Simvastatin induced cholesterol depletion reduces intracellular Mycoplasma bovis load in bovine mammary epithelial cells and murine mammary tissues

Mycoplasma bovis is a globally significant pathogen in dairy cow associated with pneumonia, arthritis, mastitis, otitis, and reproductive failure with different age, resulting in substantial economic losses in the cattle industry. M. bovis relies on host-derived cholesterol for its membrane integrity and intracellular survival, as it cannot synthesize this lipid itself. However, the specific mechanisms by which M. bovis alters host cell cholesterol metabolism remain unclear. Simvastatin, a common cholesterol-lowering drug that inhibits HMG-CoA reductase, provides a tool to investigate this host–pathogen interaction. This study aimed to: (1) determine the effect of M. bovis infection on cholesterol metabolism in bovine mammary epithelial cells (bMECs); (2) assess the role of intracellular cholesterol in bacterial proliferation; (3) investigate whether cholesterol depletion activates anti-bacterial autophagy via the AMPK–mTORC1–TFEB pathway; and (4) evaluate the anti- M. bovis efficacy of simvastatin in both in vitro and in vivo models. Results showed that in bMECs, M. bovis infection dysregulated the expression of cholesterol metabolism genes. Depleting cholesterol (using MβCD or simvastatin) significantly reduced the intracellular M. bovis load. This reduction was accompanied by the activation of autophagy and the AMPK–mTORC1–TFEB signaling pathway. In a murine mastitis model, simvastatin treatment enhanced autophagy, reduced the M. bovis burden, and mitigated infection-induced pathology. Our findings demonstrate that simvastatin exerts anti- M. bovis effects by reducing host cholesterol, activating the AMPK–mTORC1–TFEB pathway, and enhancing autophagic clearance of the bacteria. This reveals a novel potential therapeutic strategy for combating M. bovis infections.