The subgingival microbiome associated with periodontitis in type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) is a systemic disease, predisposing patients to other inflammatory conditions including periodontitis. The subgingival microbiome, a key player in periodontitis pathogenesis, is not well characterized in T2DM population. To better understand whether the subgingival microbiome is different between T2DM and systemically healthy, nondiabetic (ND) subjects, we performed a longitudinal analysis of the subgingival microbiome in T2DM patients (n = 15) compared with ND subjects (n = 16). Using metagenomic shotgun sequencing, we investigated the microbiome in the healthy periodontal state, periodontitis state, and resolved state after treatment. We found that in the periodontitis state, the shift in the subgingival microbiome from the healthy state was less prominent in T2DM compared with ND subjects, yet the clinical signs of disease were similar for both. Furthermore, we revealed highly correlated presence of pathogenic species in relative abundance not only in the periodontitis state, but also in the healthy state in T2DM, suggesting an elevated risk of progression to periodontitis in this cohort. We further investigated the functional potentials of the subgingival microbiome and identified a set of microbial marker genes associated with the clinical states. These genes were significantly enriched in 21 pathways, some of which are associated with periodontitis and some potentially link T2DM and periodontitis. This study identified the longitudinal changes of the subgingival microbiome associated with periodontitis in T2DM and suggests that T2DM patients are more susceptible to shifts in the subgingival microbiome toward dysbiosis, potentially due to impaired host metabolic and immune regulation.

Dynamic changes in the subgingival microbiome and their potential for diagnosis and prognosis of periodontitis.

UNLABELLED: The human microbiome influences and reflects the health or disease state of the host. Periodontitis, a disease affecting about half of American adults, is associated with alterations in the subgingival microbiome of individual tooth sites. Although it can be treated, the disease can reoccur and may progress without symptoms. Without prognostic markers, follow-up examinations are required to assess reoccurrence and disease progression and to determine the need for additional treatments. To better identify and predict the disease progression, we aim to determine whether the subgingival microbiome can serve as a diagnosis and prognosis indicator. Using metagenomic shotgun sequencing, we characterized the dynamic changes in the subgingival microbiome in periodontitis patients before and after treatment at the same tooth sites. At the taxonomic composition level, the periodontitis-associated microorganisms were significantly shifted from highly correlated in the diseased state to poorly correlated after treatment, suggesting that coordinated interactions among the pathogenic microorganisms are essential to disease pathogenesis. At the functional level, we identified disease-associated pathways that were significantly altered in relative abundance in the two states. Furthermore, using the subgingival microbiome profile, we were able to classify the samples to their clinical states with an accuracy of 81.1%. Follow-up clinical examination of the sampled sites supported the predictive power of the microbiome profile on disease progression. Our study revealed the dynamic changes in the subgingival microbiome contributing to periodontitis and suggested potential clinical applications of monitoring the subgingival microbiome as an indicator in disease diagnosis and prognosis. IMPORTANCE: Periodontitis is a common oral disease. Although it can be treated, the disease may reoccur without obvious symptoms. Current clinical examination parameters are useful in disease diagnosis but cannot adequately predict the outcome of individual tooth sites after treatment. A link between the subgingival microbiota and periodontitis was identified previously; however, it remains to be investigated whether the microbiome can serve as a diagnostic and prognostic indicator. In this study, for the first time, we characterized the subgingival microbiome of individual tooth sites before and after treatment using a large-scale metagenomic analysis. Our longitudinal study revealed changes in the microbiota in taxonomic composition, cooccurrence of subgingival microorganisms, and functional composition. Using the microbiome profiles, we were able to classify the clinical states of subgingival plaque samples with a high accuracy. Follow-up clinical examination of sampled sites indicates that the subgingival microbiome profile shows promise for the development of diagnostic and prognostic tools.

Composition of the adult digestive tract bacterial microbiome based on seven mouth surfaces, tonsils, throat and stool samples.

