Manganese binds to Clostridium difficile Fbp68 and is essential for fibronectin binding

Area of Science:

• Microbiology
• Biochemistry
• Infectious Diseases

Background:

• Clostridium difficile causes severe gastrointestinal infections like pseudomembranous colitis.
• Bacterial adhesion is a critical initial step in C. difficile infection.
• Colonization factors, alongside toxins, are increasingly recognized for their role in C. difficile pathogenesis.

Purpose of the Study:

• To investigate the role of Fbp68, a fibronectin-binding colonization factor, in C. difficile infection.
• To determine the effect of metal ion binding on Fbp68 structure and function.
• To elucidate the mechanism of Fbp68-mediated adhesion to host cells.

Main Methods:

• Identified Fbp68 as a manganese-binding protein using biochemical assays.
• Assessed the impact of manganese binding on Fbp68 conformation and stability via circular dichroism and differential scanning calorimetry.
• Quantified Fbp68-fibronectin interactions using isothermal titration calorimetry.
• Evaluated the role of Fbp68 in C. difficile adhesion to Caco-2 cells and fibronectin.

Main Results:

• Fbp68 binds manganese with high affinity (K(D) = 52.70 ± 1.97 nM).
• Manganese binding significantly stabilizes Fbp68 structure and enhances protease K resistance.
• Fibronectin binding activity of Fbp68 is dependent on manganese.
• The C-terminal domain of Fbp68 binds the N-terminal domain of fibronectin.
• Fbp68 partially inhibits C. difficile adhesion to Caco-2 cells, demonstrating its role in host cell interaction.

Conclusions:

• Fbp68 is a manganese-binding protein crucial for Clostridium difficile adhesion.
• Manganese-dependent structural stabilization of Fbp68 enhances its fibronectin-binding capability.
• Fbp68 plays a significant role in the initial adherence of C. difficile to host cells, contributing to infection initiation.