Transposon sequencing identifies genes impacting Staphylococcus aureus invasion in a human macrophage model

• Published in: Infection and immunity 2023-10, Vol.91 (10), p.e0022823-e0022823
• Main Authors: Lo, Hsin-Yu, Long, Dustin R, Holmes, Elizbeth A, Penewit, Kelsi, Hodgson, Taylor, Lewis, Janessa D, Waalkes, Adam, Salipante, Stephen J
• Format: Article
• Language: English
• Subjects: Cystic Fibrosis - complications; Humans; Molecular Genomics; Respiratory System; Staphylococcal Infections - metabolism; Staphylococcus aureus - metabolism; Virulence - genetics
• ISSN: 0019-9567; 1098-5522
• DOI: 10.1128/iai.00228-23

is a facultative intracellular pathogen in many host cell types, facilitating its persistence in chronic infections. The genes contributing to intracellular pathogenesis have not yet been fully enumerated. Here, we cataloged genes influencing invasion and survival within human THP-1 derived macrophages using two laboratory strains (ATCC2913 and JE2). We developed an transposition method to produce highly saturated transposon mutant libraries in 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 strains, with most ( = 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 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 mutations occurring in groups ( = 237) of clonally related isolates from the airways of chronically infected individuals with cystic fibrosis (CF) revealed significantly greater 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 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 .