Project description:Individual miRNA analyzed were successfully constructed through nanostring technology of a total of 577 mouse miRNAs in 20 number of SHAM mice and 20 number of Tannerella forsythia infected mice, which have been euthanized on the end of 16 weeks infection study.
Project description:BACKGROUND:Tannerella forsythia is a bacterial pathogen implicated in periodontal disease. Numerous virulence-associated T. forsythia genes have been described, however, it is necessary to expand the knowledge on T. forsythia's genome structure and genetic repertoire to further elucidate its role within pathogenesis. Tannerella sp. BU063, a putative periodontal health-associated sister taxon and closest known relative to T. forsythia is available for comparative analyses. In the past, strain confusion involving the T. forsythia reference type strain ATCC 43037 led to discrepancies between results obtained from in silico analyses and wet-lab experimentation. RESULTS:We generated a substantially improved genome assembly of T. forsythia ATCC 43037 covering 99% of the genome in three sequences. Using annotated genomes of ten Tannerella strains we established a soft core genome encompassing 2108 genes, based on orthologs present in >?=?80% of the strains analysed. We used a set of known and hypothetical virulence factors for comparisons in pathogenic strains and the putative periodontal health-associated isolate Tannerella sp. BU063 to identify candidate genes promoting T. forsythia's pathogenesis. Searching for pathogenicity islands we detected 38 candidate regions in the T. forsythia genome. Only four of these regions corresponded to previously described pathogenicity islands. While the general protein O-glycosylation gene cluster of T. forsythia ATCC 43037 has been described previously, genes required for the initiation of glycan synthesis are yet to be discovered. We found six putative glycosylation loci which were only partially conserved in other bacteria. Lastly, we performed a comparative analysis of translational bias in T. forsythia and Tannerella sp. BU063 and detected highly biased genes. CONCLUSIONS:We provide resources and important information on the genomes of Tannerella strains. Comparative analyses enabled us to assess the suitability of T. forsythia virulence factors as therapeutic targets and to suggest novel putative virulence factors. Further, we report on gene loci that should be addressed in the context of elucidating T. forsythia's protein O-glycosylation pathway. In summary, our work paves the way for further molecular dissection of T. forsythia biology in general and virulence of this species in particular.
Project description:Tannerella forsythia is a Gram-negative oral pathogen known to possess an O-glycosylation system responsible for targeting multiple proteins associated with virulence at the three-residue motif (D)(S/T)(A/I/L/V/M/T). Multiple proteins have been identified to be decorated with a decasaccharide glycan composed of a poorly defined core plus a species-specific portion whose biosynthesis is largely characterized. To date, the glycosylation of mainly the two S-layer glycoproteins, TfsA and TfsB has been studied yet the true extent of glycosylation within this species has not been explored. In the present study we explore the glycoproteome of T. forsythia employing FAIMS based glycopeptide enrichment of a cell membrane fraction. We demonstrate that at least 13 glycans are utilized within the T. forsythia glycoproteome varying with respect to the presence of the three terminal sugars and the presence of fucose and digitoxose branches at the reducing end. To improve the localization of glycosylation events and enhance the detection of glycopeptides we applied trifluoromethanesulfonic acid treatment to allow the selective chemical cleavage of glycans. By reducing the chemical complexity of glycopeptides this dramatically improved the number of glycopeptides identified and our ability to localize glycosylation sites by ETD fragmentation leading to the identification of 312 putative glycosylation sites in 145 glycoproteins. Glycosylation site analysis revealed that glycosylation occurs on a much broader glycosylation motif than initially reported with glycosylation found at (D)(S/T)(A/I/L/V/M/T/S/C/G/F). The data confirm earlier predictions of hundreds of possible O-glycoproteins present in this organism.
Project description:Tannerella forsythia is an oral pathogen implicated in the development of periodontitis. Here, we report the draft genome sequence of the Tannerella forsythia strain ATCC 43037. The previously available genome of this designation (NCBI reference sequence NC_016610.1) was discovered to be derived from a different strain, FDC 92A2 (= ATCC BAA-2717).
Project description:Tannerella forsythia is the only 'red-complex' bacterium covered by an S-layer, which has been shown to affect virulence. Here, outer membrane vesicles (OMVs) enriched with putative glycoproteins are described as a new addition to the virulence repertoire of T. forsythia. Investigations of this bacterium are hampered by its fastidious growth requirements and the recently discovered mismatch of the available genome sequence (92A2 = ATCC BAA-2717) and the widely used T. forsythia strain (ATCC 43037). T. forsythia was grown anaerobically in serum-free medium and biogenesis of OMVs was analyzed by electron and atomic force microscopy. This revealed OMVs with a mean diameter of ~100 nm budding off from the outer membrane while retaining the S-layer. An LC-ESI-TOF/TOF proteomic analysis of OMVs from three independent biological replicates identified 175 proteins. Of these, 14 exhibited a C-terminal outer membrane translocation signal that directs them to the cell/vesicle surface, 61 and 53 were localized to the outer membrane and periplasm, respectively, 22 were predicted to be extracellular, and 39 to originate from the cytoplasm. Eighty proteins contained the Bacteroidales O-glycosylation motif, 18 of which were confirmed as glycoproteins. Release of pro-inflammatory mediators from the human monocytic cell line U937 and periodontal ligament fibroblasts upon stimulation with OMVs followed a concentration-dependent increase that was more pronounced in the presence of soluble CD14 in conditioned media. The inflammatory response was significantly higher than that caused by whole T. forsythia cells. Our study represents the first characterization of T. forsythia OMVs, their proteomic composition and immunogenic potential.
Project description:Draft genome sequences of three clinical isolates of Tannerella forsythia isolated from sub-gingival plaque from periodontitis patients