectively analyze the co-adherence profiles of oral bacteria, and could facilitate the systematic study of interbacterial binding of oral microbial species.
doi:10.4248/IJOS11033
PMCID: PMC3469882  PMID: 21485313
membrane binding assay; polymerase chain reaction-denaturing gradient gel electrophoresis; coaggregation; Fusobacterium nucleatum; Streptococcus mutans
3.  Fusobacterium nucleatum ATCC 10953 Requires Actinomyces naeslundii ATCC 43146 for Growth on Saliva in a Three-Species Community That Includes Streptococcus oralis 34▿  
Applied and Environmental Microbiology  2009;75(10):3250-3257.
Formation of dental plaque is a developmental process involving initial and late colonizing species that form polymicrobial communities. Fusobacteria are the most numerous gram-negative bacteria in dental plaque, but they become prevalent after the initial commensal colonizers, such as streptococci and actinomyces, have established communities. The unusual ability of these bacteria to coaggregate with commensals, as well as pathogenic late colonizers, has been proposed to facilitate colonization by the latter organisms. We investigated the integration of Fusobacterium nucleatum into multispecies communities by employing two in vitro models with saliva as the sole nutritional source. In flow cell biofilms, numbers of cells were quantified using fluorescently conjugated antibodies against each species, and static biofilms were analyzed by quantitative real-time PCR (q-PCR) using species-specific primers. Unable to grow as single-species biofilms, F. nucleatum grew in two-species biofilms with Actinomyces naeslundii but not with Streptococcus oralis. However, enhanced growth of fusobacteria was observed in three-species biofilms, indicating that there was multispecies cooperation. Importantly, these community dynamics yielded an 18-fold increase in the F. nucleatum biomass between 4 h and 18 h in the flow cell inoculated with three species. q-PCR analysis of static biofilms revealed that maximum growth of the three species occurred at 24 h to 36 h. Lower numbers of cells were observed at 48 h, suggesting that saliva could not support higher cell densities as the sole nutrient. Integration of F. nucleatum into multispecies commensal communities was evident from the interdigitation of fusobacteria in coaggregates with A. naeslundii and S. oralis and from the improved growth of fusobacteria, which was dependent on the presence of A. naeslundii.
doi:10.1128/AEM.02901-08
PMCID: PMC2681650  PMID: 19286780
4.  Proteomics of Streptococcus gordonii within a model developing oral microbial community 
BMC Microbiology  2012;12:211.
Background
Streptococcus gordonii is one of several species that can initiate the formation of oral biofilms that develop into the complex multispecies microbial communities referred to as dental plaque. It is in the context of dental plaque that periodontal pathogens such as Porphyromonas gingivalis cause disease. We have previously reported a whole cell quantitative proteomics investigation of P. gingivalis in a model dental plaque community of S. gordonii, P. gingivalis, and Fusobacterium nucleatum. Here we report the adaptation of S. gordonii to the same model.
Results
1122 S. gordonii proteins were detected in S. gordonii control samples, 915 in communities with F. nucleatum, 849 with P. gingivalis, and 649 with all three organisms. Quantitative comparisons showed extensive proteome changes in association with F. nucleatum or P. gingivalis individually or both P. gingivalis and F. nucleatum together. The changes were species specific, though the P. gingivalis interaction may be dominant, indicated by large differences between the proteomes with F. nucleatum or P. gingivalis but limited changes between communities with P. gingivalis or both P. gingivalis and F. nucleatum. The results were inspected manually and an ontology analysis conducted using DAVID. Extensive changes were seen in nutrition pathways with increases in energy metabolism and changes in the resulting byproducts, while the acid and sugar repressed PTS (phosphoenolpyruvate dependent phosphotransferase system) sugar transport systems showed decreases. These results were seen across all the multispecies samples, though with different profiles according to the partner species. F. nucleatum association decreased proteins for the metabolic end products acetate and ethanol but increased lactate, the primary source of acidity from streptococcal cultures. P. gingivalis containing samples had a reduction in levels of proteins for ethanol and formate but increased proteins for both acetate and lactate production. The communities also showed increases in exopolysaccharide synthesis, amino acid biosynthesis, and oxidative stress protection and decreases in adhesion and transporter proteins.
Conclusion
This study showed that S. gordonii demonstrates species specific responses during interactions with F. nucleatum or P. gingivalis. Extensive changes were seen in energy metabolism and byproduct production implicating nutrient transfer as an important community interaction.
doi:10.1186/1471-2180-12-211
PMCID: PMC3534352  PMID: 22989070
Streptococcus gordonii; Oral biofilm; Proteomics; Model community; Porphyromonas gingivalis; Fusobacterium nucleatum
5.  Fusobacterium nucleatum Transports Noninvasive Streptococcus cristatus into Human Epithelial Cells  
Infection and Immunity  2006;74(1):654-662.
