Exploration of Bacterial Bottlenecks and Streptococcus pneumoniae Pathogenesis by CRISPRi-Seq

Streptococcus pneumoniae is an opportunistic human pathogen that causes invasive diseases, including pneumonia, with greater health risks upon influenza A virus (IAV) co-infection. To facilitate pathogenesis studies in vivo, we developed an inducible CRISPR interference system that enables genome-wide fitness testing in one sequencing step (CRISPRi-seq). We applied CRISPRi-seq to assess bottlenecks and identify pneumococcal genes important in a murine pneumonia model. A critical bottleneck occurs at 48 h with few bacteria causing systemic infection. This bottleneck is not present during IAV superinfection, facilitating identification of pneumococcal pathogenesis-related genes. Top in vivo essential genes included purA, encoding adenylsuccinate synthetase, and the cps operon required for capsule production. Surprisingly, CRISPRi-seq indicated no fitness-related role for pneumolysin during superinfection. Interestingly, although metK (encoding S-adenosylmethionine synthetase) was essential in vitro, it was dispensable in vivo. This highlights advantages of CRISPRi-seq over transposon-based genetic screens, as all genes, including essential genes, can be tested for pathogenesis potential. [Display omitted] •CRISPRi-seq enables in vivo genome-wide fitness testing in one sequencing step•Identification of a strong bottleneck in a S. pneumoniae murine pneumonia model•CRISPRi-seq reveals pneumococcal genes critical during influenza virus superinfection•CRISPRi-seq can test gene fitness of all genes, including essential genes Liu et al. developed CRISPRi-seq to enable in vivo genome-wide fitness testing of Streptococcus pneumoniae in one sequencing step. CRISPRi-seq revealed a bottleneck during murine pneumococcal infection not observed upon influenza virus co-infection, enabling identification of essential genes. By testing all genes, including essential genes, CRISPRi-seq has broad utility.