Objectives: The bacterium T. denticola is a periodontal pathogen. The ability of T. denticola to bind fibronectin (FN) may be critical to its pathogenicity. Several T. denticola FN binding proteins (Fbps) have been identified previously by Western Blotting, but have not been characterized in detail. A gene (fbp) with significant homology to known Fbps genes from other bacteria was identified from the T. denticola genome sequence. To investigate this potential virulence factor, we cloned the fbp gene and initiated characterization of the encoded protein.
Methods: The fbp gene was amplified from T. denticola strain 35405 genomic DNA by high fidelity PCR after digestion with HindIII, cloned into pRSETA with an N-terminal His tag, and verified by DNA sequencing. Fbp was then expressed in E. coli BL21(DE3) and purified by metal affinity chromatography. Interactions of Fbp with FN were analyzed in microwell-based protein-protein binding assays.
Results: The amplified fbp gene from T. denticola ATCC 35405 genomic DNA had a size of 1.4 Kb. The fbp reading frame encoded a 471 amino acid Fbp, precisely matching the GenBank database sequence (accession number AE017226.1). Recombinant Fbp expressed in soluble form with a mass of 54 kDa after induction with IPTG. Fbp was purified to a purity >90% as assessed by scanning of images of SDS-PAGE gels. Initial protein binding assays demonstrated specific interactions between the T. denticola Fbp and FN. Studies are now verifying the role of Fbp in binding T. denticola to FN.
Conclusion: These experiments cloned a gene (fbp) from T. denticola which encoded a FN binding protein (Fbp). Understanding the specific contribution of Fbp to T. denticola interactions with FN may provide important clues to the virulence mechanisms of this microorganism. Supported by NIDCR grants DE12818, DE14236, and DE016312 as well as DE14318 for CO STAR.
M. R, B, S., Z, C., A, Y., Dr. Sanjay Pal, E, K., and X, X., “Cloning and Expression of Treponema Denticola Fibronectin-binding Protein (Fbp)”, J Dent Res , vol. 86, no. A, p. 2873, 2007.