Organelle remodeling is critical for cellular homeostasis, but host factors that control organelle function during microbial infection remain largely uncharacterized. Here, a genome-scale CRISPR/Cas9 screen in intestinal epithelial cells with the prototypical intracellular bacterial pathogen Salmonella led us to discover that type I interferon (IFN-I) remodels lysosomes. Even in the absence of infection, IFN-I signaling modified the localization, acidification, protease activity and proteomic profile of lysosomes. Proteomic and genetic analyses revealed that multiple IFN-I-stimulated genes including Ifitm3 , Slc15a3 , and Cnp contribute to lysosome acidification. IFN-I-dependent lysosome acidification stimulated intracellular Salmonella virulence gene expression, leading to rupture of the Salmonella -containing vacuole and host cell death. Moreover, IFN-I signaling promoted in vivo Salmonella pathogenesis in the intestinal epithelium, where Salmonella initiates infection. Our findings explain how an intracellular bacterial pathogen co-opts epithelial IFN-I signaling. We propose that IFN-I control of lysosome function broadly impacts host defense against diverse viral and microbial pathogens.### Competing Interest StatementThe authors have declared no competing interest.

Functional remodeling of lysosomes by type I interferon modifies host defense