Analysis of the complement sensitivity of oral treponemes and the potential influence of FH binding, FH cleavage and dentilisin activity on the pathogenesis of periodontal disease.

Treponema denticola, a periopathogen, evades complement-mediated killing by binding the negative complement regulatory protein factor H (FH) to its surface via the FhbB protein. Paradoxically, bound FH is cleaved by T. denticola's dentilisin protease, a process hypothesized to trigger localized dysregulation of complement activation in periodontal pockets. The ability of other oral treponemes to evade complement-mediated killing and bind and cleave FH has not been assessed. In this report, we demonstrate that representative isolates of Treponema socranskii, Treponema medium, Treponema pectinovorum and Treponema maltophilum are also serum resistant, whereas Treponema vincentii and Treponema amylovorum are serum sensitive. Although T. denticola's ability to evade complement-mediated killing is strictly dependent on FH binding, other serum-resistant treponemal species lack FhbB and do not bind FH, indicating an FH-independent mechanism of complement evasion. To assess the influence of FhbB sequence variation on FH binding and cleavage by T. denticola, fhbB sequences were determined for 30 isolates. Three distinct phyletic types were identified. All T. denticola strains bound FH and were serum resistant, but differences in binding kinetics, dentilisin activity and FH cleavage ability were observed. Based on these analyses, we hypothesize that the composition of the T. denticola population is a determining factor that influences the progression and severity of periodontal disease.

Conservation and revised annotation of the Treponema denticola prcB-prcA-prtP locus encoding the dentilisin (CTLP) protease complex.

Interstrain differences in antigenic surface proteins may reflect immunological pressure or differences in receptor specificity of the antigen. Treponema denticola exhibits considerable interstrain variability in its major surface protein (Msp), but no studies have addressed this issue in dentilisin (CTLP), a surface protease complex that has a significant role in T. denticola-host interactions in periodontal disease. Furthermore, the genome annotation of the prcB-prcA-prtP operon encoding dentilisin contains apparent errors and lacks a deduced PrtP amino acid sequence. To address these issues we analysed the protease operon from diverse T. denticola strains, as well as clones of the ATCC 35405 Type strain from which the genome sequence and original GenBank prtP sequence were derived. 6xHis-tagging of the PrtP C-terminus in ATCC 35405 demonstrated absence of the 'authentic frameshift' in PrtP reported in the genome databases. We propose that T. denticola genome annotations be updated to reflect this new information. PrcB and the PrtP N-terminal region that includes the catalytic domain were highly conserved in common laboratory strains and clinical isolates of T. denticola. Dentilisin proteolytic activity varied considerably between strains. Antibodies against PrcB, PrcA and PrtP from the type strain recognized these proteins in most T. denticola strains. PrtP varied up to 20% over the C-terminal 270 residues between strains. The PrtP C-terminal eight-residues (DWFYVEYP) was present in all strains, with two strains containing an additional Y-residue preceding the stop codon. Such conserved PrtP domains may be required for interactions with PrcA and PrcB, or for substrate interactions.

Identification of the primary mechanism of complement evasion by the periodontal pathogen, Treponema denticola.

Treponema denticola, a periodontal pathogen, binds the complement regulatory protein Factor H (FH). Factor H binding protein B (FhbB) is the sole FH binding protein produced by T. denticola. The interaction of FhbB with FH is unique in that FH is bound to the cell and then cleaved by the T. denticola protease, dentilisin. A ∼ 50-kDa product generated by dentilisin cleavage is retained at the cell surface. Until this study, a direct role for the FhbB-FH interaction in complement evasion and serum sensitivity had not been demonstrated. Here we assess the serum resistance of T. denticola strain 35405 (Td35405wt) and isogenic mutants deficient in dentilisin (Td35405-CCE) and FhbB production (Td35405ΔfhbB), respectively. Both dentilisin and FhbB have been postulated to be key virulence factors that mediate complement evasion. Consistent with conditions in the subgingival crevice, an environment with a significant concentration of complement, Td35405wt was resistant to serum concentrations as high as 25%. Deletion of fhbB (Td35405ΔfhbB), which resulted in the complete loss of FH binding ability, but not inactivation of dentilisin activity (Td35405-CCE), rendered T. denticola highly sensitive to 25% human serum with 80% of the cells being disrupted after 4 h of incubation. Heat treatment of the serum to inactivate complement confirmed that killing was mediated by complement. These results indicate that the FH-FhbB interaction is required for serum resistance whereas dentilisin is not. This report provides new insight into the novel complement evasion mechanisms of T. denticola.

Differential gene expression in an actinorhizal symbiosis: evidence for a nodule-specific cysteine proteinase.

Nodules formed on the roots of actinorhizal plants as a consequence of nitrogen-fixing symbioses with the actinomycete Frankia appear to result from modification of the developmental pathway that leads to lateral root formation. Presently no information exists about factors that control this developmental switch or, until now, about genes that are differentially expressed as a result of an altered developmental pathway. Differential screening of an Alnus glutinosa nodule cDNA library revealed altered levels of gene expression in nodules as compared with roots and allowed isolation of host plant nodule-specific cDNA sequences. The deduced amino acid sequence of one full-length cDNA, AgNOD-CP1, represents a nodule-specific cysteine proteinase similar to cysteine proteinases of the papain superfamily. Residues critical to catalysis, active site, and disulfide bridges are conserved. Suggested roles for this enzyme are as a defense response to Frankia invasion, as a component of tissue remodeling in root and nodule tissues, as a cell cycle component, or as an element of protein turnover. Complexity of hybridization patterns revealed by Southern blot analysis suggests that the gene for AgNOD-CP1 is a member of a multigene family. Northern hybridization results indicate that this gene may have been recruited for a role specific to this symbiosis, a phenomenon observed in the Rhizobium-legume symbioses, perhaps common to many microbe-plant interactions.