Engineering Probiotic Yeast to Enhance Gut Residence Time for Inflammatory Bowel Disease Therapy

Inflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation and disruption of gut homeostasis, often accompanied by extracellular matrix (ECM) remodeling at ulcer sites. Although the probiotic yeast Saccharomyces boulardii has demonstrated therapeutic potential in IBD, its clinical efficacy is limited by rapid gastrointestinal clearance and poor mucosal retention. This study aimed to enhance the gut residence time of S. boulardii by engineering targeted adhesion to ECM proteins enriched in IBD ulcerations, fibronectin and collagen IV. Building on prior work demonstrating ECM-specific targeting via antibody-coated yeast, we developed and evaluated a dual‑labeling strategy in which surface-displayed monomeric streptavidin (mSA) enables simultaneous attachment of biotinylated antibodies against fibronectin and collagen IV. In vitro binding assays revealed that dual‑labeled yeast exhibited significantly greater adhesion to mixed ECM substrates compared to singly labeled strains, while maintaining binding capabilities on individual ECM components. Time‑course adhesion studies further demonstrated that dual labeling reduced detachment over 48 hours relative to single‑target platforms, indicating improved persistence in heterogeneous ECM environments. To address the inherent limitations of antibody-based coatings, this work also identified and computationally validated genetically encoded ECM-binding adhesins—DraE and a fibronectin‑binding subdomain of FnBPA5—for future surface-display engineering. Structural modeling and aggregation analyses supported their suitability for yeast expression. Together, these findings demonstrate that dual ECM targeting improves adhesion robustness and lay the groundwork for integrating endogenous adhesin expression to enable more durable, self‑sustaining probiotic therapeutics. This work advances strategies for localized, living therapeutics for IBD and establishes a foundation for future in vivo evaluation.