Bacillus halotolerans attenuates inflammation induced by enterotoxigenic Escherichia coli infection in vivo and in vitro based on its metabolite soyasaponin I regulating the p105-Tpl2-ERK pathway.

Soyasaponins, recognized for their anti-inflammatory and antioxidant effects, have not yet been fully explored for their role in combating enterotoxigenic Escherichia coli (ETEC) infections. Recent findings identified them in small-molecule metabolites of Bacillus , suggesting their broader biological relevance. This research screened 88 strains of B. halotolerans , identifying the strain BH M20221856 as significantly inhibitory against ETEC growth in vitro . It also reduced cellular damage and inflammatory response in IPEC-J2 cells. The antimicrobial activity of BH M20221856 was attributed to its small-molecule metabolites rather than secretory proteins. A total of 69 small molecules were identified from the metabolites of BH M20221856 using liquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS). Among these, soyasaponin I (SoSa I) represented the largest multiple change in the enrichment analysis of differential metabolites and exhibited potent anti-ETEC effects in vivo . It significantly reduced the bacterial load of E. coli in mouse intestines, decreased serum endotoxin, D-lactic acid, and oxidative stress levels and alleviated intestinal pathological damage and inflammation. SoSa I enhanced immune regulation by mediating the p105-Tpl2-ERK signaling pathway. Further evaluations using transepithelial electrical resistance (TEER) and cell permeability assays showed that SoSa I alleviated ETEC-induced damage to epithelial barrier function. These results suggest that BH M20221856 and SoSa I may serve as preventative biologics against ETEC infections, providing new insights for developing strategies to prevent and control this disease.