Analysis of human buccal epithelial cells frequently reveals an intracellular polymicrobial consortium of bacteria. Although several oral bacteria have been demonstrated to invade cultured epithelial cells, several others appear unable to internalize. We hypothesized that normally noninvasive bacteria may gain entry into epithelial cells via adhesion to invasive bacteria. Fusobacterium nucleatum is capable of binding to and invading oral epithelial cells. By contrast, Streptococcus cristatus binds weakly to host cells and is not internalized. F. nucleatum and S. cristatus coaggregate strongly via an arginine-sensitive interaction. Coincubation of KB or TERT-2 epithelial cells with equal numbers of F. nucleatum and S. cristatus bacteria led to significantly increased numbers of adherent and internalized streptococci. F. nucleatum also promoted invasion of KB cells by other oral streptococci and Actinomyces naeslundii. Dissection of fusobacterial or streptococcal adhesive interactions by using sugars, amino acids, or antibodies demonstrated that this phenomenon is due to direct attachment of S. cristatus to adherent and invading F. nucleatum. Inhibition of F. nucleatum host cell attachment and invasion with galactose, or fusobacterial-streptococcal coaggregation by the arginine homologue l-canavanine, abrogated the increased S. cristatus adhesion to, and invasion of, host cells. In addition, polyclonal antibodies to F. nucleatum, which inhibited fusobacterial attachment to both KB cells and S. cristatus, significantly decreased invasion by both species. Similar decreases were obtained when epithelial cells were pretreated with cytochalasin D, staurosporine, or cycloheximide. These studies indicate that F. nucleatum may facilitate the colonization of epithelial cells by bacteria unable to adhere or invade directly.
doi:10.1128/IAI.74.1.654-662.2006
PMCID: PMC1346643  PMID: 16369022
6.  Oral Community Interactions of Filifactor alocis In Vitro 
PLoS ONE  2013;8(10):e76271.
Filifactor alocis is a gram positive anaerobe that is emerging as an important periodontal pathogen. In the oral cavity F. alocis colonizes polymicrobial biofilm communities; however, little is known regarding the nature of the interactions between F. alocis and other oral biofilm bacteria. Here we investigate the community interactions of two strains of F. alocis with Streptococcus gordonii, Fusobacterium nucleatum, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, organisms with differing pathogenic potential in the oral cavity. In an in vitro community development model, S. gordonii was antagonistic to the accumulation of F. alocis into a dual species community. In contrast, F. nucleatum and the type strain of F. alocis formed a synergistic partnership. Accumulation of a low passage isolate of F. alocis was also enhanced by F. nucleatum. In three species communities of S. gordonii, F. nucleatum and F. alocis, the antagonistic effects of S. gordonii superseded the synergistic effects of F. nucleatum toward F. alocis. The interaction between A. actinomycetemcomitans and F. alocis was strain specific and A. actinomycetemcomitans could either stimulate F. alocis accumulation or have no effect depending on the strain. P. gingivalis and F. alocis formed heterotypic communities with the amount of P. gingivalis greater than in the absence of F. alocis. However, while P. gingivalis benefited from the relationship, levels of F. alocis in the dual species community were lower compared to F. alocis alone. The inhibitory effect of P. gingivalis toward F. alocis was dependent, at least partially, on the presence of the Mfa1 fimbrial subunit. In addition, AI-2 production by P. gingivalis helped maintain levels of F. alocis. Collectively, these results show that the pattern of F. alocis colonization will be dictated by the spatial composition of microbial microenvironments, and that the organism may preferentially accumulate at sites rich in F. nucleatum.
doi:10.1371/journal.pone.0076271
PMCID: PMC3789735  PMID: 24098460
7.  Fusobacterium nucleatum adhesin FadA binds vascular-endothelial cadherin and alters endothelial integrity 
Molecular microbiology  2011;82(6):1468-1480.
SUMMARY
Fusobacterium nucleatum is a gram-negative oral anaerobe, capable of systemic dissemination causing infections and abscesses, often in mixed-species, at different body sites. We have shown previously that F. nucleatum adheres to and invades host epithelial and endothelial cells via a novel FadA adhesin. In this study, vascular endothelial (VE)-cadherin, a member of the cadherin family and a cell-cell junction molecule, was identified as the endothelial receptor for FadA, required for F. nucleatum binding to the cells. FadA co-localized with VE-cadherin on endothelial cells, causing relocation of VE-cadherin away from the cell-cell junctions. As a result, the endothelial permeability was increased, allowing the bacteria to cross the endothelium through loosened junctions. This crossing mechanism may explain why the organism is able to disseminate systemically to colonize in different body sites and even overcome the placental and blood-brain barriers. Co-incubation of F. nucleatum and E. coli enhanced penetration of the endothelial cells by the latter in the transwell assays, suggesting F. nucleatum may serve as an “enabler” for other microorganisms to spread systemically. This may explain why F. nucleatum is often found in mixed infections. This study reveals a possible novel dissemination mechanism utilized by pathogens.
doi:10.1111/j.1365-2958.2011.07905.x
PMCID: PMC3237733  PMID: 22040113
8.  Proteomics of Fusobacterium nucleatum within a model developing oral microbial community 
MicrobiologyOpen  2014;3(5):729-751.
Fusobacterium nucleatum is a common oral organism that can provide adhesive and metabolic support to developing periodontal bacterial communities. It is within the context of these communities that disease occurs. We have previously reported whole cell proteomics analyses of Porphyromonas gingivalis and Streptococcus gordonii in early-stage communities with each other and with F. nucleatum, modeled using 18 h pellets. Here, we report the adaptation of F. nucleatum to the same experimental conditions as measured by differential protein expression. About 1210 F. nucleatum proteins were detected in single species F. nucleatum control samples, 1192 in communities with P. gingivalis, 1224 with S. gordonii, and 1135 with all three species. Quantitative comparisons among the proteomes revealed important changes in all mixed samples with distinct responses to P. gingivalis or S. gordonii alone and in combination. The results were inspected manually and an ontology analysis conducted using DAVID (Database for annotation, visualization, and integrated discovery). Extensive changes were detected in energy metabolism. All multispecies comparisons showed reductions in amino acid fermentation and a shift toward butanoate as a metabolic byproduct, although the two organism model community with S. gordonii showed increases in alanine, threonine, methionine, and cysteine pathways, and in the three species samples there were increases in lysine and methionine. The communities with P. gingivalis or all three organisms showed reduced glycolysis proteins, but F. nucleatum paired with S. gordonii displayed increased glycolysis/gluconeogenesis proteins. The S. gordonii containing two organism model also showed increases in the ethanolamine pathway while the three species sample showed decreases relative to the F. nucleatum single organism control. All of the nascent model communities displayed reduced translation, lipopolysaccharide, and cell wall biosynthesis, DNA replication and DNA repair.
