Characterization of fibrinogen-binding by Treponema denticola

Objectives: T. denticola, an organism strongly associated with human periodontal disease, binds to a variety of different substrates including fibronectin, laminin and fibrinogen. Binding to fibrinogen could play a key role in T. denticola colonisation at sites of tissue damage, such as those found in periodontal pockets. Once established, this organism may contribute to disease progression. The major surface protein (Msp) of T. denticola has previously been shown to mediate adhesion to other substrates. This study aimed to characterize fibrinogen-binding by T. denticola using the species type strain and a mutant strain deficient in Msp. Methods: All experiments utilised T. denticola strains ATCC 35405 and MHE (Msp mutant, kindly donated by J.C. Fenno, University of Michigan) grown anaerobically under standard conditions. Binding of stationary phase bacterial cells on microtitre wells to soluble fibrinogen was quantified using horse radish peroxidase (HRP)-linked anti-fibrinogen antibodies. Binding of biotinylated cells to fibrinogen, immobilised either on microtitre plate wells or Western blots, was assayed following addition of HRP-linked streptavidin. Results: Wild-type ATCC 35405 bound both soluble and immobilised fibrinogen in a dose-dependant manner. However, prior incubation with soluble fibrinogen did not inhibit binding of biotinylated wild-type cells to immobilised fibrinogen. The b-chain of fibrinogen, separated from a and g chains by SDS-PAGE and immobilised by Western blotting on nitrocellulose, was preferentially bound by wild-type cells. This binding was heat sensitive. The Msp mutant, MHE, also bound soluble and immobilised fibrinogen, though at slightly lower levels compared to the wild-type. Conclusion: T. denticola bound fibrinogen in a dose-dependant manner, and preferentially adhered to the immobilised b-chain of the protein. Though reduced compared with the wild-type, the Msp mutant bound fibrinogen, suggesting other adhesins are important in binding this substrate.