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Sequence divergence in the Treponema denticola FhbB protein and its impact on factor H binding.

ABSTRACT: Treponema denticola is an anaerobic spirochete whose abundance in the subgingival crevice correlates with the development and severity of periodontal disease. The ability of T. denticola to survive and thrive in the hostile environment of the periodontal pocket is due, at least in part, to its ability to bind factor H (FH), a negative regulator of the alternative complement pathway. The FH binding protein of T. denticola has been identified as FhbB and its atomic structure has been determined. The interaction of FH with T. denticola is unique in that FH bound to the cell surface is cleaved by the T. denticola protease, dentilisin. It has been postulated that FH cleavage by T. denticola leads to immune dysregulation in periodontal pockets. In this study, we conduct a comparative assessment of the sequence, properties, structure and ligand binding kinetics of the FhbB proteins of strains 33521 and 35405. The biological outcome of the interaction of these strains with FH could differ significantly as 33521 lacks dentilisin activity. The data presented here offer insight into our understanding of the interactions of T. denticola with the host and its potential to influence disease progression.

SUBMITTER: Miller DP

PROVIDER: S-EPMC3785937 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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Sequence divergence in the Treponema denticola FhbB protein and its impact on factor H binding.

Miller D P DP McDowell J V JV Rhodes D V DV Allard A A Caimano M M Bell J K JK Marconi R T RT

Molecular oral microbiology 20130422 4

Treponema denticola is an anaerobic spirochete whose abundance in the subgingival crevice correlates with the development and severity of periodontal disease. The ability of T. denticola to survive and thrive in the hostile environment of the periodontal pocket is due, at least in part, to its ability to bind factor H (FH), a negative regulator of the alternative complement pathway. The FH binding protein of T. denticola has been identified as FhbB and its atomic structure has been determined. T ...[more]

PMID: 23601078

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Project description:BackgroundThe oral spirochete bacterium Treponema denticola is associated with both the incidence and severity of periodontal disease. Although the biological or phenotypic properties of a significant number of T. denticola isolates have been reported in the literature, their genetic diversity or phylogeny has never been systematically investigated. Here, we describe a multilocus sequence analysis (MLSA) of 20 of the most highly studied reference strains and clinical isolates of T. denticola; which were originally isolated from subgingival plaque samples taken from subjects from China, Japan, the Netherlands, Canada and the USA.ResultsThe sequences of the 16S ribosomal RNA gene, and 7 conserved protein-encoding genes (flaA, recA, pyrH, ppnK, dnaN, era and radC) were successfully determined for each strain. Sequence data was analyzed using a variety of bioinformatic and phylogenetic software tools. We found no evidence of positive selection or DNA recombination within the protein-encoding genes, where levels of intraspecific sequence polymorphism varied from 18.8% (flaA) to 8.9% (dnaN). Phylogenetic analysis of the concatenated protein-encoding gene sequence data (ca. 6,513 nucleotides for each strain) using Bayesian and maximum likelihood approaches indicated that the T. denticola strains were monophyletic, and formed 6 well-defined clades. All analyzed T. denticola strains appeared to have a genetic origin distinct from that of 'Treponema vincentii' or Treponema pallidum. No specific geographical relationships could be established; but several strains isolated from different continents appear to be closely related at the genetic level.ConclusionsOur analyses indicate that previous biological and biophysical investigations have predominantly focused on a subset of T. denticola strains with a relatively narrow range of genetic diversity. Our methodology and results establish a genetic framework for the discrimination and phylogenetic analysis of T. denticola isolates, which will greatly assist future biological and epidemiological investigations involving this putative 'periodontopathogen'.

Project description:The gene encoding the major outer sheath protein (Msp) of the oral spirochete Treponema denticola ATCC 35405 was cloned, sequenced, and expressed in Escherichia coli. Preliminary sequence analysis showed that the 5' end of the msp gene was not present on the 5.5-kb cloned fragment described in a recent study (M. Haapasalo, K. H. Müller, V. J. Uitto, W. K. Leung, and B. C. McBride, Infect. Immun. 60:2058-2065,1992). The 5' end of msp was obtained by PCR amplification from a T. denticola genomic library, and an open reading frame of 1,629 bp was identified as the coding region for Msp by combining overlapping sequences. The deduced peptide consisted of 543 amino acids and had a molecular mass of 58,233 Da. The peptide had a typical prokaryotic signal sequence with a potential cleavage site for signal peptidase 1. Northern (RNA) blot analysis showing the msp transcript to be approximately 1.7 kb was consistent with the identification of a promoter consensus sequence located optimally upstream of msp and a transcription termination signal found downstream of the stop codon. The entire msp sequence was amplified from T. denticola genomic DNA and cloned in E. coli by using a tightly regulated T7 RNA polymerase vector system. Expression of Msp was toxic to E. coli when the entire msp gene was present. High levels of Msp were produced as inclusion bodies when the putative signal peptide sequence was deleted and replaced by a vector-encoded T7 peptide sequence. Recombinant Msp purified to homogeneity from a clone containing the full-length msp gene adhered to immobilized laminin and fibronectin but not to bovine serum albumin. Attachment of recombinant Msp was decreased in the presence of soluble substrate. Attachment of T. denticola to immobilized laminin and fibronectin was increased by pretreatment of the substrate with recombinant Msp. These studies lend further support to the hypothesis that Msp mediates the extracellular matrix binding activity of T. denticola.

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Sequence divergence in the Treponema denticola FhbB protein and its impact on factor H binding.

Publications

Sequence divergence in the Treponema denticola FhbB protein and its impact on factor H binding.

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