BACKGROUND: To understand the relationship between our bacterial microbiome and health, it is essential to define the microbiome in the absence of disease. The digestive tract includes diverse habitats and hosts the human body's greatest bacterial density. We describe the bacterial community composition of ten digestive tract sites from more than 200 normal adults enrolled in the Human Microbiome Project, and metagenomically determined metabolic potentials of four representative sites. RESULTS: The microbiota of these diverse habitats formed four groups based on similar community compositions: buccal mucosa, keratinized gingiva, hard palate; saliva, tongue, tonsils, throat; sub- and supra-gingival plaques; and stool. Phyla initially identified from environmental samples were detected throughout this population, primarily TM7, SR1, and Synergistetes. Genera with pathogenic members were well-represented among this disease-free cohort. Tooth-associated communities were distinct, but not entirely dissimilar, from other oral surfaces. The Porphyromonadaceae, Veillonellaceae and Lachnospiraceae families were common to all sites, but the distributions of their genera varied significantly. Most metabolic processes were distributed widely throughout the digestive tract microbiota, with variations in metagenomic abundance between body habitats. These included shifts in sugar transporter types between the supragingival plaque, other oral surfaces, and stool; hydrogen and hydrogen sulfide production were also differentially distributed. CONCLUSIONS: The microbiomes of ten digestive tract sites separated into four types based on composition. A core set of metabolic pathways was present across these diverse digestive tract habitats. These data provide a critical baseline for future studies investigating local and systemic diseases affecting human health.

The role of aggregation in Fusobacterium nucleatum- induced immune cell death.

INTRODUCTION: Fusobacterium nucleatum, an anaerobic oral bacterium, has been shown to be highly abundant in endodontic infections. Its role in these infections remains unclear. Previous studies have shown that F. nucleatum could aggregate immune cells. We have demonstrated that F. nucleatum can induce significant apoptosis in peripheral blood mononuclear cells (PBMCs). In this in vitro study, we sought to determine what role this aggregation phenomenon has on the induction of apoptosis in PBMCs. METHODS: F. nucleatum bacteria were treated as follows: formaldehyde-fixed, heat-treated, or sonicated before co-culturing with PBMCs. Cell aggregation and apoptosis of the PBMCs were assessed under light microscopy and analyzed by flow cytometry, respectively. PBMCs were then immobilized with a Matrigel matrix before treatment with F. nucleatum. Aggregation and apoptosis were assessed as before. Surface staining of activation marker CD69 was assessed by flow cytometry. The apoptosis and CD69 data underwent one-way analysis of variance, followed by post hoc Bonferroni test and χ(2) test, respectively, to determine statistical significance. RESULTS: Viable and formaldehyde-treated but not sonicated or heat-treated F. nucleatum bacteria were able to cause severe aggregation and apoptosis of the immune cells. Disruption of F. nucleatum mediated aggregation by immobilization of the cells with a Matrigel matrix resulted in a significant diminution of cell death but not cell activation when assessed by using surface expression of CD69 early activation antigen. CONCLUSIONS: F. nucleatum's ability to induce cell death in immune cells appears to be mediated through the immune cells being aggregated, which might have important implications for its pathogenesis.

Fusobacterium nucleatum outer membrane proteins Fap2 and RadD induce cell death in human lymphocytes.

Bacterially induced cell death in human lymphocytes is an important virulence factor for pathogenic bacteria. Previously discovered mechanisms of bacterially induced cell death are predominantly based on the transfer of bacterial proteins to the target host cell, such as the toxins secreted through type I, II, and VI secretion systems or effector proteins injected through type III, IV, and Vb secretion systems. Here, we report a mechanism employed by the Gram-negative oral pathogen Fusobacterium nucleatum for cell death induction of human lymphocytes via two outer membrane proteins (OMPs), Fap2 and RadD, which share regions homologous to autotransporter secretion systems (type Va secretion systems). Genetic and physiological studies established that inactivation of the two OMPs led to significantly reduced ability to trigger cell death in Jurkat cells, while the corresponding double mutant was almost completely attenuated. Additional biochemical and molecular analyses demonstrated that cell-free F. nucleatum membranes are sufficient to induce cell death in Jurkat cells, suggesting that no active process or effector protein transfer was necessary to induce eukaryotic cell death.

