Structural Fine‐Tuning of Clostridioides difficile Binary Toxin Components for Therapeutic Applications

Abstract

Therapeutic solutions to combat the Clostridioides difficile infection (CDI) are high in-demand due to antibiotic resistance and limited treatment options. C. difficile transferase (CDT) binary toxin is associated with hypervirulence, in addition to the major clostridial toxins. CDT consists of an enzymatic component (CDTa), and a pore-forming binding component (CDTb). Cytotoxicity of the host cell is initiated by the formation of CDTa-CDTb binary toxin complex and subsequent conformational shift in the CDTb to enforce translocation of the CDTa into the cell. This results in destruction of the host cell cytoskeleton through ADP-ribosylation of the actin filaments. Molecular level characterization of full length active wild-type CDTb by Cryogenic Electron Microscopy (CryoEM) revealed existence of two novel di-heptamer units in the absence of full-length CDTa. The ‘pore’ state is represented by extended β-barrel assembly in the AsymCDTb structure and the ‘pre-pore’ state or the SymCDTb form consisted identical heptamer domains. Detailed analysis of the structure depicted the presence of different domains including two receptor binding domains (RBD). The RBD1 domain represents a discrete non-homologous structure that was not identified in other binary toxins and the RBD2 domain is crucial for di-heptamer assembly and physiological activity of the toxin. A unique calcium-binding site in RBD1 in the X-ray crystal structure of AsymCDTb pinpointed the significant role of calcium in the structural stability and the protein folding mechanisms. Historically, potent calcium-containing therapeutic agents have been able to neutralize the activity of large clostridial toxins. Therefore, we explored the structural features of the CDTbD623/734A double mutant which disrupted the calcium binding site in the RBD1. The 2D-structural details revealed unique conformational changes in the heptameric organization of the mutant which indicated potential disturbances to the formation of β-barrel. We compared the toxicity of wild type CDTb and CDTbD623/734A in the presence of CDTa using a Vero cell assay. CDTbD623/734A showed a significant decrease in toxicity (TC50 CDTb 80±6pM; CDTbD623/734A 637±20pM), supporting the idea that this novel calcium-binding site is required for function. Calcium plays a pivotal role in the structure-activity relationship and toxin machinery. Therefore, ablation of the divalent interaction and targeting the binding site stand out as promising strategies in the drug development for CDI. Unraveling the distinctive conformational features in different mutant constructs of the binary toxin can be beneficial in fine-tuning these potential therapeutic agents.