Fig. 8: Model for membrane association and pore-formation by CDTb.

The change in orientation of the β-barrel domain (purple) within a single monomer of the CDTb heptamer is presented as CDTb transitions from an extracellular (A) to an early endosomal environment (B) and mature endosomal environment (C). In the extracellular environment, Ca²⁺ is bound to RBD1 and HD1. In a Ca²⁺-bound state, CDTb alone is unable to bind lipid bilayers or form pores. Following its uptake, dissociation of Ca²⁺ from the RBD1 domain of CDTb (^CaK_D = 40 ± 10 µM) occurs as Ca²⁺ is exported out of the endosome, resulting in the destabilization of the RBD1 domain as it enters conformational exchange. At this point (i), residues associated with the β-barrel domain begin extending into a “pre-pore” conformation of heptameric CDTb. (ii) The β-barrel domain extends fully, creating pores through the endosomal membrane at low micromolar [Ca²⁺] (i.e., <40 µM). (iii) Pore formation can begin to serve as channels for CDTa to enter the cytosol, leading to cytotoxic effects and the disruption of the intestinal epithelium that facilitate the increased severity of CDT-associated CDI. However, a more complete mechanism of action for CDTa delivery by CDTb requires further examination.