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The genes contributing to intracellular pathogenesis have not yet been fully enumerated. Here, we cataloged genes influencing\n                    <jats:italic>S. aureus<\/jats:italic>\n                    invasion and survival within human THP-1 derived macrophages using two laboratory strains (ATCC2913 and JE2). We developed an\n                    <jats:italic>in vitro<\/jats:italic>\n                    transposition method to produce highly saturated transposon mutant libraries in\n                    <jats:italic>S. aureus<\/jats:italic>\n                    and performed transposon insertion sequencing (Tn-Seq) to identify candidate genes with significantly altered abundance following macrophage invasion. While some significant genes were strain-specific, 108 were identified as common across both\n                    <jats:italic>S. aureus<\/jats:italic>\n                    strains, with most (\n                    <jats:italic>n<\/jats:italic>\n                    = 106) being required for optimal macrophage infection. We used CRISPR interference (CRISPRi) to functionally validate phenotypic contributions for a subset of genes. Of the 20 genes passing validation, seven had previously identified roles in\n                    <jats:italic>S. aureus<\/jats:italic>\n                    virulence, and 13 were newly implicated. Validated genes frequently evidenced strain-specific effects, yielding opposing phenotypes when knocked down in the alternative strain. Genomic analysis of\n                    <jats:italic>de novo<\/jats:italic>\n                    mutations occurring in groups (\n                    <jats:italic>n<\/jats:italic>\n                    = 237) of clonally related\n                    <jats:italic>S. aureus<\/jats:italic>\n                    isolates from the airways of chronically infected individuals with cystic fibrosis (CF) revealed significantly greater\n                    <jats:italic>in vivo<\/jats:italic>\n                    purifying selection in conditionally essential candidate genes than those not associated with macrophage invasion. This study implicates a core set of genes necessary to support macrophage invasion by\n                    <jats:italic>S. aureus,<\/jats:italic>\n                    highlights strain-specific differences in phenotypic effects of effector genes, and provides evidence for selection of candidate genes identified by Tn-Seq analyses during chronic airway infection in CF patients\n                    <jats:italic>in vivo<\/jats:italic>\n                    .\n                  <\/jats:p>","DOI":"10.1128\/iai.00228-23","type":"journal-article","created":{"date-parts":[[2023,9,7]],"date-time":"2023-09-07T09:01:39Z","timestamp":1694077299000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":13,"title":["Transposon sequencing identifies genes impacting\n                    <i>Staphylococcus aureus<\/i>\n                    invasion in a human macrophage model"],"prefix":"10.1128","volume":"91","author":[{"given":"Hsin-Yu","family":"Lo","sequence":"first","affiliation":[{"name":"Department of Laboratory Medicine and Pathology, University of Washington School of Medicine , Seattle, Washington, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dustin R.","family":"Long","sequence":"additional","affiliation":[{"name":"Division of Critical Care Medicine, Department of Anesthesiology and Pain Medicine, University of Washington School of Medicine , Seattle, Washington, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elizbeth A.","family":"Holmes","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine and Pathology, University of Washington School of Medicine , Seattle, Washington, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kelsi","family":"Penewit","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine and Pathology, University of Washington School of Medicine , Seattle, Washington, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Taylor","family":"Hodgson","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine and Pathology, University of Washington School of Medicine , Seattle, Washington, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Janessa D.","family":"Lewis","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine and Pathology, University of Washington School of Medicine , Seattle, Washington, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Adam","family":"Waalkes","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine and Pathology, University of Washington School of Medicine , Seattle, Washington, 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