Characterising the Clostridioides difficile-host interface during infection

Clostridioides difficile is recognised as the aetiological cause of antibiotic-associated gastrointestinal infections, with a recent rise in community-acquired cases exacerbating the burden on global healthcare services. C. difficile’s inherent antibiotic resistance and propensity for recurrence underscores the need to better understand the bacterial and host factors that contribute to colonisation and subsequent infection. This project aimed to characterise the role of Cwp10, a minor S-layer protein upregulated during early infection and MUC13, a transmembrane mucin, in C. difficile pathogenesis Deletion of cwp10 in C. difficile leads to a reduced adherence of the isogenic mutants to intestinal epithelial cells and host glycoproteins in vitro and attenuation of the virulence of the hypervirulent R20291 strain in vivo, implicating Cwp10 as an important protein in C. difficile infection dynamics. From the host side, CRISPR/Cas9 generated MUC13-knockdown intestinal cells were observed to have an increased association with C. difficile, potentially due to either impaired decoy mechanisms or reduced occlusion of adhesion sites. In vivo, Muc13-deficient mice exhibited altered inflammatory responses during infection, though definitive mechanistic conclusions were limited by experimental constraints. To address gaps in our understanding of the roles of uncharacterised bacterial and host factors that are modulated during C. difficile infection, a 3D-printed millifluidic prototype was developed. Here, computational fluid dynamics simulations was utilised to optimise the device design to replicate physiologically relevant levels of fluid-derived mechanical forces. This novel system was designed to support extended anaerobic co-culture studies while mitigating metabolic byproduct accumulation, offering a tool for the real-time analysis of C. difficile infection progression. These findings highlight Cwp10 as a potential therapeutic target and suggest a role for MUC13 in modulating host-pathogen interactions. The fluidic gut model provides a physiologically relevant tool for dissecting colonisation mechanisms, informing effective interventions to disrupt C. difficile pathogenesis and mitigate its clinical impact.