Membrane binding and pore formation is Ca ²⁺ -dependent for the Clostridioides difficile binary toxin

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Clostridioides difficile binary toxin (CDT) is an AB-type toxin that enters host cells via endosomal pathways.
• Many binary toxins utilize pH changes within endosomes to activate their cell-binding components.
• The precise mechanism by which CDT interacts with and permeabilizes endosomal membranes remains incompletely understood.

Purpose of the Study:

• To elucidate the molecular mechanism by which the cell-binding component of CDT, CDTb, mediates pore formation in lipid bilayers.
• To determine the role of calcium ions (Ca2+) and pH in the activation of CDTb.
• To identify the structural basis for CDTb-induced membrane permeabilization.

Main Methods:

• Cryoelectron microscopy (Cryo-EM) for structural analysis.
• Nuclear magnetic resonance (NMR) spectroscopy to study protein dynamics and interactions.
• Surface plasmon resonance (SPR) and electrochemical impedance spectroscopy (EIS) to assess binding and pore formation kinetics.
• Site-directed mutagenesis to probe the function of specific residues.
• Toxicity studies to evaluate the biological activity of CDT.

Main Results:

• CDTb binds to lipid bilayers and forms pores specifically upon depletion of free Ca2+ ions, not due to a decrease in pH.
• Ca2+ dissociation from a single site in receptor binding domain 1 (RBD1) of CDTb acts as a trigger.
• This Ca2+ dissociation induces a conformational change in CDTb, enabling membrane binding and pore formation.
• Structural and biophysical data support a model where decreasing Ca2+ concentrations during endosomal delivery activate CDTb.

Conclusions:

• The mechanism of CDT-mediated cell entry relies on Ca2+ depletion-induced conformational changes in CDTb, leading to pore formation.
• This Ca2+ -dependent mechanism differs from the pH-dependent activation observed in other binary toxins.
• Understanding this unique activation pathway provides insights into C. difficile pathogenesis and potential therapeutic targets.