Membrane binding and pore formation is Ca2+-dependent for the Clostridioides difficile binary toxin

The C. difficile binary toxin (CDT) enters host cells via endosomal delivery like many other AB-type binary toxins. In this study, the cell-binding component of CDT, termed CDTb, was found to bind and form pores in lipid bilayers upon depleting free Ca2+ ion concentrations, and not by lowering pH, as found for other binary toxins (i.e., anthrax). Cryoelectron microscopy, nuclear magnetic resonance spectroscopy, surface plasmon resonance, electrochemical impedance spectroscopy, CDT toxicity studies, and site directed mutagenesis show that dissociation of Ca2+ from a single site in receptor binding domain 1 (RBD1) of CDTb is consistent with a molecular mechanism in which Ca2+ dissociation from RBD1 induces a "trigger" via conformational exchange that enables CDTb to bind and form pores in endosomal membrane bilayers as free Ca2+ concentrations decrease during CDT endosomal delivery. One-Sentence SummaryCa2+ dissociation and unfolding of a receptor binding domain in CDTb activates pore-formation by the C. difficile binary toxin.