Liam Keogh

Clostridioides difficile infection (CDI) is the leading cause of healthcare-associated infectious diarrhea and remains a major burden to healthcare systems worldwide. The development of novel therapeutics for CDI requires robust and reproducible preclinical models. However, the microbiota has emerged as a major source of variability in animal studies. Here, we found that genetically similar mice obtained from two commercial vendors, Jackson Laboratory (JAX) and Charles River Laboratories (CRL), exhibited marked differences in susceptibility to CDI, with JAX mice developing fulminant disease and CRL mice remaining resistant. Using full-length 16S rRNA gene sequencing, we show that JAX and CRL mice harboured distinct gut microbiota, and that cohousing susceptible JAX mice with resistant CRL mice was sufficient to shift the JAX microbiota toward the CRL community structure and confer resistance to CDI. Differential abundance analysis identified taxa distinguishing resistant and susceptible mice, providing candidates for future mechanistic investigation. These findings demonstrate that vendor-derived variation in the gut microbiota drives differential susceptibility to CDI in mice, and that this phenotype is transferable via cohousing, highlighting the importance of accounting for the microbiota when designing and interpreting animal models of infectious disease. IMPORTANCE: Mice are widely used to study Clostridioides difficile infection, but animals purchased from different commercial vendors can respond differently to infection. This study shows that mice from two commonly used suppliers differ substantially in their susceptibility to C. difficile , and that this difference is caused by variation in their gut microbiota, as opposed to genetic differences. By housing susceptible and resistant mice together, we found that the protective bacterial community could be transferred to susceptible mice, making them resistant to severe infection. This work underscores that the choice of animal vendor is a critical experimental variable that can substantially influence infection outcomes, highlighting the importance of accounting for vendor source when designing, interpreting, and comparing mouse studies of C. difficile infection.

SUMMARY: Early postnatal life is a highly dynamic period in which the intestine is inundated with billions of microbes that not only battle for colonization, but also strongly influence immune system function and long-term health. This brief period of development provides a window of opportunity to imprint resilience against disease, yet the intercellular dialogue that dictates tissue protection are only beginning to be discovered. By performing a kinetic analysis of immune cell activation in the colon of mice from early life to adulthood, we identified transient activation of a microbiota-dependent type 3 immune response during the weaning period. This response was characterized by a selective increase in IL-17 production by fetal-derived γδ T cells that occurred in an IL-1 receptor-dependent, cell-intrinsic manner. The subsequent differentiation of IL-10 producing Rorγt+ T regulatory cells extinguished IL-17 production by γδ T cells and prevented immunopathology. Microbial gain-of-function approaches determined that colonic γδ T cell activation occurred in response to passive acquisition of C. difficile at birth. In turn, IL-17 limited C. difficile growth and intestinal barrier breach by the commensal microbiota. Collectively, our results reveal how an agile immune cell network meets the demands of a maturing microbiota to support gut health during early life. Highlights: - Colonic γδ17 T cells are activated during early life in a microbiota-specific manner - Fetal-derived γδ17 T cells require IL-1 signals for activation - Passive acquisition of C.difficile induces γδ17 T cell activation - IL-17 limits C. difficile growth and systemic dissemination of commensals

Clostridioides difficile is a bacterium that causes life-threatening intestinal infections. Infection symptoms are mediated by a toxin secreted by the bacterium. Toxin pathogenesis is modulated by the intracellular molecule, inositol-hexakisphosphate (IP6). IP6 binds to a cysteine protease domain (CPD) on the toxin, inducing auto-proteolysis, which liberates a virulence factor in the cell cytosol. We developed second-generation IP6 analogs designed to induce auto-proteolysis in the gut lumen, prior to toxin uptake, circumventing pathogenesis. We synthesized a panel of thiophosphate-/sulfate-containing IP6 analogs, and characterized their toxin binding affinity, auto-proteolysis induction and cation interactions. Our top candidate was soluble in extracellular cation concentrations, unlike IP6. The IP6 analogs were more negatively charged than IP6, which improved affinity and stabilization of the CPD, enhancing toxin auto-proteolysis. Our data illustrate the optimization of IP6 with thiophosphate biomimetics which are more capable of inducing toxin auto-proteolysis than the native ligand, warranting further studies in vivo.