Genome-wide identification of tissue-specific fitness genes in murine models of Staphylococcus aureus infection

Staphylococcus aureus must dynamically rewire its metabolism to persist within distinct host tissues during infection. We applied in vivo transposon-directed insertion site sequencing (TraDIS) in murine models of skin, kidney, and spleen infections to define tissue-specific fitness landscapes for the epidemic USA300 lineage. We identified 46, 76, and 69 fitness genes in the skin, kidney, and spleen, respectively. The core gluconeogenesis gene fbp was essential across all tissues, whereas pckA and gapB showed organ-specific essentiality in the kidney and spleen. Skin infection required oxidative stress and DNA repair genes (ahpC, ahpF, dps, uvrC, and xseA), consistent with elevated genotoxic pressure. In contrast, kidney and spleen relied on branched-chain amino acid catabolism (bkdAB), lipid metabolism (SAUSA300_0355), and putative polyamine biosynthesis (SAUSA300_0458). Competition assays in vivo and under oxidative (H₂O₂) and gluconeogenic (M9) conditions validated these tissue-specific dependencies. These results reveal how S. aureus remodels metabolic networks and identifies context-specific vulnerabilities for therapeutic targeting.