Nanobodies against Clostridioides difficile CDTb provide a toolkit for potent toxin neutralization and highly sensitive quantitation

Clostridioides difficile is a pathogenic bacterium and a leading cause of antibiotic-associated diarrhea. Symptoms of the infection arise because of the production of large clostridial toxins that disrupt the intestinal barrier and cause an acute host inflammatory response. Epidemic C. difficile strains also produce the C. difficile transferase toxin (CDT), a binary toxin consisting of separate enzymatically active (CDTa) and cell-binding (CDTb) components. However, the role of CDT during C. difficile pathogenesis remains poorly understood. We created a CDTb nanobody (Nb) clone library and identified and purified five clones with promising CDTb-binding properties. Studies using the Carterra LSA^XT platform revealed high-affinity binding interactions between the Nbs and three distinct CDTb epitopes. Functionally, these Nbs potently neutralize cellular cytopathic effects of CDT at equimolar concentrations in vitro. We further identified two distinct neutralization mechanisms-inhibition of CDTb heptamer formation and inhibition of cell surface binding, both of which are crucial for CDTa delivery into the host cell. These Nbs were used in a sandwich ELISA assay to monitor CDTb levels between 1- and 7-day post R20291 infection in the cecal material of infected mice. Notably, levels of CDTb spiked during days 3 and 4, with monomers constituting the majority of CDTb. We anticipate that these reagents will allow researchers to further expand toxin intervention and monitoring strategies to obtain a deeper understanding of the CDT mechanism of action.