Host–Pathogen Dual Targeting With Repurposed Drugs Identifies a Synergistic Therapy for Intracellular Staphylococcus aureus

Staphylococcus aureus is a major cause of severe infections, including pneumonia and sepsis, partly due to its ability to survive within host cells where many antibiotics are ineffective. Drug repurposing offers a rapid strategy to identify compounds that enhance intracellular antibacterial activity by modulating host pathways. Here, a high-throughput screen of 6297 clinically approved compounds in S. aureus-infected A549 cells identified 5-fluoro-2'-deoxycytidine (5-FdC) as an effective intracellular inhibitor. When combined with rifapentine (5FR), 5-FdC displayed synergistic activity across community- and hospital-acquired MRSA and MSSA strains, as well as in different host cell types, including non-tumorigenic bronchial cells. Metabolomic and host RNA-sequencing analyses showed that 5-FdC treatment activated host stress-response and DNA damage response (DDR) pathways while restoring infection-induced metabolic imbalances, particularly in amino acid and central carbon metabolism. These transcriptional and metabolic changes correlated with reduced intracellular bacterial markers. In vivo, the 5FR combination significantly decreased bacterial loads in Galleria mellonella and murine pneumonia models without detectable toxicity. This study presents the largest repurposing screen performed against intracellular S. aureus and identifies a synergistic host- and pathogen-targeted combination that enhances bacterial clearance through coordinated modulation of host DDR, stress, and metabolic responses.