Fibronectin-binding protein TDE1579 affects cytotoxicity of Treponema denticola

Abstract

While FbpA, a family of bacterial fibronectin (FN) binding proteins has been studied in several gram-positive bacteria, the gram-negative Treponema denticola, an anaerobic periodontal pathogen, also has an overlooked fbp gene (tde1579). In this research, we confirm that recombinant Fbp protein (rFbp) of T. denticola binds human FN with a Kdapp of 1.5 × 10(-7) M and blocks the binding of T. denticola to FN in a concentration-dependent manner to a level of 42%. The fbp gene was expressed in T. denticola. To reveal the roles of fbp in T. denticola pathogenesis, an fbp isogenic mutant was constructed. The fbp mutant had 51% reduced binding ability to human gingival fibroblasts (hGF). When hGF were challenged with T. denticola, the fbp mutant caused less cell morphology change, had 50% reduced cytotoxicity to hGF, and had less influence on the growth of hGF cells.

Keywords: Fbp; Fibronectin binding protein; Gene mutant; Periodontal disease; Treponema denticola.

Fig. 1

Analysis of fbp gene of T. denticola shows significant homology to other bacterial Fbp proteins in the FbpA family. A. Multiple alignment of FbpA proteins from T. denticola, T. vincentii, B. burgdorferi, S. pyogenes (Fbp54) and S. pneumoniae (PavA) by ClustalW. “*”; residues are identical in all sequences. “:”;conserved substitutions have been observed. “.”;semi-conserved substitutions are observed. B. Schematic diagram of identified domain structure of 471 residue Fbp protein. The FbpA domain is located in the N-terminal region with an internal deletion region. A domain of unknown function (DUF814) is located at C-terminal region.

Fig. 2

Recombinant Fbp binds to human FN in a concentration dependent manner, and preferentially binds type I and type II FN modules. A. SDS-PAGE shows expression of recombinant Fbp in E. coli/pFBP. B. The rFbp was purified from E. coli. (rFbp indicated by arrow). C. The purified rFbp binds FN in a concentration-dependent manner with K_dapp of 1.5 × 10⁻⁷ M. Each value is the average and SD of three independent assays. D. Eight recombinant FN fragments were illustrated as rectangular boxes with full length FN in a schematic diagram. E. The binding of rFbp to recombinant FN fragments was determined. Each value is the average and SD of three independent assays.

Fig. 3

A. Fbp was present in membrane and cytoplasmic fractions of wildtype T. denticola, but not in fbp mutant. B. T. denticola membrane treated at indicated temperature demonstrated that membrane proteins dissociated with heating. C. Fbp in T. denticola membrane was detected when T. denticola membrane was treated at temperature greater than 56 °C.

Fig. 4

Confirmation of T. denticola fbp deletion mutant by PCR. Wildtype strain and fbp mutant with various primer pairs showed that fbp gene in T. denticola mutant was replaced by erm cassette.

Fig. 5

A. The binding of T. denticola to human FN and gingival fibroblast cells. No difference in bacterial FN binding between the wildtype strain and the fbp mutant was identified. Each value is the average and SD of three independent assays. B. The fbp mutant showed 51% reduced binding to hGF compared to wildtype. Each value is the average and SD of four independent assays.

Fig. 6

Cytotoxic effects of T. denticola on human gingival fibroblasts. The cultured hGF cells were challenged by T. denticola and were viewed by phase-contrast microscopy after 48 h. A. Unchallenged cells serve as negative control. B. hGF cells challenged with wildtype manifested a rounded shape. C. hGF cells challenged with fbp mutant showed less morphologic changes than those infected with wildtype. D. Survival of hGF cells were determined 5 days after challenge with T. denticola. The fbp mutant caused less cell death at concentration of 0.37 × 10⁸ and 1.1 × 10⁸ (*P <0.05 by one-tail t-test). Data is the average and SD of 3 independent experiments. E. The growth of hGF cells in co-culture with T. denticola after passage of cells on day 7 was determined. The fbp mutant had less effect on the growth of hGF cells than wildtype strain (*P <0.05 by one-tail t-test). Data is the average and SD of 3 independent experiments.