doi:10.1002/mbo3.204
PMCID: PMC4234264  PMID: 25155235
Biofilm; Fusobacterium nucleatum; microbial proteomics; microbiome; oral microbial ecology
9.  Role of Fusobacterium nucleatum and Coaggregation in Anaerobe Survival in Planktonic and Biofilm Oral Microbial Communities during Aeration 
Infection and Immunity  1998;66(10):4729-4732.
Coaggregation is a well-characterized phenomenon by which specific pairs of oral bacteria interact physically. The aim of this study was to examine the patterns of coaggregation between obligately anaerobic and oxygen-tolerant species that coexist in a model oral microbial community. Obligate anaerobes other than Fusobacterium nucleatum coaggregated only poorly with oxygen-tolerant species. In contrast, F. nucleatum was able to coaggregate not only with both oxygen-tolerant and other obligately anaerobic species but also with otherwise-noncoaggregating obligate anaerobe–oxygen-tolerant species pairs. The effects of the presence or absence of F. nucleatum on anaerobe survival in both the biofilm and planktonic phases of a complex community of oral bacteria grown in an aerated (gas phase, 200 ml of 5% CO2 in air · min−1) chemostat system were then investigated. In the presence of F. nucleatum, anaerobes persisted in high numbers (>107 · ml−1 in the planktonic phase and >107 · cm−2 in 4-day biofilms). In an equivalent culture in the absence of F. nucleatum, the numbers of black-pigmented anaerobes (Porphyromonas gingivalis and Prevotella nigrescens) were significantly reduced (P ≤ 0.001) in both the planktonic phase and in 4-day biofilms, while the numbers of facultatively anaerobic bacteria increased in these communities. Coaggregation-mediated interactions between F. nucleatum and other species facilitated the survival of obligate anaerobes in aerated environments.
PMCID: PMC108582  PMID: 9746571
10.  Novel Model for Multispecies Biofilms That Uses Rigid Gas-Permeable Lenses ▿  
Applied and Environmental Microbiology  2011;77(10):3413-3421.
Oral biofilms comprise complex multispecies consortia aided by specific inter- and intraspecies interactions occurring among commensals and pathogenic bacterial species. Oral biofilms are primary initiating factors of periodontal disease, although complex multifactorial biological influences, including host cell responses, contribute to the individual outcome of the disease. To provide a system to study initial stages of interaction between oral biofilms and the host cells that contribute to the disease process, we developed a novel in vitro model system to grow biofilms on rigid gas-permeable contact lenses (RGPLs), which enable oxygen to permeate through the lens material. Bacterial species belonging to early- and late-colonizing groups were successfully established as single- or three-species biofilms, with each group comprising Streptococcus gordonii, Streptococcus oralis, and Streptococcus sanguinis; S. gordonii, Actinomyces naeslundii, and Fusobacterium nucleatum; or S. gordonii, F. nucleatum, and Porphyromonas gingivalis. Quantification of biofilm numbers by quantitative PCR (qPCR) revealed substantial differences in the magnitude of bacterial numbers in single-species and multispecies biofilms. We evaluated cell-permeable conventional nucleic acid stains acridine orange, hexidium iodide, and Hoechst 33258 and novel SYTO red, blue, and green fluorochromes for their effect on bacterial viability and fluorescence yield to allow visualization of the aggregates of individual bacterial species by confocal laser scanning microscopy (CLSM). Substantial differences in the quantity and distribution of the species in the multispecies biofilms were identified. The specific features of these biofilms may help us better understand the role of various bacteria in local challenge of oral tissues.
doi:10.1128/AEM.00039-11
PMCID: PMC3126442  PMID: 21421785
11.  Strain-Specific Colonization Patterns and Serum Modulation of Multi-Species Oral Biofilm Development 
Anaerobe  2012;18(4):459-470.
Periodontitis results from an ecological shift in the composition of subgingival biofilms. Subgingival community maturation is modulated by inter-organismal interactions and the relationship of communities with the host. In an effort to better understand this process, we evaluated biofilm formation, with oral commensal species, by three strains of the subgingivally prevalent microorganism Fusobacterium nucleatum and four strains of the periodontopathogen Porphyromonas gingivalis. We also tested the effect of serum, which resembles gingival exudates, on subgingival biofilms. Biofilms were allowed to develop in flow cells using salivary medium. We found that although not all strains of F. nucleatum were able to grow in mono-species biofilms, forming a community with health-associated partners Actinomyces oris and Veillonella parvula promoted biofilm growth of all F. nucleatum strains. Strains of P. gingivalis also showed variable ability to form mono-species biofilms. P. gingivalis W50 and W83 did not form biofilms, while ATCC 33277 and 381 formed biofilm structures, but only strain ATCC 33277 grew over time. Unlike the enhanced growth of F. nucleatum with the two health-associated species, no strain of P. gingivalis grew in three-species communities with A. oris and V. parvula. However, addition of F. nucleatum facilitated growth of P. gingivalis ATCC 33277 with health-associated partners. Importantly, serum negatively affected the adhesion of F. nucleatum, while it favored biofilm growth by P. gingivalis. This work highlights strain specificity in subgingival biofilm formation. Environmental factors such as serum alter the colonization patterns of oral microorganisms and could impact subgingival biofilms by selectively promoting pathogenic species.
