Streptococcus pneumoniae remains a major cause of bacterial pneumonia and is a frequent cause of severe secondary bacterial infections after Influenza A virus (IAV). The genetic determinants that enable pneumococci to attach to host cells and to survive inside host phagocytes, especially in the context of viral co-infection, are incompletely defined. The authors used an inducible genome-wide CRISPRi library to systematically probe pneumococcal gene fitness during two key infection contexts: epithelial attachment and intracellular survival in macrophages, under both single bacterial infection and IAV co-infection.
An inducible, genome-wide CRISPR interference platform was applied to S. pneumoniae to suppress gene expression across the genome and determine genes required for fitness during interaction with host cells. Screens were performed across multiple host cell types and with different viral strains to capture condition-specific dependencies. Results from these pooled functional genetic screens were used to identify genes whose knockdown altered bacterial attachment to epithelial cells or intracellular survival in macrophages, both in single-infection settings and during IAV co-infection.
The CRISPRi screens identified 42 pneumococcal genes that influence host-cell attachment. These loci represent genetic contributors to initial host colonization and adherence under the experimental conditions tested. The abstract reports the number of genes discovered but does not list individual gene names beyond selected pathways; full gene-level results were not provided in the source abstract.
A separate set of 63 genes was found to modulate intracellular survival in macrophages. These genes reflect functions required for persistence or replication within phagocytic cells. As with attachment, specific gene identities beyond highlighted pathways are not detailed in the abstract; the full dataset is described as a genome-scale resource in the report.
While the global patterns of gene fitness were largely conserved between single infection and IAV co-infection, the screens revealed selective reshaping of genetic dependencies under co-infection. Rather than a wholesale change in pathogenicity programs, influenza exposure selectively increased the importance of certain bacterial genes and pathways. This indicates that viral–bacterial interactions can alter which pneumococcal functions are most critical for successful infection in host-altered environments.
Among the pathways with increased importance during co-infection were genes involved in cell envelope biogenesis. The undecaprenyl pyrophosphate phosphatase uppP is specifically highlighted as becoming more critical in the presence of IAV. This enzyme plays a role in cell wall precursor recycling and envelope homeostasis; its elevated importance during co-infection suggests that maintenance of the bacterial envelope is a vulnerability when host conditions are altered by viral infection.
The authors report that pharmacological inhibition of cell wall biosynthesis reproduced several of the genetic phenotypes observed in the CRISPRi screens. In particular, bacitracin sensitivity was enhanced in virally altered host environments, linking the genetic dependency on envelope homeostasis to an actionable antimicrobial vulnerability. The abstract indicates that drug perturbation recapitulated genetic findings, but specific experimental parameters, concentrations, or quantitative effects were not provided in the source text.
Integration of the CRISPRi screening results with untargeted metabolomics revealed extensive remodeling of nucleotide metabolism during infection. Metabolic profiling indicated that infection-associated changes in host or bacterial metabolism intersect with genetic requirements, implicating nucleotide pathways as central to the bacterial response during interaction with host cells and during co-infection.
Disruption of nucleoside transport in pneumococci altered multiple phenotypes: bacterial fitness, cell morphology, capsule expression, and host-cell attachment. These observations link nucleotide uptake and processing to structural and virulence-associated traits. The screens and metabolomics indicated that uridine availability emerged as a key regulator connecting metabolic adaptation to virulence phenotypes. The authors describe a trade-off in which uridine-mediated metabolic changes influence the balance between bacterial growth and adherence.
Collectively, the findings prioritize cell envelope homeostasis and nucleotide metabolism as central regulators of pneumococcal virulence under both single-infection and IAV co-infection contexts. The genome-scale resource of fitness determinants generated by these CRISPRi screens can guide future efforts to develop targeted antimicrobials or anti-virulence approaches that exploit vulnerabilities exposed during viral co-infection. Enhanced susceptibility to agents such as bacitracin in virally altered environments suggests therapeutic avenues for combination or context-specific treatments.
This report is a preprint and has not undergone peer review. The abstract summarizes major experimental findings but does not provide full experimental details, lists of all identified genes, quantitative metrics, or specific metabolomics datasets. Those elements are presumably contained in the full preprint; they were not reported in the abstract text supplied as the source. Readers should consult the full preprint for detailed methods, complete gene lists, statistical analyses, and supporting data before applying findings to experimental design or clinical inference.