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 °C; differential scanning calorimetry, T(m) = 59-66 °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.
Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, USA.
Yi-Pin
YP
Lin
Chih-Jung
CJ
Kuo
Xhelil
X
Koleci
Sean P
SP
McDonough
Yung-Fu
YF
Chang
D001422:Bacterial Adhesion / Q000187:drug effects*; D001426:Bacterial Proteins / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites; D018938:Caco-2 Cells; D002448:Cell Adhesion; D016360:Clostridioides difficile / Q000502:physiology*; D019286:Endopeptidase K; D005353:Fibronectins / Q000378:metabolism*; D006801:Humans; D008345:Manganese / Q000378:metabolism*; D011485:Protein Binding; D055550:Protein Stability
D001426:Bacterial Proteins; D005353:Fibronectins; D008345:Manganese; D019286:Endopeptidase K