Targeted inactivation of protein disulfide-isomerase 1 by Lactobacillus reuteri-derived reuterin promotes intestinal stem cell proliferation via ROS signaling

Enteric Lactobacillus plays a crucial role in maintaining the intestinal barrier integrity. Lactobacillus reuteri , one of the most described probiotic species of Lactobacillus , has been shown to alleviate enteritis-associated intestinal mucosal damage by stimulating the expansion of intestinal stem cells (ISCs). However, the underlying ligand-protein interactions remain unexplored. Therefore, this study aimed to elucidate the specific molecular mechanisms by which L. reuteri and its metabolites govern ISC stemness. We employed broiler enteritis model and organoid culture to compare the probiotic effect of a wild-type (WT) L. reuteri strain and a reuterin-deficient isogenic mutant. Compared to the WT strain, the deficient strain exhibited an impaired ability to alleviate inflammation, restore the intestinal barrier, and stimulate ISC proliferation both in vivo and ex vivo, indicating that reuterin is the essential effector. Furthermore, bioorthogonal click chemistry combined with in silico molecular docking were utilized to map the direct host protein targets. We identified protein disulfide-isomerase 1 (PDIA1), an endoplasmic reticulum (ER)-resident redox-active protein, as the direct binding partner of reuterin, characterized by a strong binding affinity. The inactivation of PDIA1 by reuterin binding increased ER-derived reactive oxygen species (ROS) level, leading to the upregulation of Wnt/β-catenin pathway and the maintenance of ISCs. In conclusion, by inactivating the ER-resident PDIA1, L. reuteri -derived reuterin promoted ISC proliferation and ameliorated intestinal mucosal damage.