Mobilization and prevalence of a Fusobacterial plasmid.

Fusobacterium nucleatum is a Gram-negative anaerobic rod found in dental plaque biofilms, and is an opportunistic pathogen implicated in periodontitis as well as a wide range of systemic abscesses and infections. Genomic analyses of F. nucleatum indicate considerable genetic diversity and a prominent role for horizontal gene transfer in the evolution of the species. Several plasmids isolated from F. nucleatum, including pFN1, harbor relaxase gene homologs that may function in plasmid mobilization. In this investigation we examined the RP4-mediated mobilization properties of pFN1 and the prevalence of pFN1-related sequences in a panel of F. nucleatum clinical isolates. The fusobacterial plasmid pFN1 was mobilized by RP4 at a high frequency. Deletion analyses were used to delineate the core mobilon of pFN1, which consisted of the relaxase gene (rlx), an upstream open reading frame ORF4 and a region of DNA upstream of ORF4 with potential nic sites. To examine the prevalence of pFN1 in a panel of clinical isolates, total DNA isolated from the strains was hybridized with pFN1 replication (repA) and rlx gene probes. DNA from strains harboring plasmids known to be homologous to pFN1 hybridized with both the repA and rlx probes. Five additional strains were rlx-positive but repA-negative, indicating a greater prevalence of rlx-related genes in comparison with repA-related genes. Plasmid or plasmid-related sequences were identified in 11.5% of the strains examined. These findings demonstrate mobilization properties of a fusobacterial plasmid that may be important in horizontal gene transfer.

A novel vaccine targeting Fusobacterium nucleatum against abscesses and halitosis.

An abscess in a gum pocket, resulting from bacterial infection, is a common source of chronic halitosis. Although antibiotics are generally prescribed for abscesses, they require multiple treatments with risks of creating resistant bacterial strains. Here we develop a novel vaccine using ultraviolet-inactivated Fusobacterium nucleatum (F. nucleatum), a representative oral bacterium for halitosis. A gum pocket model, established by continuous inoculation of F. nucleatum, was employed to validate the vaccine potency. Mice immunized with inactivated F. nucleatum effectively minimized the progression of abscesses, measured by swollen tissues of gum pockets. Most notably, the immunized mice were capable of eliciting neutralizing antibodies against the production of volatile sulfur compounds of F. nucleatum. The novel vaccine inducing protective immunity provides an alternative option to conventional antibiotic treatments for chronic halitosis associated with abscesses.

The Fusobacterium nucleatum outer membrane protein RadD is an arginine-inhibitable adhesin required for inter-species adherence and the structured architecture of multispecies biofilm.

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.

Genome sequence of Fusobacterium nucleatum subspecies polymorphum - a genetically tractable fusobacterium.

Fusobacterium nucleatum is a prominent member of the oral microbiota and is a common cause of human infection. F. nucleatum includes five subspecies: polymorphum, nucleatum, vincentii, fusiforme, and animalis. F. nucleatum subsp. polymorphum ATCC 10953 has been well characterized phenotypically and, in contrast to previously sequenced strains, is amenable to gene transfer. We sequenced and annotated the 2,429,698 bp genome of F. nucleatum subsp. polymorphum ATCC 10953. Plasmid pFN3 from the strain was also sequenced and analyzed. When compared to the other two available fusobacterial genomes (F. nucleatum subsp. nucleatum, and F. nucleatum subsp. vincentii) 627 open reading frames unique to F. nucleatum subsp. polymorphum ATCC 10953 were identified. A large percentage of these mapped within one of 28 regions or islands containing five or more genes. Seventeen percent of the clustered proteins that demonstrated similarity were most similar to proteins from the clostridia, with others being most similar to proteins from other gram-positive organisms such as Bacillus and Streptococcus. A ten kilobase region homologous to the Salmonella typhimurium propanediol utilization locus was identified, as was a prophage and integrated conjugal plasmid. The genome contains five composite ribozyme/transposons, similar to the CdISt IStrons described in Clostridium difficile. IStrons are not present in the other fusobacterial genomes. These findings indicate that F. nucleatum subsp. polymorphum is proficient at horizontal gene transfer and that exchange with the Firmicutes, particularly the Clostridia, is common.

