Binding and internalization of Clostridium botulinum C2 toxin

Area of Science:

• Microbiology
• Cell Biology
• Toxicology

Background:

• Clostridium botulinum C2 toxin is a binary toxin.
• It comprises an enzymatic component (C2I) and a binding component (C2II).
• Activated C2IIa forms heptamers, facilitating toxin uptake via receptor-mediated endocytosis.

Purpose of the Study:

• To investigate the binding and internalization mechanisms of C2IIa in cells.
• To elucidate the role of lipid rafts and signaling pathways in C2 toxin entry.
• To understand the sequential events leading to C2 toxin-induced cellular effects.

Main Methods:

• Utilized MDCK cells to study C2IIa binding and oligomerization on lipid rafts.
• Employed methyl-beta-cyclodextrin to inhibit C2IIa binding and cellular responses.
• Applied surface plasmon resonance to analyze C2I-C2IIa interactions.
• Investigated the involvement of phosphatidylinositol 3-kinase (PI3K) and Akt signaling pathways using specific inhibitors (LY294002, Akt inhibitor X).

Main Results:

• C2IIa monomers oligomerized on lipid rafts in MDCK cell membranes.
• Methyl-beta-cyclodextrin treatment inhibited C2IIa binding and C2 toxin-induced cell rounding.
• C2I bound to C2IIa oligomers, not monomers, and co-localized with rafts upon C2IIa presence.
• PI3K-Akt pathway activation was triggered by C2I binding to C2IIa oligomers, preceding C2 toxin internalization and cell rounding.
• Inhibition of PI3K blocked internalization and cell rounding, while Akt inhibition only affected cell rounding.

Conclusions:

• Lipid raft-dependent oligomerization of C2IIa is crucial for C2 toxin binding to cells.
• The binding of C2I to C2IIa oligomers on lipid rafts initiates PI3K-Akt signaling.
• PI3K-Akt pathway activation is essential for the endocytosis of C2 toxin and subsequent cellular effects.