Highly Saturated Transposon Sequencing identifies genes impacting Staphylococcus aureus pathogenesis in macrophages

Abstract

Staphylococcus aureus 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 S. aureus invasion and survival within human macrophages using two laboratory strains (ATCC2913 and JE2). We developed an in vitro transposition method to produce saturated transposon mutant libraries in S. aureus, and performed Tn-Seq to identify candidate genes with significantly altered abundance following macrophage invasion. While some significant genes were strain-specific, 107 were identified in common across both S. aureus strains, with most (n=105) being required for optimal macrophage infection. We used CRISPR interference (CRISPRi) to functionally validate phenotypic contributions for a select subset of genes. Of the 20 genes passing validation, 7 had a previously identified role in S. aureus 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 de novo mutations occurring in groups (n=237) of clonally-related S. aureus isolates from the airways of chronically infected individuals with cystic fibrosis (CF) revealed significantly greater rates of in vivo selection in candidate genes than factors not associated with macrophage invasion. This study implicates a core set of genes necessary to support macrophage invasion by S. aureus, 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 in vivo.