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Background Disruption of the intestinal epithelial barrier is a pivotal event in neonatal enteric infections, facilitating inflammation, immune dysregulation, and systemic dissemination of pathogens such as Escherichia coli (E. coli) K1. Lactobacillus rhamnoses GG (LGG) is a well-characterized probiotic with established benefits in gut homeostasis and host defense; however, the precise mechanisms by which LGG protects against E. coli K1–induced barrier injury remain incompletely defined.Methods An in vitro intestinal epithelial model was employed to examine the effects of LGG-conditioned supernatant (LCS) on E. coli K1 adhesion, invasion, and transepithelial electrical resistance (TEER). Expression and localization of the barrier-associated proteins ZO-1 and MUC2 were analyzed by Western blotting and immunofluorescence. Bioactive components within LCS were identified using LC-MS/MS. The functional effects of the recombinant LGG-derived protein HM0539 were further evaluated in LPS-stimulated macrophages and in dextran sulfate sodium (DSS)-induced colitis models to delineate its anti-inflammatory and barrier-protective mechanisms.Results Pre-treatment with LCS significantly inhibited E. coli K1 adhesion, invasion, and epithelial translocation, while preserving TEER and up-regulating ZO-1 and MUC2 expression in vitro. Proteomic analysis identified HM0539 as a key bioactive component of LCS. Recombinant HM0539 recapitulated the barrier-protective effects of LGG, reducing E. coli K1 colonization, ameliorating colitis, and attenuating secondary liver injury in vivo. Mechanistically, HM0539 down-regulated TLR4 and MyD88 expression, suppressed NF-κB activation, and reduced the production of inflammatory mediators including COX-2, iNOS, PGE2, and nitric oxide. Concurrently, HM0539 enhanced the expression of the barrier-reinforcing proteins MUC2 and ZO-1, thereby decreasing epithelial permeability and limiting bacterial transmigration.Conclusions The LGG-derived postbiotic protein HM0539 protects against E. coli K1 infection through a dual mechanism that integrates reinforcement of intestinal epithelial barrier integrity with suppression of TLR4/MyD88/NF-κB-mediated inflammatory signaling. These findings identify HM0539 as a mechanistically defined postbiotic with translational potential for the prevention of neonatal enteric infection and barrier-associated inflammatory disorders. (Acknowledgements: This study was supported by Undergraduate Training Program for Innovation and Entrepreneurship of Southern Medical University, No. 202512121297)