Chymotrypsin-like protease of Treponema denticola also acts as an adhesin

Treponema denticola, a proteolytic, highly motile, oral spirochaete strongly associated with human periodontal disease, expresses several surface adhesins that bind host proteins in important virulence mechanisms. The specific bacterial adhesins responsible for binding fibrinogen and fibronectin have not yet been clearly elucidated. Objectives: This study aimed to analyse interactions of T. denticola cells with fibrinogen and fibronectin by investigation of adhesive and degradative properties of outer membrane proteins. Understanding such mechanisms may result in novel strategies to reduce spirochaete-associated oral diseases. Methods: T. denticola ATCC 35405 and a chymotrypsin-like protease (CTLP) deficient strain CKE (kindly donated by J.C. Fenno, University of Michigan) were grown anaerobically in modified New Oral Spirochaete medium. Outer membrane proteins, extracted using Triton X-114 solution, were analysed by affinity chromatography, SDS-PAGE, fibrinogen zymography, and Western blot overlay. Biotin-labelled spirochaete cells were assayed for binding to fibrinogen or fibronectin immobilised to wells of plastic microtitre plates. Results: A protein band of ≈100 kDa was eluted following affinity chromatography of ATCC 35405 outer membrane extracts with fibrinogen-linked 4B CNBr sepharose. Heating, prior to SDS-PAGE, resolved the protein to 80, 48 and 45 kDa polypeptides, and destroyed fibrinogenolytic activity. Separated outer membrane proteins overlaid with fibrinogen also revealed a predominant ≈100 kDa adhesin, and binding was similarly abolished by heating. Parallels between these data and reported work suggest the principle fibrinogen adhesin is the CTLP. Pre-incubation with phenylmethylsulfonyl fluoride (PMSF), which inhibits CTLP activity, enhanced binding of ATCC 35405 cells to fibrinogen, but reduced adhesion to fibronectin. CTLP-deficient CKE was reduced in binding to both fibrinogen and fibronectin compared with the parent strain, with PMSF having no effect on either interaction. Conclusion: The CTLP is the principle fibrinogen-binding protein of T. denticola and may also play a role, with other adhesins, in binding different host substrates such as fibronectin.