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Although some factors governing<jats:italic>Staphylococcus aureus<\/jats:italic>pathogenicity have been identified and studied, a comprehensive genomic analysis of virulence functions will be a prerequisite for developing a global understanding of interactions between this pathogen and its human host. In this study, we describe a genetic screening strategy and demonstrate its use in screening a collection of 6,300<jats:italic>S. aureus<\/jats:italic>insertion mutants for virulence attenuation in a murine model of systemic infection. Ninety-five attenuated mutants were identified, reassembled into new pools, and rescreened using the same murine model. This effort identified 24 highly attenuated mutants, each of which was further characterized for virulence attenuation in vivo and for growth phenotypes in vitro. Mutants were recovered in numbers up to 1,200-fold less than wild type in the spleens of systemically infected animals and up to 4,000-fold less than wild type in localized abscess infections. Genetic analysis of the mutants identified insertions in 23 unique genes. The largest gene classes represented by these mutants encoded enzymes involved in small-molecule biosynthesis and cell surface transmembrane proteins involved in small-molecule binding and transport. Additionally, three insertions defined two histidine kinase sensor-response regulator gene pairs important for<jats:italic>S. aureus<\/jats:italic>in vivo survival. Our findings extend the understanding of pathogenic mechanisms employed by<jats:italic>S. aureus<\/jats:italic>to ensure its successful growth and survival in vivo. Many of the gene products we have identified represent attractive new targets for antibacterial chemotherapy.<\/jats:p>","DOI":"10.1128\/jb.186.24.8478-8489.2004","type":"journal-article","created":{"date-parts":[[2004,12,2]],"date-time":"2004-12-02T23:48:13Z","timestamp":1102031293000},"page":"8478-8489","update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":103,"title":["Large-Scale Identification of Genes Required for Full Virulence of<i>Staphylococcus aureus<\/i>"],"prefix":"10.1128","volume":"186","author":[{"given":"Bret M.","family":"Benton","sequence":"first","affiliation":[{"name":"Essential Therapeutics, Inc., Mountain View, California"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"J. 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