Two Bile Salt Hydrolase Homologues Drive Bile Resistance in Lactobacillus johnsonii ATCC 43121

The role of bile salt hydrolase (BSH) in Lactobacillus johnsonii ’s intestinal adaptability remains unclear. Here, we elucidate the functions and mechanisms of three BSH homologues (BSHA, BSHB, and BSHC) in mediating bile resistance in Lb. johnsonii ATCC 43121, using bsh knockout and complementation mutants and functional assays under simulated human small intestinal pH conditions. Functional tests showed BSHA and BSHC significantly enhanced the strain’s resistance to conjugated bile acids, whereas BSHB was ineffective due to low enzymatic activity. Mechanistically, BSHA and BSHC hydrolyze intracellular conjugated bile acids into free forms with high export efficiency, preserving membrane integrity and the homeostasis of the transmembrane pH gradient, transmembrane potential, and intracellular ATP and GTP. Molecular docking and molecular dynamics simulations revealed that steric hindrance in BSHB’s active pocket prevents substrate entry, whereas BSHA and BSHC accommodate all six tested conjugated bile acids, forming stable interactions specifically with the three glycine-conjugated bile acids.