Activation of botulinum C2 toxin by trypsin

Area of Science:

• Microbiology
• Toxicology
• Protein Biochemistry

Background:

• Clostridium botulinum types C and D produce C2 toxin (C2T), a binary toxin.
• C2T comprises two distinct protein components: component I and component II.
• The biological activity of C2T is known to be potentiated by trypsin treatment.

Purpose of the Study:

• To elucidate the mechanism of C2T activation by trypsin.
• To characterize the molecular changes in component II upon trypsinization.
• To determine the optimal conditions for C2T activation and its effect on lethality.

Main Methods:

• Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to determine molecular weights.
• Gel filtration chromatography to separate and analyze components.
• Assays for hemagglutination and hemolytic activities.
• Lethality assays in conjunction with component mixing.

Main Results:

• Maximum C2T lethality achieved by mixing untrypsinized component I and trypsinized component II under specific conditions (10:1 ratio, 30 min, 35°C, pH 7.5).
• Trypsin activation of component II resulted in a molecular weight shift from 101,000 to 88,000 (SDS-PAGE).
• Gel filtration separated trypsinized component II into two active species (365,000 and 74,000 MW) with similar lethality and hemagglutination activity, but only the larger species possessed hemolytic activity.

Conclusions:

• C2T activation by trypsin is a molecular conversion of component II to an 88,000 MW form.
• Trypsin-activated component II (88,000 MW) can form oligomers.
• Both high and low molecular weight forms of activated component II contribute to C2T's lethality when combined with component I.