Proteomic and Functional Analyses Reveal the Stress Tolerance Network in <i>Saccharomyces boulardii</i> during Gastrointestinal Challenge

The probiotic yeast <i>Saccharomyces boulardii</i> exhibits remarkable resilience to gastrointestinal stress, yet its underlying adaptive mechanisms remain incompletely understood. This study employed multiomics approaches to investigate its stress response systematically. Electron microscopy revealed significant morphological perturbations, including cell shape deformation and compromised cell wall integrity under the stress conditions. Concomitantly, Na⁺/K⁺-ATPase activity exhibited a progressive increase from 0.84 to 3.1 μmol/mg protein, suggesting active ion homeostasis regulation. Proteomic analysis identified 75 differentially expressed proteins under gastric stress and 2470 under intestinal conditions; enhanced pathway enrichment revealed three interconnected regulatory modules. These include upregulated pyruvate metabolism enzymes (e.g., <i>ADE</i> family) for nucleotide/energy production, remodeled nucleocytoplasmic transport for stress-molecule shuttling, and condition-specific hubs, chaperones (<i>APJ1</i>, <i>SSA3</i>) in gastric environments, and glycolytic enzymes (<i>PGK1</i>, <i>TDH3</i>) in intestinal conditions. These findings collectively demonstrate <i>S. boulardii</i>'s sophisticated, multifaceted adaptation to gastrointestinal challenges, providing new insights into its probiotic functionality at the molecular level.