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

<i>Staphylococcus aureus</i> must dynamically rewire its metabolism to persist within distinct host tissues during infection. We applied <i>in vivo</i> 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 <i>fbp</i> was essential across all tissues, whereas <i>pckA</i> and <i>gapB</i> showed organ-specific essentiality in the kidney and spleen. Skin infection required oxidative stress and DNA repair genes (<i>ahpC</i>, <i>ahpF</i>, <i>dps</i>, <i>uvrC</i>, and <i>xseA</i>), consistent with elevated genotoxic pressure. In contrast, kidney and spleen relied on branched-chain amino acid catabolism (<i>bkdAB</i>), lipid metabolism (<i>SAUSA300_0355</i>), and putative polyamine biosynthesis (<i>SAUSA300_0458</i>). Competition assays <i>in vivo</i> and under oxidative (H₂O₂) and gluconeogenic (M9) conditions validated these tissue-specific dependencies. These results reveal how <i>S. aureus</i> remodels metabolic networks and identifies context-specific vulnerabilities for therapeutic targeting.