Treponema denticola is a dominant microorganism in human periodontal lesions. One of the major virulence factors of this microorganism is its chymotrypsinlike surface protease, dentilisin. The purpose of this study was to evaluate the effect of dentilisin on human polymorphonuclear leukocytes (PMNs). We used chemiluminescence to assess production of O 2 -by PMNs against T. denticola ATCC 35405 and dentilisin deficient mutant K1. T. denticola ATCC 35405 induced production of O 2 -, whereas dentilisin deficient K1 did not. We found that chymostatin, a protease inhibitor, strongly reduced the ability of T. denticola ATCC 35405 to induce production of, O 2 -, whereas K1 was relatively unaffected. We also usedImmunoblot and ELISA to evaluate the activation of complement by this microorganism in relation to PMNs. T. denticola ATCC 35405 hydrolyzed the α-chain of C3, producing iC3b.Furthermore, strain ATCC 35405 induced a larger release of MMP-9 from PMNs than strain K1. Dentilisin, activated PMNs via complement pathways, and may play a role in establishing periodontal lesions.

Dentilisin is a major surface protease and virulence factor of the bacterium Treponema denticola. In this study, we found that T. denticola reduced inflammatory cytokines, including interleukin-1␤ (IL-1␤), IL-6, and tumor necrosis factor alpha, in peripheral blood mononuclear cells through degradation by dentilisin.Chronic periodontitis is an infectious disease characterized by the accumulation of inflammatory cells in extravascular gingival connective tissue, which finally causes the breakdown of periodontal tissue and tooth loss (21,28,35,36,38). Treponema denticola has been identified as a major pathogen in human periodontal disease (1,18,20,25). Previous studies have revealed that a number of inflammatory cytokines are synthesized in response to periodontopathic bacteria and their products, thus inducing and maintaining an inflammatory response in the periodontium (3,29). Inflammatory cytokines, such as interleukin-1␤ (IL-1␤), IL-6, and tumor necrosis factor alpha (TNF-␣), are closely associated with the development of periodontal lesions (9, 16, 23, 30-32, 34, 40). It has been shown that T. denticola induces the production of various cytokines, including IL-1␤, IL-6, IL-8, and TNF-␣ from various cell types (2,6,15,26,33). On the other hand, it has also been suggested that T. denticola hydrolyzes 26). This microorganism possesses a surface protease, dentilisin, which hydrolyzes host proteins and is believed to be a major pathogen of this microorganism (12, 13, 37). Proteases from periodontopathic bacteria such as Porphyromonas gingivalis have been reported to modulate immunoresponses by hydrolyzing inflammatory mediators (3, 5). The aim of this study was to verify the ability of dentilisin to degrade inflammatory cytokines, including IL-1␤, IL-6, and TNF-␣.In this study, we used T. denticola ATCC 35405 and dentilisin-deficient mutant K1 (12). T. denticola was propagated in TYGVS medium (27) and incubated at 37°C for 4 days under anaerobic conditions as described previously (10).In order to estimate the effect of T. denticola on the production of inflammatory cytokines, we determined the amount of cytokines produced in human peripheral blood mononuclear cells (PBMCs) after exposure to T. denticola. Peripheral blood was obtained from three healthy adult volunteers by venipuncture after informed consents were obtained. The PBMCs were separated by density gradient centrifugation using a Lymphoprep tube (Axis-Shield, Oslo, Norway) according to the manufacturer's instructions. The cells obtained were washed with phosphatebuffered saline (PBS [pH 7.4]; Nissui, Tokyo, Japan) and resuspended in RPMI 1640 medium containing 2 mM L-glutamine (Nissui) and 10% fetal bovine serum (Bio-Whittaker, Maryland). The PBMCs (5 ϫ 10 8 cells/ml) were then seeded in 96-well microtiter plates at 200 l/well, and a 20-l T. denticola cell suspension was then added at 1 ϫ 10 7 cells/well. Cells were incubated at 37°C for 24 h in humidified air containing 5% CO 2 . After incubation, the amounts of cytokines in the supernatants were measure...

Treponema denticola is a major pathogen of chronic periodontitis. Analysis of the T. denticola genome revealed a gene orthologous with a gene encoding a cysteine protease from Streptococcus pyogenes (IdeS). IdeS interferes with IgG-dependent opsonophagocytosis by specific cleavage of IgG molecules. Analysis of this gene (termed ideT) revealed it to encode a two domain protein. The N-terminus of this protein is composed of a tandem, immunoglobulin-like domain, followed by a C-terminally located IdeS-like protease domain. We show here that during secretion the IdeT protein is processed into an N-terminal fragment which remains associated with the cell, and a C-terminal portion released into the medium. Although this secreted domain of IdeT, termed dentipain, shows only 25% identity with the IdeS protease, the putative catalytic cysteine and histidine residues are strongly conserved. Analysis of recombinant dentipain revealed that it cleaved the insulin β-chain, an activity which was inhibited by E-64, a diagnostic inhibitor of cysteine proteases. Apart from insulin no cleavage of other protein substrates was detected, suggesting that dentipain has oligopeptidase activity. A mutant strain was constructed bearing a modified ideT whose dentipain domain was deleted. This mutant was found to be significantly reduced in its abscess forming activity compared to the parental strain in a murine abscess model, suggesting that dentipain contributes to the virulence of T. denticola.

Background and Objectives: High levels of colonization by periodontopathic bacteria and a

Treponema denticola, a major pathogen of periodontitis, has also been detected in the lesions of atherosclerosis. The aim of this study was to investigate induction of chemokine production in human umbilical vein endothelial cells (HUVECs) by T. denticola and determine whether those chemokines were degraded by a protease, dentilisin. T. denticola ATCC35405 or dentilisin-deficient mutant K1 were added to HUVECs and levels of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) in the culture supernatants were determined by enzyme-linked immunosorbent assay. T. denticola ATCC35405 induced production of IL-8 in a time-dependent manner, with both production of IL-8 and expression of IL-8 mRNA showing higher levels than with exposure to dentilisin-deficient mutant K1. Although exposure to ATCC35405 induced expression of MCP-1 mRNA in the HUVECs, MCP-1 levels were remained similar to that in unstimulated cells. IL-8 and MCP-1 showed partial hydrolysis with exposure to T. denticola ATCC35405, but not with T. denticola K1. These results suggest that T. denticola can evade host defense mechanisms by modulating production of IL-8 and MCP-1, and that this play a role in the development of chronic infections such as periodontitis. The association of T. denticola infection to atherosclerosis was also discussed based on the present study.-3 -