ABSTRACT Clostridioides difficile is a clinically important opportunistic pathogen that exploits disruptions in the commensal microbiome of the gastrointestinal tract. C. difficile infection (CDI) is characterized by colitis and diarrhea, which are largely caused by two secreted toxins, TcdA and TcdB. These primary virulence factors bind to the cell surface via their CROPs and internalization domains; upon cell internalization - they glycosylate Rho-family proteins, thus disrupting Rho-dependent cellular processes, ultimately leading to inflammation and increased epithelial permeability. The threat of CDI is rising due to the prevalence of hypervirulent and antibiotic resistant strains. The increasing risk of CDI, combined with the shortcomings of conventional antibiotic and fecal transplantation treatment options, poses an urgent need for novel therapies. Biotherapeutic approaches using monoclonal anti-TcdA/TcdB antibodies indicated a promising route, complementary to antibiotics; however, they suffer from cumbersome administration and limitations in biodistribution. Responding to these challenges, we developed neutralizing peptides that inhibit both TcdA and TcdB and engineered probiotic yeast as a delivery vector of anti-toxin peptides in the colon. In our preliminary work, we computationally designed and evaluated two experimentally effective neutralizing peptides (SA1 and SB6), which demonstrated anti-TcdA and TcdB biorecognition and conferred epithelial protection in primary derived human colonic epithelial monolayers. Additionally, we developed a yeast strain yielding >5 g/L peptide during fermentation after knockout of key proteases. In this work, we propose to implement this toolbox to develop a comprehensive strategy against TcdB-mediated C. difficile infection. To this end, we will computationally design peptides to bind to several domains on TcdB and evaluate a broader set of therapeutic hypotheses at the molecular, cellular, and organismal scales. First, we will elucidate the molecular mechanism of TcdB inhibition by SB6. This knowledge will inform a concerted computational-experimental design of peptides that target the TcdB active domain and neutralize its Rho glycosylation activity. Next, we will use recently published crystal structures and known receptor-binding domains of TcdB to develop peptides that block TcdB’s entry into the cell. These peptides will be inspired by the proteins to which TcdB natively binds on the cell surface, as well as the reference CROP-blocking antibody Bezlotoxumab. Finally, we will engineer probiotic yeast to secrete these peptides, and characterize their efficacy on human colonic epithelial cell and murine animal C. difficile infection models. Collectively, these efforts will elucidate the mechanisms by which inhibitory peptides can block TcdB activity and use this knowledge to develop optimized peptides that inhibit CDI by integrating expertise at the atomic, molecular, cellular, and organismal sc...