doi:10.1016/j.anaerobe.2012.06.003
PMCID: PMC3410037  PMID: 22771792
oral multi-species communities; subgingival; Fusobacterium nucleatum; Porphyromonas gingivalis; strain variability; serum; biofilms
12.  Development of a Multispecies Oral Bacterial Community in a Saliva-Conditioned Flow Cell 
Microbial communities within the human oral cavity are dynamic associations of more than 500 bacterial species that form biofilms on the soft and hard tissues of the mouth. Understanding the development and spatial organization of oral biofilms has been facilitated by the use of in vitro models. We used a saliva-conditioned flow cell, with saliva as the sole nutritional source, as a model to examine the development of multispecies biofilm communities from an inoculum containing the coaggregation partners Streptococcus gordonii, Actinomyces naeslundii, Veillonella atypica, and Fusobacterium nucleatum. Biofilms inoculated with individual species in a sequential order were compared with biofilms inoculated with coaggregates of the four species. Our results indicated that flow cells inoculated sequentially produced biofilms with larger biovolumes compared to those biofilms inoculated with coaggregates. Individual-species biovolumes within the four-species communities also differed between the two modes of inoculation. Fluorescence in situ hybridization with genus- and species-specific probes revealed that the majority of cells in both sequentially and coaggregate-inoculated biofilms were S. gordonii, regardless of the inoculation order. However, the representation of A. naeslundii and V. atypica was significantly higher in biofilms inoculated with coaggregates compared to sequentially inoculated biofilms. Thus, these results indicate that the development of multispecies biofilm communities is influenced by coaggregations preformed in planktonic phase. Coaggregating bacteria such as certain streptococci are especially adapted to primary colonization of saliva-conditioned surfaces independent of the mode of inoculation and order of addition in the multispecies inoculum. Preformed coaggregations favor other bacterial strains and may facilitate symbiotic relationships.
doi:10.1128/AEM.70.7.4340-4348.2004
PMCID: PMC444820  PMID: 15240317
13.  Aggregatibacter actinomycetemcomitans Builds Mutualistic Biofilm Communities with Fusobacterium nucleatum and Veillonella Species in Saliva▿  
Infection and Immunity  2009;77(9):3542-3551.
Human oral bacterial pathogens grow in attached multispecies biofilm communities. Unattached cells are quickly removed by swallowing. Therefore, surface attachment is essential for growth, and we investigated multispecies community interactions resulting in mutualistic growth on saliva as the sole nutritional source. We used two model systems, saliva-coated transferable solid-phase polystyrene pegs (peg biofilms) and flow cells with saliva-coated glass surfaces. Fluorescent antibody staining and image analysis were used to quantify the biomass in flow cells, and quantitative real-time PCR with species-specific primers was used to quantify the biomass in peg biofilms. Veillonella sp. strain PK1910, Aggregatibacter actinomycetemcomitans JP2, and Fusobacterium nucleatum ATCC 10953 were unable to grow as single species in flow cells. Only A. actinomycetemcomitans grew after 36 h when peg biofilms remained submerged in saliva from the time of inoculation. Mixed-species coaggregates were used for two- and three-species inoculation. The biomass in two-species biofilms increased in both systems when Veillonella sp. strain PK1910 was present as one of the partners. Enhanced growth of all strains was observed in three-species biofilms in flow cells. Interestingly, in flow cells F. nucleatum and A. actinomycetemcomitans exhibited mutualism, and, although F. nucleatum was unable to grow with either of the other species in the peg system, F. nucleatum stimulated the growth of Veillonella sp. and together these two organisms increased the total biomass of A. actinomycetemcomitans in three-species peg biofilms. We propose that mutualistic two-species and multispecies oral biofilm communities form in vivo and that mutualism between commensal veillonellae and late colonizing pathogens, such as aggregatibacteria, contributes to the development of periodontal disease.
doi:10.1128/IAI.00345-09
PMCID: PMC2738031  PMID: 19564387
14.  Autoaggregation Response of Fusobacterium nucleatum▿ †  
Applied and Environmental Microbiology  2009;75(24):7725-7733.
Fusobacterium nucleatum is a gram-negative oral bacterial species associated with periodontal disease progression. This species is perhaps best known for its ability to adhere to a vast array of other bacteria and eukaryotic cells. Numerous studies of F. nucleatum have examined various coaggregation partners and inhibitors, but it is largely unknown whether these interactions induce a particular genetic response. We tested coaggregation between F. nucleatum ATCC strain 25586 and various species of Streptococcus in the presence of a semidefined growth medium containing saliva. We found that this condition could support efficient coaggregation but, surprisingly, also stimulated a similar degree of autoaggregation. We further characterized the autoaggregation response, since few reports have examined this in F. nucleatum. After screening several common coaggregation inhibitors, we identified l-lysine as a competitive inhibitor of autoaggregation. We performed a microarray analysis of the planktonic versus autoaggregated cells and found nearly 100 genes that were affected after only about 60 min of aggregation. We tested a subset of these genes via real-time reverse transcription-PCR and confirmed the validity of the microarray results. Some of these genes were also found to be inducible in cell pellets created by centrifugation. Based upon these data, it appears that autoaggregation activates a genetic program that may be utilized for growth in a high cell density environment, such as the oral biofilm.
doi:10.1128/AEM.00916-09
PMCID: PMC2794088  PMID: 19837836
15.  Interactions between Periodontal Bacteria and Human Oral Epithelial Cells: Fusobacterium nucleatum Adheres to and Invades Epithelial Cells 
Infection and Immunity  2000;68(6):3140-3146.
