Characterization of a novel family of fibronectin-binding proteins with M23 peptidase domains from Treponema denticola

Abstract

Interactions with fibronectin are important in the virulence strategies of a range of disease-related bacteria. The periodontitis-associated oral spirochaete Treponema denticola expresses at least two fibronectin-binding proteins, designated Msp (major surface protein) and OppA (oligopeptide-binding protein homologue). To identify other T. denticola outer membrane fibronectin-binding proteins, the amino acid sequence of the Treponema pallidum fibronectin-binding protein Tp0155 was used to survey the T. denticola genome. Seven T. denticola genes encoding orthologous proteins were identified. All but two were expressed in Escherichia coli and purified recombinant proteins bound fibronectin. Using antibodies to the N-terminal region of Tp0155, it was demonstrated that T. denticola TDE2318, with highest homology to Tp0155, was cell surface localized. Like Tp0155, the seven T. denticola proteins contained an M23 peptidase domain and four (TDE2318, TDE2753, TDE1738, TDE1297) contained one or two LysM domains. M23 peptidases can degrade peptidoglycan whereas LysM domains recognize carbohydrate polymers. In addition, TDE1738 may act as a bacteriocin based on homology with other bacterial lysins and the presence of an adjacent gene encoding a putative immunity factor. Collectively, these results suggest that T. denticola expresses fibronectin-binding proteins associated with the cell surface that may also have cell wall modifying or lytic functions.

Figure 1

(A) Predicted identifiable peptide motifs (www.ebi.ac.uk/InterProScan/) of Treponema pallidum fibronectin-binding protein Tp0155 and potential orthologues identified from the Treponema denticola genome using BLAST similarity searches. LysM domains are shown with vertical bars (A) or dots (B). Treponema pallidum and T. denticola M23 peptidase domains (diagonal bars) retain 37% identity. Potential signal sequences (solid black) were identified using http://bioinformatics.leeds.ac.uk/prot_analysis/signal.html. (B) Alignments of LysM(A) and LysM(B) domain sequences from T. pallidum Tp0155 and potential T. denticola orthologues showing high degree of consensus across the different peptide sequences. Degree of identity is shown from highest (e.g. ) to lowest (e.g. H). The LysM motif YxxxxGxx(Hyd) (Turner et al., 2004) is boxed. Asterisk indicates residues that do not fit the specified motif.

Figure 2

Recombinant Treponema denticola His-tagged proteins (5μg), which show homology to fibronectin-binding protein Tp0155 of Treponema pallidum, purified using nickel affinity chromatography, analysed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) stained with silver nitrate (A), and by Western blot, using anti-tetra His antibodies to detect each protein (B) or antibodies to the T. pallidum protein Tp0155, which had first been pre-adsorbed with T. phagedenis (Kazan) (C). Treponema denticola ATCC35405 outer membrane proteins extracted with 1% Triton X-114 incubated at 20°C or 100°C before separation by SDS–PAGE analyses (d) exposed to antibodies to T. pallidum Tp0155 (E). Molecular mass standards (kDa) are indicated to the side of the figure.

Figure 3

Adhesion of recombinant Treponema denticola proteins to matrix (grey bars) or plasma fibronectin (black bars). Recombinant protein (5 μg) was added to each well, except for TDE2753 and TDE1738, which appeared to bind non-specifically to the substrates when more than 1.25μg was added. Stars denote significant difference (P < 0.05) between adhesion to either matrix or plasma fibronectin and bovine serum albumin-blocked wells. Error bars indicate ± SD of the mean from triplicates of a representative experiment.

Figure 4

Detection of proteins on the surface of Treponema denticola ATCC 35405 using antibodies to the Treponema pallidum protein Tp0155 which had first been pre-adsorbed against Treponema phagedenis (Kazan). (A, C, E, G, I, K and M) Phase-contrast images; (B, D, F, H, J, L and N) corresponding immunofluorescence. (A, B) Preimmune serum for Tp0155-specific antibodies; (C, D), Tp0155-specific antibodies, non-detergent-treated T. denticola; (E, F), Tp0155-specific antibodies, Triton X-114-treated T. denticola; (G, H), preimmune serum for Tp0249-specific antibodies; (I, J), Tp0249-specific antibodies, non-detergent-treated T. denticola (negative control); (K, L), Tp0249-specific antibodies, Triton X-114-treated T. denticola; (M, N), anti-T. denticola antibodies (control) (Edwards et al., 2005).

Figure 5

(A) Alignments of M23 peptidase domains from TDE1738 of Treponema denticola and homologous regions of peptidoglycan-degrading enzymes lysostaphin from Staphylococcus simulans biovar staphylolyticus, zoocin A from Streptococcus equi subsp zooepidemicus and ALE-1 from Staphylococcus capitis. The M23 peptidase domain of TDE1738 retains 43% identity to lysostaphin, 43% identity to zoocin A and 40% identity to ALE-1 with conservation across the HxGxD and HLH active sites. (B) Alignments of a 92 amino acid (aa) residue portion regions of TDE1739 of T. denticola and lysostaphin immunity factor from S. simulans biovar staphylolyticus, demonstrating homology (29% identity) between these peptides. Sequence consensus is shown as is degree of identity from highest (e.g. ) to lowest (e.g. A). (C) Diagram depicting genes encoding potential and known peptidoglycan degradation enzymes and adjacent immunity factors which provide protection from autolysis by the peptidoglycanolytic enzyme. (i) The chromosomal gene TDE1738 from T. denticola encodes for a potential peptidoglycan-degrading enzyme, and TDE1739 for a potential immunity factor. (ii) Lysostaphin and adjacent lysostaphin immunity factor (Lif/Epr) are encoded on the plasmid pACK1 of S. simulans biovar staphylolyticus. (iii) The gene encoding zoocin A and corresponding zoocin A immunity factor (Zif) is on the chromosome of Streptococcus equi subsp zooepidemicus, while (iv) ale-1 and adjacent endopeptidase resistance (epr) genes are located on a plasmid in Staphylococcus capitis. (v) Tp0706 from the Treponema pallidum chromosome, with homology to TDE1738 may also be an enzyme with peptidoglycan-degrading activity and the adjacent gene Tp0705 is annotated as a pencillin-binding protein in the same manner as zoocin A immunity factor (Zif).