Project Summary Clostridioides difficile is a gram-positive, spore-forming anaerobe, a leading cause of nosocomial infection in the United States, and a significant problem for Veterans receiving care in Veterans Health Administration (VHA) facilities. The disease state is most often preceded by disruption of the host microbiome in response to antibiotic treatment and is characterized by mild to severe (and often recurrent) diarrhea. Left untreated, C. difficile infection (CDI) can be life threatening with sequelae that include antibiotic-associated pseudomembranous colitis, toxic megacolon, and sepsis. CDI is dependent on the secretion of one or more AB-type toxins: toxin A (TcdA), toxin B (TcdB), and the C. difficile transferase toxin (CDT, or binary toxin). While TcdA and TcdB are considered the primary virulence factors, multiple studies suggest that CDT increases the severity of CDI. The increasing prevalence of CDT containing clinical isolates and the potential for developing CDT as an effective vaccine antigen necessitate a deeper understanding of the CDT mechanism of action. CDT belongs to the Iota family of binary toxins and consists of two proteins: an ADP-ribosyltransferase (CdtA) and a cell binding and pore-forming protein (CdtB). CdtB engages host cells by binding the lipolysis stimulated lipoprotein receptor (LSR). Proteolytic cleavage promotes CdtB oligomerization into a prepore which allows for CdtA binding. The CdtA-CdtB prepore complex is internalized by endocytosis, and endosome acidification leads to CdtB pore formation and CdtA translocation into the cell. CdtA-mediated disruption of actin cytoskeletal structure has been associated with the formation of microtubule protrusions that could play a role in C. difficile adherence to the epithelium. CDT can also synergize with TcdA/TcdB to promote inflammasome activation and suppression of an otherwise protective eosinophil response. We have obtained structures of the CdtB prepore and CdtB pore by cryo-electron microscopy. We propose to define the structures of CdtB bound to CdtA and the LSR receptor (Aim 1), and to dissect the structural features involved in epithelial cell intoxication (Aim 1) and inflammasome priming (Aim 2) using structure-guided mutagenesis and functional studies. In parallel, we will generate a panel of CdtB specific nanobodies that can be used to define the important epitopes needed for high affinity binding and neutralization (Aim 3). The over-arching goal is to provide the mechanistic foundation for understanding the role of CDT function in pathogenesis and the tools to advance this knowledge into effective vaccine development strategies.