IFN-β blocks the thymidine salvage pathway in Staphylococcus aureus through high-affinity binding to thymidine kinase.

The escalating crisis of antimicrobial resistance necessitates the exploration of new treatment strategies, particularly against priority pathogens such as Staphylococcus aureus (S. aureus). While interferon-beta (IFN-β) is known for its immunomodulatory effects, its potential direct antibacterial activity remains poorly understood. This study uncovers a new, non-immune mechanism by which IFN-β inhibits S. aureus by directly blocking thymidine kinase (TK), a key enzyme in the nucleotide salvage pathway. We first identified a strong, spontaneous interaction between IFN-β and TK through fluorescence quenching and isothermal titration calorimetry. Further enzyme kinetics showed that IFN-β acts as a potent, concentration-dependent inhibitor of TK ATPase activity, with an IC₅₀ of 10.06 μM. In vitro growth experiments demonstrated that IFN-β significantly reduces bacterial growth, an effect completely reversed by thymidine addition at both the start of culture and after 9 h, confirming TK-mediated thymidine auxotrophy as the primary mechanism. Computer analyses offered a structural explanation for this inhibition. Protein-protein docking and molecular dynamics simulations revealed that IFN-β forms a stable complex with TK, engaging a strategic interface that includes the ATP-binding P-loop. This interaction, stabilized by electrostatic and hydrophobic forces, blocks substrate access and reduces catalytic function. Overall, our results change the view of IFN-β from a solely immune-modulating agent to a host-derived antimicrobial that directly targets an essential bacterial metabolic enzyme. This discovery reveals a new aspect of host-pathogen interaction. It highlights the TK pathway as a vulnerable target, laying the groundwork for the development of innovative, host-inspired treatments to fight drug-resistant staphylococcal infections.