Project Summary (Project 3) Clostridioides difficile is the leading nosocomial infection in the US, and is characterized by recurrent infection and poor generation of antibacterial antibody responses. Identifying how C. difficile evades the adaptive immune system is critical to develop rational treatments for patients with C. difficile infections and to prevent recurrence. Interestingly, the major risk factors for C. difficile infection (broad spectrum antibiotic usage, proton pump inhibitor treatment, inflammatory bowel disease) all result in either a loss or dysfunction of nerves in the gut. Infection with C. difficile also results in a profound loss of nerves in the colon of mice. It is currently unknown if a loss of innervation in the gut can be a primary driver of CDI susceptibility. Additionally, there is increasing awareness of the outsized role of the nervous system in promoting immune function. Sensory nerves directly sense pathogens, cellular damage, and noxious chemicals in order to allow organisms to respond and eliminate damage. Adrenergic nerves in the spleen and lymph nodes are necessary for optimal B cell responses to vaccination and infection. We posit that sensory nerve signaling from the site of infection or vaccination communicates through the nervous system to the adrenergic nerves in lymphoid organs to promote immune responses. Given the important role for neuronal signaling in promoting immunity, is it perhaps unsurprising that numerous pathogens across a wide diversity of species specifically target the nervous system. Significantly, the C. difficile exotoxins TcdA and TcdB both have neuronal activity in addition to their well described function in the inactivation of Rho GTPases by glucosylation. In collaboration with Dr. Mark Lang, we have found that TcdA inhibits the development of antigen-specific antibody responses during vaccination, and this inhibition was dependent upon sensory nerve signaling; desensitization of these nerves during vaccination restored antibody responses even in the presence of TcdA. Combined with our finding that CDI induces profound neuronal loss in the gut, We hypothesize that TcdA and TcdB manipulation and deletion of nerves is necessary for C. difficile infection and immune evasion. In Specific Aim 1 we will determine the role of neuronal signaling in C. difficile infection and immune evasion. In Specific Aim 2 we will identify the mechanism by which TcdA hyperactivation of sensory nerves impairs humoral immunity. These studies will advance the C. difficile vaccination field by revealing specific mechanisms that limit successful humoral immune responses to vaccination and infection, and determining whether neuropreservation is a critical correlate of protection for CDI.