3-Epicabraleadiol inhibits LmrS protein of Staphylococcus aureus isolated from cow mastitis and enhances antibiotic susceptibility.

Staphylococcus aureus (S. aureus) is a major pathogen of bovine mastitis, and its antimicrobial resistance poses challenges to livestock and public health. The lincomycin resistance protein (LmrS) contributes significantly to this resistance. However, many efflux pump inhibitors (EPIs) are limited clinically due to toxicity, highlighting the need for low-toxicity EPIs. We introduced site-directed single and double mutations at N29 and Q30 within the Motif-D1 region of the LmrS substrate-binding domain. Mutant efflux activity was assessed via broth microdilution and ethidium bromide accumulation/efflux assays. Molecular docking was used to screen a plant-derived EPI, 3-Epicabraleadiol. Its inhibitory effects on LmrS-mediated efflux were further examined in engineered E. coli and wild-type S. aureus isolates XJ50, XJNA10, and XJ69. Functional analysis revealed that N29/Q30 single and double mutations lowered the MICs of all tested antibiotics to empty-vector control levels. Notably, alanine substitutions at N29 and Q30 nearly abolished ethidium bromide efflux. Docking predicted that 3-Epicabraleadiol binds to N62 and Q30 of LmrS. Antibiotic susceptibility tests showed that 3-Epicabraleadiol effectively blocks efflux of lincomycin hydrochloride, kanamycin sulfate, and fusidic acid in both engineered and wild-type strains. At ≥2 μg/mL, 3-Epicabraleadiol reduced the MICs of these antibiotics against wild-type S. aureus by half.