Dataset Information

Degradation of chondroitin sulfate A by a PUL-like operon in Tannerella forsythia.

ABSTRACT: Advanced periodontitis has been shown to have strong association with the residence of the bacterial consortia known as the red complex comprised by Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. T. forsythia shares a distant genetic linkage to Bacteroidetes thetaiotaomicron and may therefore produce analogous polysaccharide utilization loci (PUL) which enable complex carbohydrate degradation, import, and use, although this capacity has yet to be demonstrated. Chondroitin sulfate A is a linear, sulfated carbohydrate linked to periodontal disease as the principal species of glycosaminoglycan appended on the surface of cortical bone of teeth and in supporting dental ligaments. Through genomic comparisons with B. thetaiotaomicron, a new PUL-like operon (Bfo2285-Bfo2295, and Bfo3043) was identified in T. forsythia and the crystal structure of two proteins from this PUL-like operon, Bfo2290 and Bfo2294, were reported using X-ray crystallography. Enzyme kinetics for Bfo2290 were reported using a pH-dependent assay and suggested a Km of 0.75 mg/ml ± 0.60 mg/ml, Kcat of 3.74 min-1 ± 0.88 min-1, and Vmax of 7.48 μM/min ± 1.76 μM/min with partially degraded chondroitin sulfate A. Fluorophore-assisted carbohydrate electrophoresis was used to show the processive degradation of chondroitin sulfate A by the proteins encoded in T. forsythia PUL-like operon, and revealed Bfo2291 and Bfo2290 to be an endolytic chondroitin sulfate A lyase and exolytic ΔDi-4S chondroitin sulfate A sulfatase, respectively.

SUBMITTER: Nguyen P

PROVIDER: S-EPMC9481042 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Degradation of chondroitin sulfate A by a PUL-like operon in Tannerella forsythia.

Nguyen Peter P Eshaque Rony R Garland Barbara Anne BA Dang Anthony A Suits Michael D L MDL

PloS one 20220916 9

Advanced periodontitis has been shown to have strong association with the residence of the bacterial consortia known as the red complex comprised by Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. T. forsythia shares a distant genetic linkage to Bacteroidetes thetaiotaomicron and may therefore produce analogous polysaccharide utilization loci (PUL) which enable complex carbohydrate degradation, import, and use, although this capacity has yet to be demonstrated. Chondroit ...[more]

PMID: 36112631

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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 partially characterized species-specific section. To date, glycosylation studies have focused mainly on the two S-layer glycoproteins, TfsA and TfsB, so the true extent of glycosylation within this species has not been fully explored. In the present study, we characterize the glycoproteome of T. forsythia by 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 residues at the reducing end. To improve the localization of glycosylation events and enhance the detection of glycopeptides, we utilized trifluoromethanesulfonic acid treatment to allow the selective chemical cleavage of glycans. Reducing the chemical complexity of glycopeptides 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 motif than initially reported, with glycosylation found at (D)(S/T)(A/I/L/V/M/T/S/C/G/F). The prevalence of this broader glycosylation motif in the genome suggests the existence of hundreds of potential O-glycoproteins in this organism. IMPORTANCE Tannerella forsythia is an oral pathogen associated with severe forms of periodontal disease characterized by destruction of the tooth's supporting tissues, including the bone. The bacterium releases a variety of proteins associated with virulence on the surface of outer membrane vesicles. There is evidence that these proteins are modified by glycosylation, and this modification is essential for virulence in producing disease. We have utilized novel techniques coupled with mass spectrometry to identify over 13 glycans and 312 putative glycosylation sites in 145 glycoproteins within T. forsythia. Glycosylation site analysis revealed that this modification occurs on a much broader motif than initially reported such that there is a high prevalence of potential glycoproteins in this organism that may help to explain its role in periodontal disease.

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:IntroductionDegradation of host proteins by bacterial proteases leads to the subversion of the host response and disruption of oral epithelial integrity, which is considered an essential factor in the progression of periodontitis. High-temperature requirement A (HtrA) protease, which is critical for bacterial survival and environmental adaptation, is found in several oral bacteria, including the periodontal pathogen Tannerella forsythia. This study investigated the proteolytic activity of HtrA from T. forsythia and its ability to modulate the host response.MethodsHtrA of T. forsythia was identified bioinformatically and produced as a recombinant protein. T. forsythia mutants with depleted and restored HtrA production were constructed. The effect of T. forsythia wild-type, mutants and recombinant HtrA on the degradation of casein and E-cadherin was tested in vitro. Additionally, the responses of human gingival fibroblasts and U937 macrophages to the different HtrA-stimuli were investigated and compared to those triggered by the HtrA-deficient mutant.ResultsT. forsythia wild-type producing HtrA as well as the recombinant enzyme exhibited proteolytic activity towards casein and E-cadherin. No cytotoxic effect of either the wild-type, T. forsythia mutants or rHtrA on the viability of host cells was found. In hGFB and U937 macrophages, both T. forsythia species induced an inflammatory response of similar magnitude, as indicated by gene and protein expression of interleukin (IL)-1β, IL-6, IL-8, tumour necrosis factor α and monocyte chemoattractant protein (MCP)-1. Recombinant HtrA had no significant effect on the inflammatory response in hGFBs, whereas in U937 macrophages, it induced a transient inflammatory response at the early stage of infection.ConclusionHtrA of T. forsythia exhibit proteolytic activity towards the host adhesion molecule E-cadherin and has the potential to influence the host response. Its role in the progression of periodontitis needs further clarification.

Dataset Information

Degradation of chondroitin sulfate A by a PUL-like operon in Tannerella forsythia.

Publications

Degradation of chondroitin sulfate A by a PUL-like operon in Tannerella forsythia.

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