Abstract
<title>Abstract</title> <p>Bacillus subtilis is widely used as a probiotic to mitigate alcohol-induced gut-liver injury, yet its therapeutic potential is limited by poor survival under gastrointestinal and ethanol stress, often necessitating encapsulation for viable delivery. Here, we isolated food-borne B. subtilis strains carrying DNA phosphorothioation (PT)—a DNA backbone modification in which sulfur replaces a non-bridging oxygen. The PT strain exhibited markedly enhanced tolerance to multiple stresses compared with the PT-deficient mutant, an effect we attribute to PT-mediated neutralization of reactive oxygen species (ROS) generated by ethanol and gastrointestinal conditions. In an acute mouse model of alcohol-induced injury, the PT strain showed a favorable safety profile and superior efficacy in reducing oxidative damage. In a chronic weaned piglet model, the PT strain achieved higher gastrointestinal survival and conferred stronger hepato-intestinal protection, reversing gut microbiota dysbiosis, preserving mucosal barrier integrity, and attenuating hepatic steatosis. Mechanistically, B. subtilis converts luminal taurochenodeoxycholic acid (TCDCA) into the cytoprotective bile acid tauroursodeoxycholic acid (TUDCA), which activates host TGR5 signaling to reinforce the intestinal barrier. This barrier fortification blocks lipopolysaccharide (LPS) translocation into the portal circulation, thereby suppressing the hepatic IFN-γ/JAK-STAT cytokine cascade and ameliorating liver damage. Our findings establish DNA phosphorothioation as a built-in stress shield that enables robust probiotic delivery in the alcohol-exposed gastrointestinal tract, providing a strategy for engineering resilient live biotherapeutics against gut-liver axis disorders.</p>