Host-pathogen dual targeting with repurposed drugs identifies a synergistic therapy for intracellular Staphylococcus aureus

Background: Staphylococcus aureus causes persistent infections by surviving intracellularly, where it escapes immune defenses and antibiotics. Repurposing clinically approved drugs provides a rapid strategy to expand therapies against intracellular pathogens. Methods: A high-throughput screen of 5,599 approved compounds was performed in A549 cells infected with S. aureus USA300 LAC. Hits were validated across CA-MRSA, HA-MRSA, and MSSA strains and in non-tumorigenic bronchial epithelial cells. Mechanistic studies combined metabolomics, dual RNA sequencing, and DNA repair mutant panels. Efficacy was assessed in Galleria mellonella and a murine pneumonia model. Results: The nucleoside analog 5-fluoro-2′-deoxycytidine (5-FdC) and rifapentine formed a synergistic combination (5FR) effective across strains and host cell types. 5-FdC induced structural DNA damage beyond thymineless death, confirmed in DNA repair mutants, and triggered host DNA damage responses and metabolic rewiring. Dual RNA-seq revealed bacterial growth arrest alongside host stress pathway activation. The 5FR combination significantly reduced bacterial burdens in larvae and in murine lungs and spleen. Conclusions: This integrative pipeline, from screening to in vivo validation, identifies 5FR as a promising repurposed drug combination against intracellular S. aureus. Targeting both host and pathogen pathways may overcome monotherapy limitations, reduce resistance risk, and expand therapeutic options.