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The Chymotrypsin-Like Protease Complex of Treponema denticola ATCC 35405 Mediates Fibrinogen Adherence and Degradation

Oral Microbiology Unit, Department of Oral and Dental Science, University of Bristol, Lower Maudlin Street, Bristol BS1 2LY, United Kingdom,¹ Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan, Ann Arbor, Michigan²

Received 16 February 2007/ Returned for modification 25 April 2007/ Accepted 15 June 2007

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 Bß 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, 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-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.

Published ahead of print on 25 June 2007.

Editor: J. N. Weiser