Staphylococcus aureus Mnh1 cation–proton antiporter promotes phagocytic uptake and intracellular survival in human monocytes

While Staphylococcus aureus is predominantly an extracellular human commensal, it can also exhibit an intracellular lifestyle that enables its persistence within diverse host cell types, including monocytes and macrophages. This capacity complicates treatment, contributing to chronic infections and therapeutic failure. Employing combined flow cytometry and microscopy, we show that monocyte internalization is associated with markedly increased antibiotic survival of intracellular S. aureus . To identify staphylococcal genetic determinants important for its intracellular lifestyle, we utilized a genome-wide CRISPRi-seq screening strategy using a co-infection model with human-derived THP-1 cells. This screen revealed a set of genes specifically required for intracellular fitness, confirming established virulence factors as well as identifying novel contributors to uptake and survival within host phagocytes. Among these, we identified the cation–proton antiporter Mnh1 and demonstrated that it is particularly important for phagocytic entry. Time-lapse microscopy demonstrated that its depletion significantly reduced uptake by THP-1 cells leading to reduced intracellular bacterial load. Functional assays further showed that the Mnh1 operon plays a critical role in the intracellular survival of multiple S. aureus lineages in THP-1 cells. Consistent with this finding, levofloxacin treatment of infected monocytes cleared knockdown strains more efficiently than the control strain. Our findings underscore the intracellular niche as a critical reservoir for bacteria and suggest that selective inhibition of Mnh1, in combination with clinically relevant antibiotics, offers a promising therapeutic approach for the treatment of intracellular S. aureus infections.