In vivo CRISPRi-seq identifies essential pneumococcal vulnerabilities during meningitis

Streptococcus pneumoniae is an important human pathogen and the leading cause of community-acquired bacterial meningitis. However, the bacterial determinants required for survival within the central nervous system (CNS) are poorly understood. Here, we performed genome-wide CRISPRi screening in a zebrafish model to map pneumococcal fitness determinants during meningitis. The screen identified 244 genes whose repression significantly reduced bacterial fitness in vivo, representing pathways involved in virulence, metabolism, cell-envelope biogenesis, translation, and stress adaptation. Comparative analysis with in vitro CRISPRi-seq datasets showed that metabolic genes such as purA, proABC, thrC, glyA, and manLMN, as well as those involved in peptidoglycan and capsule biosynthesis (pbp3, cps2 operon) and oxidative stress response (nox, dpr), become selectively more important during meningitis. Functional validation confirmed roles for these pathways in virulence and nutrient-dependent growth during meningitis and supported a contribution of oxidative stress-related processes to pneumococcal fitness in vivo. Untargeted metabolomic profiling of infected zebrafish embryos revealed selective remodelling of host carbohydrate, amino acid, nucleotide, and lipid-associated metabolites, including marked glucose depletion consistent with hypoglycorrhachia, and alterations that aligned with pathways identified as essential by CRISPRi-seq. Integration of the in vivo essentialome with antibiotic-target annotations uncovered both established and previously unrecognised vulnerabilities, including aminoacyl-tRNA synthetases such as leucyl-tRNA synthetase, whose inhibition by the benzoxaborole epetraborole improved host survival, highlighting its potential as a new therapeutic strategy for multidrug-resistant (MDR) S. pneumoniae. Together, these findings demonstrate that pneumococcal persistence in the CNS depends on metabolic flexibility, endogenous biosynthesis, cell-envelope maintenance, and oxidative stress management in a nutrient-restricted environment. This fitness map advances our understanding of pneumococcal adaptation in the CNS and identifies promising targets for developing therapies against S. pneumoniae.