Bacteria are causative agents of periodontal diseases. Interactions between oral bacteria and gingival epithelial cells are essential aspects of periodontal infections. Using an in vitro tissue culture model, a selected group of gram-negative anaerobic bacteria frequently associated with periodontal diseases, including Bacteroides forsythus, Campylobacter curvus, Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedia, were examined for their ability to adhere to and invade primary cultures of human gingival epithelial cells (HGEC). The effects of these bacteria on the production of interleukin-8 (IL-8), a proinflammatory chemokine, were also measured. These studies provided an initial demonstration that F. nucleatum adhered to and invaded HGEC and that this was accompanied by high levels of IL-8 secretion from the epithelial cells. The attachment and invasion characteristics of F. nucleatum were also tested using KB cells, an oral epithelial cell line. The invasion was verified by transmission electron microscopy and with metabolic inhibitors. Invasion appeared to occur via a “zipping” mechanism and required the involvement of actins, microtubules, signal transduction, protein synthesis, and energy metabolism of the epithelial cell, as well as protein synthesis by F. nucleatum. A spontaneous mutant, lam, of F. nucleatum, isolated as defective in autoagglutination, was unable to attach to or invade HGEC or KB cells, further indicating the requirement of bacterial components in these processes. Sugar inhibition assays indicated that lectin-like interactions were involved in the attachment of F. nucleatum to KB cells. Investigation of these new virulence phenotypes should improve our understanding of the role of F. nucleatum in periodontal infections.
PMCID: PMC97547  PMID: 10816455
16.  Insertional inactivation of binding determinants of Streptococcus crista CC5A using Tn916 
Oral microbiology and immunology  1995;10(4):220-226.
Intermicrobial binding plays an important role in the ecology of the oral cavity because it represents one mechanism by which specific bacteria colonize dental plaque. The formation of “corncobs”, a morphologically distinct microbial unit composed of Streptococcus crista and Fusobacterium nucleatum, is a highly specific binding interaction that depends on the presence of polar tufts of fimbriae on the streptococci. We have used a genetic approach to examine the role of streptococcal cell surface components involved in the binding of S. crista to F. nucleatum. Such binding may be an important component of corncob formation. A method for the genetic transformation of S. crista was used to transfer the broad host range transposon, Tn916, into the bacteria. Cells were grown to early log phase in brain heart infusion broth containing 10% fetal calf serum. The competent cells were mixed with purified DNA from pDL916, a plasmid construct consisting of Tn916 and the streptococcal/Escherichia coli shuttle vector pDL278. Over 300 transformants were screened for a reduction in binding to F. nucleatum. Five of the transformants showed a change in binding ranging from 59% to 29% of the positive control values. Southern blots revealed that the binding-deficient transformants contained the Tn916 element integrated into one of 4 different sites in the chromosome. The transposon, integrated into 4 different sites, appeared to be stable in the absence of selective pressure. Based on these findings, it appears that some strains of S. crista are naturally competent and that insertional inactivation methods can be used to facilitate the study of binding receptors in this group of oral streptococci.
PMCID: PMC3534806  PMID: 8602334
Streptococcus crista; Fusobacterium nucleatum; corncob; transposon; transformation; dental plaque
17.  Mutualistic Biofilm Communities Develop with Porphyromonas gingivalis and Initial, Early, and Late Colonizers of Enamel▿  
Journal of Bacteriology  2009;191(22):6804-6811.
Porphyromonas gingivalis is present in dental plaque as early as 4 h after tooth cleaning, but it is also associated with periodontal disease, a late-developing event in the microbial successions that characterize daily plaque development. We report here that P. gingivalis ATCC 33277 is remarkable in its ability to interact with a variety of initial, early, middle, and late colonizers growing solely on saliva. Integration of P. gingivalis into multispecies communities was investigated by using two in vitro biofilm models. In flow cells, bacterial growth was quantified using fluorescently conjugated antibodies against each species, and static biofilm growth on saliva-submerged polystyrene pegs was analyzed by quantitative real-time PCR using species-specific primers. P. gingivalis could not grow as a single species or together with initial colonizer Streptococcus oralis but showed mutualistic growth when paired with two other initial colonizers, Streptococcus gordonii and Actinomyces oris, as well as with Veillonella sp. (early colonizer), Fusobacterium nucleatum (middle colonizer), and Aggregatibacter actinomycetemcomitans (late colonizer). In three-species flow cells, P. gingivalis grew with Veillonella sp. and A. actinomycetemcomitans but not with S. oralis and A. actinomycetemcomitans. Also, it grew with Veillonella sp. and F. nucleatum but not with S. oralis and F. nucleatum, indicating that P. gingivalis and S. oralis are not compatible. However, P. gingivalis grew in combination with S. gordonii and S. oralis, demonstrating its ability to overcome the incompatibility when cultured with a second initially colonizing species. Collectively, these data help explain the observed presence of P. gingivalis at all stages of dental plaque development.
doi:10.1128/JB.01006-09
PMCID: PMC2772475  PMID: 19749049
18.  Coaggregation of Candida dubliniensis with Fusobacterium nucleatum 
Journal of Clinical Microbiology  1999;37(5):1464-1468.
