Abstract

--- - Staphylococcus aureus is an important pathogen that leads to high mor-bidity and mortality. Although S. aureus produces many factors important for patho-genesis, few have been validated as playing a role in the pathogenesis of S. aureus pneumonia. To gain a better understanding of the genetic elements required for S. aureus pathogenesis in the airway, we performed an unbiased genome-wide transposon sequencing (Tn-seq) screen in a model of acute murine pneumonia. We identified 136 genes important for bacterial survival during infection, with a high proportion involved in metabolic processes. Phenotyping 80 individual dele-tion mutants through diverse in vitro and in vivo assays demonstrated that me-tabolism is linked to several processes, which include biofilm formation, growth, and resistance to host stressors. We further validated the importance of 23 muta-tions in pneumonia. Multivariate and principal-component analyses identified two key metabolic mechanisms enabling infection in the airway, growth (e.g., the abil-ity to replicate and form biofilms) and resistance to host stresses. As deep valida-tion of these hypotheses, we investigated the role of pyruvate carboxylase, which was important across multiple infection models and confirmed a connection between growth and resistance to host cell killing. Pathogenesis is conventionally understood in terms of the host-pathogen interactions that enable a pathogen to neutralize a host's immune response. We demonstrate with the important bacte-rial pathogen S. aureus that microbial metabolism influences key traits important for in vivo infection, independent from host immunomodulation. - IMPORTANCE Staphylococcus aureus is an important bacterial pathogen that causes significant morbidity and mortality, infecting numerous bodily sites, including the re-spiratory tract. To identify the bacterial requirements for lung infection, we con-ducted a genome-wide screen in a mouse model of acute pneumonia. We discov-ered that metabolic genes were overrepresented in those required for lung infection. In contrast to the conventional view of pathogenesis focusing on immuno-modulation, we demonstrate through phenotyping of deletion mutants in several functional assays that replicative ability and tolerance against host defenses form two key metabolic dimensions of bacterial infection. These dimensions are independ-ent for most pathways but are coupled in central carbon metabolism and highlight the critical role of bacterial metabolism in survival against host defenses during infection.