The Chymotrypsin-Like Protease Complex ofTreponema denticolaATCC 35405 Mediates Fibrinogen Adherence and Degradation

Abstract

Treponema denticola is an anaerobic spirochete strongly associated with human periodontal disease. T. denticola bacteria interact with a range of host tissue proteins, including fibronectin, laminin, and fibrinogen. The latter localizes in the extracellular matrix where tissue damage has occurred, and interactions with fibrinogen may play a key role in T. denticola colonization of the damaged sites. T. denticola ATCC 35405 showed saturable binding of fluid-phase fibrinogen to the cell surface and saturable adherence to immobilized fibrinogen. Levels of fibrinogen binding were enhanced in the presence of the serine protease inhibitor phenylmethylsulfonyl fluoride. The A␣ and B␤ chains of fibrinogen, but not the ␥ chains, were specifically recognized by T. denticola. Following fibrinogen affinity chromatography analysis of cell surface extracts, a major fibrinogen-binding component (polypeptide molecular mass, ϳ100 kDa), which also degraded fibrinogen, was purified. Upon heating at 100°C, the polypeptide was dissociated into three components (apparent molecular masses, 80, 48, and 45 kDa) that did not individually bind or degrade fibrinogen. The native 100-kDa polypeptide complex was identified as chymotrypsin-like protease (CTLP), or dentilisin. In an isogenic CTLP ؊ mutant strain, CKE, chymotrypsin-like activity was reduced >90% compared to that in the wild type and fibrinogen binding and hydrolysis were ablated. Isogenic mutant strain MHE, deficient in the production of Msp (major surface protein), showed levels of CTLP reduced 40% relative to those in the wild type and exhibited correspondingly reduced levels of fibrinogen binding and proteolysis. Thrombin clotting times in the presence of wild-type T. denticola cells, but not strain CKE (CTLP ؊ ) cells, were extended. These results suggest that interactions of T. denticola with fibrinogen, which may promote colonization and modulate hemostasis, are mediated principally by CTLP.

Dentilisin involvement in coaggregation between Treponema denticola and Tannerella forsythia

“…In the present study, treatment of T. denticola with PMSF did not affect coaggregation. Similar results were reported with respect to adherence during biofilm formation by T. denticola [37,38]. Dentilisin-deficient mutants of T. denticola reduce binding to fibrinogen, whereas adherence of T. denticola to fibrinogen is not affected by PMSF treatment [37].…”

Treponema denticola biofilm-induced expression of a bacteriophage, toxin–antitoxin systems and transposases

“…Dentilisin is an immunogenic, outer-membrane lipoprotein complex that is the primary mediator of fibrinogen binding by T. denticola. It is capable of preventing blood clot formation, has cytopathic properties and is implicated in penetration of epithelial layers (Bamford et al, 2007;Fenno et al, 1998;Grenier et al, 1990;Uitto et al, 1995). All three genes encoding the dentilisin complex were significantly upregulated in biofilm cells, with two genes being upregulated by more than 1.5-fold.…”

Treponema denticola chymotrypsin-like proteinase (CTLP) integrates spirochaetes within oral microbial communities

“…One of these is a surface-localized prolyl-phenylalanine-specific chymotrypsin-like protease (CTLP) with a broad repertoire of cytopathic activities (Fenno et al, 1998b;Ellen et al, 2000). The CTLP, also designated dentilisin (Ishihara et al, 1996), has been reported to degrade a range of host proteins (Bamford et al, 2007;McDowell et al, 2009), hydrolyse bioactive peptides (Mäkinen et al, 1995;Miyamoto et al, 2006) and help spirochaetes penetrate epithelial cell monolayers (Fenno et al, 1998b;Ellen et al, 2000;Chi et al, 2003). Formation of the CTLP functional complex on the cell surface requires expression of three genes, prcB, prcA and prtP (Godovikova et al, 2010).…”

“…Cell suspensions were incubated with substrate solution (1.0 mM SAAPFNA in 0.5 M Tris/HCl, pH 7.9, containing 2 mM DTT) for 1 h at 37 uC (Bamford et al, 2007). Bacteria were sedimented by centrifugation and the A 405 of the supernatant was measured.…”