The binding of microorganisms to each other and oral surfaces contributes to the progression of microbial infections in the oral cavity. Candida dubliniensis, a newly characterized species, has been identified in human immunodeficiency virus-seropositive patients and other immunocompromised individuals. C. dubliniensis phenotypically resembles Candida albicans in many respects yet can be identified and differentiated as a unique Candida species by phenotypic and genetic profiles. The purpose of this study was to determine oral coaggregation (CoAg) partners of C. dubliniensis and to compare these findings with CoAg of C. albicans under the same environmental conditions. Fifteen isolates of C. dubliniensis and 40 isolates of C. albicans were tested for their ability to coaggregate with strains of Fusobacterium nucleatum, Peptostreptococcus micros, Peptostreptococcus magnus, Peptostreptococcus anaerobius, Porphyromonas gingivalis, and Prevotella intermedia. When C. dubliniensis and C. albicans strains were grown at 37°C on Sabouraud dextrose agar, only C. dubliniensis strains coaggregated with F. nucleatum ATCC 49256 and no C. albicans strains showed CoAg. However, when the C. dubliniensis and C. albicans strains were grown at 25 or 45°C, both C. dubliniensis and C. albicans strains demonstrated CoAg with F. nucleatum. Heating the C. albicans strains (grown at 37°C) at 85°C for 30 min or treating them with dithiothreitol allowed the C. albicans strains grown at 37°C to coaggregate with F. nucleatum. CoAg at all growth temperatures was inhibited by mannose and α-methyl mannoside but not by EDTA or arginine. The CoAg reaction between F. nucleatum and the Candida species involved a heat-labile component on F. nucleatum and a mannan-containing heat-stable receptor on the Candida species. The CoAg reactions between F. nucleatum and the Candida species may be important in the colonization of the yeast in the oral cavity, and the CoAg of C. dubliniensis by F. nucleatum when grown at 37°C provides a rapid, specific, and inexpensive means to differentiate C. dubliniensis from C. albicans isolates in the clinical laboratory.
PMCID: PMC84803  PMID: 10203506
19.  Intergeneric coaggregation of oral Treponema spp. with Fusobacterium spp. and intrageneric coaggregation among Fusobacterium spp. 
Infection and Immunity  1995;63(12):4584-4588.
A total of 22 strains of Treponema spp. including members of all four named human oral species were tested for coaggregation with 7 strains of oral fusobacteria, 2 strains of nonoral fusobacteria, and 45 strains of other oral bacteria, which included actinobacilli, actinomyces, capnocytophagae, eubacteria, porphyromonads, prevotellae, selenomonads, streptococci, and veillonellae. None of the treponemes coaggregated with any of the latter 45 oral strains or with the two nonoral fusobacteria. All treponemes, eight Treponema denticola strains, eight T. socranskii strains, four oral pectinolytic treponemes, one T. pectinovorum strain, and one T. vincentii strain coaggregated with at least one strain of the fusobacteria tested as partners. The partners consisted of one strain of Fusobacterium periodonticum, five F. nucleatum strains including all four subspecies of F. nucleatum, and a strain of F. simiae obtained from the dental plaque of a monkey. In the more than 100 coaggregations observed, the fusobacterial partner was heat inactivated (85 degrees C for 30 min), while the treponemes were unaffected by the heat treatment. Furthermore, the fusobacteria were usually inactivated by proteinase K treatment, and the treponemes were not affected. Only the T. denticola coaggregations were inhibited by lactose and D-galactosamine. None were inhibited by any of 23 other different sugars or L-arginine. Intragenic coaggregations were seen among the subspecies of F. nucleatum and with F. periodonticum, and none were inhibited by any of the sugars tested or by L-arginine. No intrageneric coaggregations were observed among the treponemes. These data indicate that the human oral treponemes show a specificity for oral fusobacteria as coaggregation partners. Such cell-to cell contact may facilitate efficient metabolic communication and enhance the proliferation of each cell in the progressively more severe stages of periodontal disease.
PMCID: PMC173658  PMID: 7591109
20.  Setup of an In Vitro Test System for Basic Studies on Biofilm Behavior of Mixed-Species Cultures with Dental and Periodontal Pathogens 
PLoS ONE  2010;5(10):e13135.
Background
Caries and periodontitis are important human diseases associated with formation of multi-species biofilms. The involved bacteria are intensively studied to understand the molecular basis of the interactions in such biofilms. This study established a basic in vitro single and mixed-species culture model for oral bacteria combining three complimentary methods. The setup allows a rapid screening for effects in the mutual species interaction. Furthermore, it is easy to handle, inexpensive, and reproducible.
Methods
Streptococcus mitis, S. salivarius and S. sanguinis, typical inhabitants of the healthy oral cavity, S. mutans as main carriogenic species, and Porphyromonas gingivalis, Fusobacterium nucleatum, Parvimonas micra, S. intermedius and Aggregatibacter actinomycetemcomitans as periodontitis-associated bacteria, were investigated for their biofilm forming ability. Different liquid growth media were evaluated. Safranin-staining allowed monitoring of biofilm formation under the chosen conditions. Viable counts and microscopy permitted investigation of biofilm behavior in mixed-species and transwell setups.
