Genome-wide CRISPRi maps pneumococcal attachment and intracellular survival during single infection and Influenza co-infection

Streptococcus pneumoniae remains a leading cause of bacterial pneumonia and frequently causes severe secondary infections following influenza A virus (IAV) infection. Yet, the genetic requirements underlying pneumococcal pathogenicity during viral co-infection remain incompletely understood. Here, we employed an inducible genome-wide CRISPR interference (CRISPRi) library to systematically investigate pneumococcal gene fitness during epithelial attachment and intracellular survival in macrophages under both single-infection and IAV co-infection conditions. Screening across multiple host cell types and viral strains identified 42 pneumococcal genes affecting host-cell attachment and 63 genes influencing intracellular survival. While global patterns of gene fitness were largely conserved between single infection and co-infection, genes involved in cell envelope biogenesis, including the undecaprenyl pyrophosphate phosphatase uppP, became increasingly important during co-infection. Pharmacological inhibition of cell wall biosynthesis recapitulated several genetic phenotypes and revealed enhanced sensitivity to bacitracin in virally altered host environments. Integration of CRISPRi screening with untargeted metabolomics uncovered extensive remodeling of nucleotide metabolism during infection. Disruption of nucleoside transport altered bacterial fitness, morphology, capsule expression, and host-cell attachment. Uridine availability emerged as a key regulator linking metabolic adaptation to virulence-associated traits, highlighting a trade-off between bacterial growth and adherence. Taken together, our study provides a genome-scale resource of pneumococcal fitness determinants during infection and demonstrates that influenza co-infection selectively reshapes bacterial genetic dependencies rather than globally altering pathogenicity programs. These findings identify cell envelope homeostasis and nucleotide metabolism as central regulators of pneumococcal virulence and reveal potential targets for future antimicrobial and anti-virulence strategies.