Multispecies transcriptomics reveals influenza A virus modulation of <i>Streptococcus pneumoniae</i> EF3030 infection in human lung epithelium and murine lung

<i>Streptococcus pneumoniae</i> (Spn) is typically an asymptomatic colonizer of the nasopharynx, but it also causes pneumonia and disseminated disease affecting various host anatomical sites. To delineate host-pathogen transcriptional interactions during pneumococcal (EF3030) and influenza A (pH1N1) coinfection, we used primary differentiated human bronchial epithelial cells (HBEC-three human donors) in a transwell monolayer model at an air-liquid interface, and a mouse pneumonia model, profiled with multispecies deep RNA-seq and NanoString nCounter as complementary models. Distinct pneumococcal gene expression profiles were observed in the presence and absence of influenza in HBEC infection. Influenza coinfection enabled significantly greater pneumococcal growth and triggered the differential expression of bacterial genes corresponding to multiple metabolic pathways. Notably, a fundamentally altered bacterial metabolic state and a greater nutrient availability were observed when coinfecting with influenza. Downregulation/deletion of sialic acid utilization genes promoted EF3030 proliferation during mono/coinfection with pH1N1 on HBEC. Surprisingly, HBEC transcriptomes were only modestly perturbed by infection with EF3030 alone relative to changes resulting from influenza A infection or coinfection. Influenza-infected HBEC transcriptomes showed significant loss of ciliary function, with changes in host defense, microtubules, and extracellular matrix (ECM). Some of these findings were confirmed in the murine lung infection model. Influenza-mediated changes in the host epithelium transcriptome also contribute to bacterial invasiveness. This included downregulation of genes involved in expressing cilia and increased ECM degradation. Ultimately, we identified novel genes and pathways involved in the dynamics of epithelium-influenza-pneumococcus coinfection, such as EF3030 metabolic regulons (NanR, LacR, etc.) and epithelial protein families (keratins and matrix metalloproteases). We conclude that influenza infection promotes a pneumococcal metabolic shift, allowing for transition from colonization to disseminated disease and an exacerbated breakdown in the epithelium, potentially permitting enhanced EF3030 infection and dissemination.IMPORTANCETransition from pneumococcal colonization to invasive disease is not well understood. Studies have shown that such a transition can occur as a result of influenza A virus (IAV) coinfection. We investigated the pneumococcal (serotype 19F, strain EF3030, and isogenic mutants) and airway epithelial transcriptomes with and without IAV (A/California/07 2009 pH1N1) infection. Pneumococcus and influenza coinfection leads to enhanced bacterial transcriptional programs related to growth, nutrient availability, and energy biosynthesis, suggesting conversion to an invasive phenotype. Influenza-induced secondary EF3030 infection influences human bronchial epithelial cell (HBEC) microtubules and extracellular matrix. Notably, sialic acid (NanR) utilization is a central regulon in EF3030 mono/coinfection with pH1N1 on HBEC. Downregulation of sialic acid utilization during influenza coinfection improved Spn pathogenicity <i>ex vivo</i> but did not alter disease <i>in vivo,</i> suggesting other metabolic cues are also important. This study uncovers critical metabolic features of the EF3030-pH1N1 interface to inform how Spn proliferates during IAV coinfection.