Findings
S. mitis, F. nucleatum, P. gingivalis and P. micra failed to form biofilm structures. S. mutans, S. sanguinis, S. intermedius and S. salivarius established abundant biofilm masses in CDM/sucrose. A. actinomycetemcomitans formed patchy monolayers. For in depth analysis S. mitis, S. mutans and A. actinomycetemcomitans were chosen, because i) they are representatives of the physiological-, cariogenic and periodontitis-associated bacterial flora, respectively and ii) their difference in their biofilm forming ability. Microscopic analysis confirmed the results of safranin staining. Investigation of two species combinations of S. mitis with either S. mutans or A. actinomycetemcomitans revealed bacterial interactions influencing biofilm mass, biofilm structure and cell viability.
Conclusions
This setup shows safranin staining, microscopic analysis and viable counts together are crucial for basic examination and evaluation of biofilms. Our experiment generated meaningful results, exemplified by the noted S. mitis influence, and allows a fast decision about the most important bacterial interactions which should be investigated in depth.
doi:10.1371/journal.pone.0013135
PMCID: PMC2948514  PMID: 20957048
21.  Coaggregation of Fusobacterium nucleatum, Selenomonas flueggei, Selenomonas infelix, Selenomonas noxia, and Selenomonas sputigena with strains from 11 genera of oral bacteria. 
Infection and Immunity  1989;57(10):3194-3203.
Twenty-eight strains of Fusobacterium nucleatum and 41 Selenomonas strains, including S. sputigena (24 strains), S. flueggei (10 strains), S. infelix (5 strains), and S. noxia (2 strains), were tested for their ability to coaggregate with each other and with 49 other strains of oral bacteria representing Actinobacillus, Actinomyces, Bacteroides, Capnocytophaga, Gemella, Peptostreptococcus, Porphyromonas, Propionibacterium, Rothia, Streptococcus, and Veillonella species. Selenomonads coaggregated with fusobacteria and with Actinomyces naeslundii PK984 but not with any of the other bacteria, including other selenomonads. In contrast, fusobacteria coaggregated with members of all genera, although not with all strains of each species tested. Each fusobacterium strain appeared to have its own set of partners and coaggregation properties, unlike their partners, whose coaggregation properties in earlier surveys delineated distinct coaggregation groups. Coaggregations of fusobacteria with the 63 gram-negative strains were usually inhibited by EDTA, whereas those with the 27 gram-positive strains were usually not inhibited. Likewise, lactose-inhibitable coaggregations were common among some strains of fusobacteria and some strains from each of the genera containing gram-negative partners but were rarely observed with gram-positive partners. Heating the fusobacteria at 85 degrees C for 30 min completely prevented coaggregation with most partners, suggesting the involvement of a protein on the fusobacteria. Heat treatment of many of the gram-negative partners not only enhanced their coaggregation with the fusobacteria but also changed lactose-sensitive coaggregations to lactose-insensitive coaggregations. Although fusobacteria coaggregated with a broader variety of oral partner strains than any other group of oral bacteria tested to date, each fusobacterium exhibited coaggregation with only a certain set of partner strains, and none of the fusobacteria adhered to other strains of fusobacteria, indicating that recognition of partner cell surfaces is selective. The strains of F. nucleatum are heterogeneous and cannot be clustered into distinct coaggregation groups. Collectively, these results indicate that coaggregation between fusobacteria and many gram-negative partners is significantly different from their coaggregation with gram-positive partners. The contrasting variety of partners for fusobacteria and selenomonads supports the concept of coaggregation partner specificity that has been observed with every genus of oral bacteria so far examined.
Images
PMCID: PMC260789  PMID: 2777378
22.  Identification of a Fusobacterium nucleatum PK1594 galactose-binding adhesin which mediates coaggregation with periopathogenic bacteria and hemagglutination. 
Infection and Immunity  1997;65(12):5231-5237.
Attachment of Fusobacterium nucleatum to various oral surfaces is mediated by several adhesins anchored on its outer surface. Monoclonal antibodies (MAbs) were prepared and used to identify the putative galactose-binding adhesin of F. nucleatum PK1594. Four unique MAbs, 8G7, 26B9, 28G11, and 29D4, were isolated on the basis of their ability to inhibit coaggregation of F. nucleatum PK1594 with Porphyromonas gingivalis PK1924. All four MAbs were also capable of inhibiting galactose-inhibitable interactions of F. nucleatum PK1594 with other oral gram-negative bacteria and with erythrocytes. Preincubation of F. nucleatum PK1594 with MAb 26B9 or its Fab fragments at concentrations lower than 1 microg/ml resulted in complete inhibition of coaggregation with P. gingivalis PK1924 or hemagglutination. F. nucleatum PK1594 surface components prepared by mild sonication or by extracting whole cells with detergents were subjected to Western blot analysis. None of the MAbs were able to recognize any polypeptide in these experiments. Therefore, detergent extracts of F. nucleatum PK1594 surface components were subjected to three experimental procedures: (i) separation by ion-exchange chromatography and testing of fractions for reaction with MAb 26B9 in an enzyme-linked immunosorbent assay (ELISA), (ii) lactose-Sepharose affinity chromatography and testing of the lactose eluate in ELISA with MAb 26B9, and (iii) immunoseparation with either MAb 26B9 or 8G7. Collectively, the results suggest that the putative adhesin is a 30-kDa outer membrane polypeptide which mediates the coaggregation with P. gingivalis PK1924 as well as other galactose-sensitive interactions of F. nucleatum PK1594.
