Characterization of Dentipain, a Streptococcus-pyogenes-IdeS-protease homologue in Treponema denticola

Objectives: Treponema denticola is frequently isolated from lesions of chronic periodontitis, and exhibits a variety of proteolytic activities considered to be major inflammation-inducing virulence factors in periodontal tissue. The purpose of this study was to search and characterize the homologue of Streptococcus-pyogenes-IdeS-protease (IdeS) in T. denticola.

Methods: The IdeS homologue was obtained by a homology search against the genome sequence of T. denticola in the Oral Pathogen Sequence Database. The protease domain of this gene was cloned and overexpressed. Proteolytic activity of the recombinant protein was evaluated using insulin as a substrate. The gene was inactivated using the ermF-ermAM cassette by electroporation. Pathogenicity was evaluated by abscess formation test. T. denticola (7 x 10⁹ cells) was injected into the lateral abdomen of balb/c mice, and the size of the ensuing abscess measured.

Results: Analysis of the T. denticola genome revealed an ORF whose product was orthologous to IdeS. The N-terminus of this protein was composed of tandem, immunoglobulin-like domains, followed by a unique sequence linking to the C-terminally located IdeS-like protease domain. The obtained gene was designated as dentipain (ideT). Although this protein showed only a 25% identity with the IdeS protease, the putative catalytic cysteine and histidine residues were strongly conserved. The recombinant dentipain was found to degrade the insulin β-chain. This activity was inhibited by E-64. Analysis of the products degraded revealed specificity for small aliphatic amino acid residues at the penultimate position upstream of the hydrolyzed bond. Despite digestion of insulin β-chain, no cleavage of other protein substrates, including the IgGs, was detected, indicating a highly specific mode of action for this protease. In dentipain-deficient mutant, abscess-forming ability was significantly reduced in comparison with the parental strain

Conclusion: These results demonstrate that dentipain plays an important role in the virulence of T. denticola.