Title: Pseudomonas aeruginosa essential gene perturbations confer vulnerability to the mammalian host environment
Multi-drug resistant Pseudomonas aeruginosa causes highly morbid infections that are challenging to treat. While antibiotics reduce bacterial populations during infection, the host environment also plays a key role in inhibiting and eliminating pathogens. Identifying genetic targets that create vulnerabilities to the host environment may uncover strategies to synergize with nutrient limitation or inherent immune processes to clear bacterial infections. Here, we screened a partial knockdown library targeting P. aeruginosa essential and conditionally essential genes in a murine pneumonia model to identify genes with increased vulnerability in the host environment. We found that partial CRISPR interference (CRISPRi) knockdown of 178 genes showed significant fitness defects in mice relative to axenic culture. We validated two important outliers: ispD, encoding a key enzyme in isoprenoid precursor biosynthesis, and pgsA, encoding an enzyme involved in phospholipid synthesis that is strongly upregulated in human infections. Partial knockdown of both genes showed decreased virulence in a mouse survival assay but had little impact on in vitro growth. The use of CRISPRi screening to uncover genetic vulnerabilities represents a promising strategy to prioritize antibacterial targets that interact with the host environment.