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.

Characterization of the novel Fusobacterium nucleatum plasmid pKH9 and evidence of an addiction system.

Fusobacterium nucleatum is an important oral anaerobic pathogen involved in periodontal and systemic infections. Studies of the molecular mechanisms involved in fusobacterial virulence and adhesion have been limited by lack of systems for efficient genetic manipulation. Plasmids were isolated from eight strains of F. nucleatum. The smallest plasmid, pKH9 (4,975 bp), was characterized and used to create new vectors for fusobacterial genetic manipulation. DNA sequence analysis of pKH9 revealed an open reading frame (ORF) encoding a putative autonomous rolling circle replication protein (Rep), an ORF predicted to encode a protein homologous to members of the FtsK/SpoIIIE cell division-DNA segregation protein family, and an operon encoding a putative toxin-antitoxin plasmid addiction system (txf-axf). Deletion analysis localized the pKH9 replication region in a 0.96-kbp fragment. The pKH9 rep gene is not present in this fragment, suggesting that pKH9 can replicate in fusobacteria independently of the Rep protein. A pKH9-based, compact Escherichia coli-F. nucleatum shuttle plasmid was constructed and found to be compatible with a previously described pFN1-based fusobacterial shuttle plasmid. Deletion of the pKH9 putative addiction system (txf-axf) reduced plasmid stability in fusobacteria, indicating its addiction properties and suggesting it to be the first plasmid addiction system described for fusobacteria. pKH9, its genetic elements, and its shuttle plasmid derivatives can serve as useful tools for investigating fusobacterial properties important in biofilm ecology and pathogenesis.

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

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

Discrete proteolysis of focal contact and adherens junction components in Porphyromonas gingivalis-infected oral keratinocytes: a strategy for cell adhesion and migration disabling.

Adhesive interactions of cells are critical to tissue integrity. We show that infection with Porphyromonas gingivalis, a major pathogen in the periodontal disease periodontitis, interferes with both cell-matrix and cell-cell adhesion in the oral keratinocyte cell line HOK-16. Thus, infected cells showed reduced adhesion to extracellular matrix, changes in morphology from spread to rounded, and impaired motility on purified matrices in Transwell migration assays and scratch assays. Western blot analysis of P. gingivalis-challenged HOK-16 cells revealed proteolysis of focal contact components (e.g., focal adhesion kinase), adherens junction proteins (e.g., catenins), and adhesion signaling molecules (e.g., the tyrosine kinase SRC). Proteolysis was selective, since important components of adherens junctions (E-cadherin) or signaling molecules (extracellular signal-regulated kinases ERK1/2) were not degraded. The virulence factors gingipains, cysteine proteinases expressed by P. gingivalis, are likely responsible for this proteolytic attack, since they directly digested specific proteins in pull-down experiments, and their proteolytic activity was blocked by the cysteine proteinase inhibitor N-alpha-p-tosyl-L-lysine chloromethyl ketone and also by a caspase inhibitor. Proteolysis was strain dependent, such that ATCC 33277 and 381 had high proteolytic potential, whereas W50 showed almost no proteolytic activity. These findings may help explain the formation of gingival pockets between cementum and periodontal epithelium, a hallmark of periodontitis. Furthermore, they illustrate a new pathogenetic paradigm of infection whereby bacteria may disrupt the integrity of epithelia.