PMCID: PMC175753  PMID: 9393820
23.  Identification and Characterization of a Novel Adhesin Unique to Oral Fusobacteria 
Journal of Bacteriology  2005;187(15):5330-5340.
Fusobacterium nucleatum is a gram-negative anaerobe that is prevalent in periodontal disease and infections of different parts of the body. The organism has remarkable adherence properties, binding to partners ranging from eukaryotic and prokaryotic cells to extracellular macromolecules. Understanding its adherence is important for understanding the pathogenesis of F. nucleatum. In this study, a novel adhesin, FadA (Fusobacterium adhesin A), was demonstrated to bind to the surface proteins of the oral mucosal KB cells. FadA is composed of 129 amino acid (aa) residues, including an 18-aa signal peptide, with calculated molecular masses of 13.6 kDa for the intact form and 12.6 kDa for the secreted form. It is highly conserved among F. nucleatum, Fusobacterium periodonticum, and Fusobacterium simiae, the three most closely related oral species, but is absent in the nonoral species, including Fusobacterium gonidiaformans, Fusobacterium mortiferum, Fusobacterium naviforme, Fusobacterium russii, and Fusobacterium ulcerans. In addition to FadA, F. nucleatum ATCC 25586 and ATCC 49256 also encode two paralogues, FN1529 and FNV2159, each sharing 31% identity with FadA. A double-crossover fadA deletion mutant, F. nucleatum 12230-US1, was constructed by utilizing a novel sonoporation procedure. The mutant had a slightly slower growth rate, yet its binding to KB and Chinese hamster ovarian cells was reduced by 70 to 80% compared to that of the wild type, indicating that FadA plays an important role in fusobacterial colonization in the host. Furthermore, due to its uniqueness to oral Fusobacterium species, fadA may be used as a marker to detect orally related fusobacteria. F. nucleatum isolated from other parts of the body may originate from the oral cavity.
doi:10.1128/JB.187.15.5330-5340.2005
PMCID: PMC1196005  PMID: 16030227
24.  Lectinlike interactions of Fusobacterium nucleatum with human neutrophils. 
Infection and Immunity  1989;57(11):3601-3611.
Fusobacterium nucleatum expresses lectinlike adherence factors which mediate binding to a variety of human tissue cells. Adherence is selectively inhibited by galactose, lactose, and N-acetyl-D-galactosamine. In this study, adherence of F. nucleatum to human peripheral blood polymorphonuclear neutrophils (PMNs) was investigated. The results indicated that the fusobacteria adhered to live and metabolically inactivated or fixed PMNs. Adherence of F. nucleatum resulted in activation of PMNs as determined by PMN aggregation, membrane depolarization, increased intracellular free Ca2+, superoxide anion production, and lysozyme release. Transmission electron micrographs showed that F. nucleatum was phagocytized by the PMNs. Microbicidal assays indicated that greater than 98% of F. nucleatum organisms were killed by PMNs within 60 min. Adherence to and activation of PMNs by F. nucleatum were inhibited by N-acetyl-D-galactosamine or lactose greater than galactose, whereas equal concentrations of glucose, N-acetyl-D-glucosamine, mannose, and fucose had little or no effect on F. nucleatum-PMN interactions. Pretreatment of the fusobacteria with heat (80 degrees C, 20 min) or proteases inhibited adherence to and activation of PMNs, but superoxide production was also stimulated by heated bacteria. The results indicate that interaction of F. nucleatum with PMNs is lectinlike and is probably mediated by fusobacterial proteins which bind to other human tissue cells. Adherence of F. nucleatum to PMNs in the absence of serum opsonins, such as antibodies and complement, may play an important role in PMN recognition and killing of F. nucleatum in the gingival sulcus and in the subsequent release of PMN factors associated with tissue destruction.
Images
PMCID: PMC259874  PMID: 2553609
25.  Fusobacterium nucleatum Induces Premature and Term Stillbirths in Pregnant Mice: Implication of Oral Bacteria in Preterm Birth  
Infection and Immunity  2004;72(4):2272-2279.
Fusobacterium nucleatum is a gram-negative anaerobe ubiquitous to the oral cavity. It is associated with periodontal disease. It is also associated with preterm birth and has been isolated from the amniotic fluid, placenta, and chorioamnionic membranes of women delivering prematurely. Periodontal disease is a newly recognized risk factor for preterm birth. This study examined the possible mechanism underlying the link between these two diseases. F. nucleatum strains isolated from amniotic fluids and placentas along with those isolated from orally related sources invaded both epithelial and endothelial cells. The invasive ability may enable F. nucleatum to colonize and infect the pregnant uterus. Transient bacteremia caused by periodontal infection may facilitate bacterial transmission from the oral cavity to the uterus. To test this hypothesis, we intravenously injected F. nucleatum into pregnant CF-1 mice. The injection resulted in premature delivery, stillbirths, and nonsustained live births. The bacterial infection was restricted inside the uterus, without spreading systemically. F. nucleatum was first detected in the blood vessels in murine placentas. Invasion of the endothelial cells lining the blood vessels was observed. The bacteria then crossed the endothelium, proliferated in surrounding tissues, and finally spread to the amniotic fluid. The pattern of infection paralleled that in humans. This study represents the first evidence that F. nucleatum may be transmitted hematogenously to the placenta and cause adverse pregnancy outcomes. The results strengthen the link between periodontal disease and preterm birth. Our study also indicates that invasion may be an important virulence mechanism for F. nucleatum to infect the placenta.
doi:10.1128/IAI.72.4.2272-2279.2004
PMCID: PMC375172  PMID: 15039352

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