A niche-specific role for ArlRS enables Staphylococcus aureus growth within Kupffer cells
Staphylococcus aureus bloodstream infections cause severe morbidity and mortality despite antibiotic therapy. We previously identified Kupffer cells (KCs) as the primary hepatic macrophages that rapidly clear circulating S. aureus, yet a subset of bacteria survives intracellularly and seeds systemic infection. Here, using spinning-disk intravital and super-resolution microscopy, we show that bacterial two-component system (TCS) signaling is required for intracellular replication within KCs in vivo. A global mutant lacking all non-essential TCSs failed to form intracellular microcolonies, and targeted analysis identified the ArlRS system as specifically required for KC replication. {Delta}arlRS bacteria remained as single non-replicating organisms within KCs, exhibited reduced dissemination and kidney abscess formation, and showed attenuated virulence with improved host survival. Complementation with arlRS restored the wildtype phenotype. Notably, the intracellular replication defect was not observed in primary or cultured macrophages, indicating that ArlRS-dependent replication is highly niche-specific and not reproduced by conventional in vitro models. Furthermore, restricting the KC reservoir with vancomycin treatment after infection further reduced renal infection by {Delta}arlRS bacteria. These findings identify ArlRS as a critical regulator of intracellular replication within KCs and establish the KC intracellular reservoir as a key driver of S. aureus pathogenesis and a potential therapeutic target during bloodstream infection.