AbstractStreptococcus 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 vitroandin vivo, but still grew to high titers in both naïve 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 coinfection with these bacterial mutants. Immunohistochemical staining also revealed altered neutrophil phenotype and distribution in the lungs of animals coinfected with knockouts. These studies demonstrate a critical role for specific bacterial genes in driving virulence and immune function during influenza-associated bacterial pneumonia.Author SummaryStreptococcus pneumoniae(pneumococcus) is a common coinfecting pathogen that increases morbidity and mortality during influenza epidemics and pandemics. It is known that the strain, dose, and timing of bacterial coinfection influence the likelihood of severe pneumonia, but the specific bacterial genes that contribute to bacterial pathogenicity during influenza coinfection remain unknown. Using a genome-wide analysis, we identified the pneumococcal genes that exacerbate disease during influenza-bacterial coinfection. Most of these have a role in metabolism. To better understand their contribution to this lethal disease, we generated 5 mutants that lacked a single gene. The strains grew to high titers in the lungs and blood of both healthy and influenza-infected animals yet mortality was significantly reduced. In influenza-infected animals, there was also significantly lower inflammatory immune responses, and lung pathology. These important pneumococcal adaptations largely facilitate lethality during influenza-pneumococcal coinfection. Investigating whether similar metabolic adaptations are conserved among bacterial species that complicate influenza could yield broadly effective therapies that abrogate lethal post-influenza bacterial infections.
Targeted control of toxin production by a mobile genetic element in Streptococcus pneumoniae
