Physical Characterization of Clostridium Difficile Toxins and Toxoids: Effect of the Formaldehyde Crosslinking on Thermal Stability

Nosocomial diarrhea and pseudomembranous colitis causing toxins A and B from Clostridium difficile were studied at pH 5-8 and over the temperature range of 10-85°C. The proteins were crosslinked with formaldehyde to inactivate them to toxoid forms and permit their use as vaccines. Structural changes and aggregation behavior were monitored with circular dichroism, intrinsic and extrinsic (ANS) fluorescence spectroscopy, turbidity measurements, high-resolution UV absorbance spectroscopy and dynamic light scattering. The combined results were summarized in empirical phase diagrams. Toxins A and B had similar secondary structure with a combination of helical, β-sheet and unordered character and were partially unfolded at pH 5-5.5. Upon heating, toxin A at all pH values partially unfolded at ∼45°C and formed insoluble aggregates at ∼50°C. Toxin B partially unfolded at ∼40°C, and upon heating to ∼50°C precipitated at pH 5.0-6.0 and formed soluble aggregates at pH 6.5-7.5. The thermal stability of the toxins was pH-dependent with the proteins more thermally stable at higher pH. Similar studies of formaldehyde crosslinked toxoids A and B revealed enhanced thermal stability, in which secondary and tertiary structure changes as well aggregation were delayed by about 10°C. These studies reveal both similarities and differences between C. difficile toxins A and B and demonstrate the stabilizing effect of formaldehyde crosslinking on the thermal stability of their corresponding toxoids.