Manganese Binds to Clostridium difficile Fbp68 and Is Essential for Fibronectin Binding

Clostridium difficile is an etiological agent of pseudomembranous colitis and antibiotic-associated diarrhea. Adhesion is the crucial first step in bacterial infection. Thus, in addition to toxins, the importance of colonization factors in C. difficile-associated disease is recognized. In this study, we identified Fbp68, one of the colonization factors that bind to fibronectin (Fn), as a manganese-binding protein (K-D = 52.70 +/- 1.97 nM). Furthermore, the conformation of Fbp68 changed dramatically upon manganese binding. Manganese binding can also stabilize the structure of Fbp68 as evidenced by the increased T-m measured by thermodenatured circular dichroism and differential scanning calorimetry (CD, T-m = 58-65 degrees C; differential scanning calorimetry, T-m = 59-66 degrees C). In addition, enhanced tolerance to protease K also suggests greatly improved stability of Fbp68 through manganese binding. Fn binding activity was found to be dependent on manganese due to the lack of binding by manganese-free Fbp68 to Fn. The C-terminal 194 amino acid residues of Fbp68 (Fbp68C) were discovered to bind to the N-terminal domain of Fn (Fbp68C-NTD, K-D = 233 +/- 10 nM, obtained from isothermal titration calorimetry). Moreover, adhesion of C. difficile to Caco-2 cells can be partially blocked if cells are pretreated with Fbp68C, and the binding of Fbp68C on Fn siRNA-transfected cells was significantly reduced. These results raise the possibility that Fbp68 plays a key role in C. difficile adherence on host cells to initiate infection.