Dynamic pneumococcal genetic adaptations support bacterial growth and inflammation during coinfection with influenza

Streptococcus pneumoniae (pneumococcus) is one of the primary bacterial pathogens that complicates influenza virus infections. These secondary infections increase influenza-associated morbidity and mortality through a number of immunological and viral-mediated mechanisms. However, little is known about how specific bacterial genes contribute to post-influenza pathogenicity. Thus, we used genome-wide transposon mutagenesis(Tn-Seq) to reveal bacterial genes conferring improved fitness in influenza infected hosts. The majority of the 32 identified genes are involved in bacterial metabolism, including nucleotide biosynthesis, amino acid biosynthesis, protein translation, and membrane transport. We investigated five of the genes in detail: SPD1414, SPD2047 (cbiO1), SPD0058 (purD), SPD1098, and SPD0822 (proB). Single-gene deletion mutants showed slight growth attenuationsin vitro and in vivo, but still grew to high titers in both naive and influenza-infected murine hosts. Despite high bacterial loads in the lung and sustained bacteremia, mortality was significantly reduced or delayed with each of the knockouts. Reductions in pulmonary neutrophils, inflammatory macrophages, and select proinflammatory cytokines and chemokines were observed at discrete times after coinfectionwith these bacterial mutants. Immunohistochemical staining also revealed altered neutrophil phenotype and distribution in the lungs of animals coinfectedwith knockouts. These studies demonstrate a critical role for specific bacterial genes in driving virulence and immune function during influenza-associated bacterial pneumonia.