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

Abstract

Treponema denticolais an anaerobic spirochete strongly associated with human periodontal disease.T. denticolabacteria 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 inT. denticolacolonization of the damaged sites.T. denticolaATCC 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 Aα and Bβ chains of fibrinogen, but not the γ chains, were specifically recognized byT. denticola. Following fibrinogen affinity chromatography analysis of cell surface extracts, a major fibrinogen-binding component (polypeptide molecular mass, ∼100 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-typeT. denticolacells, but not strain CKE (CTLP⁻) cells, were extended. These results suggest that interactions ofT. denticolawith fibrinogen, which may promote colonization and modulate hemostasis, are mediated principally by CTLP.