Parallel Genome‐Wide CRISPR Screens Reveal SORL1 and ZFYVE19 as Sequential Host Determinants of Salmonella Infection

To distinguish how Salmonella invades cells vs how it survives long‐term, a parallel CRISPR screening platform is developed. This approach reveals the host proteins that the bacterium exploits at different stages of infection. The study identifies SORL1 as a novel host factor for invasion and demonstrates that blocking it with an antibody effectively prevents infection, revealing a novel host‐directed therapeutic strategy. Abstract Salmonella enterica, a major cause of gastroenteritis and typhoid fever, hijacks host machinery to invade cells, and replicate within a specialized niche. While some host factors are known, a comprehensive, temporally‐resolved understanding of the host‐pathogen interface has been hindered by a lack of suitable genome‐wide methodologies. To address this, a parallel CRISPR screening platform is developed to identify host determinants for distinct infection stages. An invasion screen captured factors for bacterial entry, while a fitness screen identified factors governing long‐term survival. The screens reveal a temporal switch in host dependency, from endosomal trafficking in early infection to cell cycle and DNA damage response pathways governing host cell fitness in long‐term infection. Notably, the approach uncovers two novel host factors with stage‐specific roles, SORL1 as a mediator of bacterial invasion and ZFYVE19 as a factor supporting intracellular proliferation. Genetic disruption of SORL1 or ZFYVE19 validate these roles, leading to impaired invasion or replication, respectively. Importantly, antibody‐mediated blockade of SORL1 effectively prevented Salmonella entry, highlighting it as a novel host‐directed therapeutic target. Together, the screening strategy provides a powerful framework for the temporal dissection of host‐pathogen interactions, revealing novel biology and promising therapeutic targets. To distinguish how Salmonella invades cells vs how it survives long-term, a parallel CRISPR screening platform is developed. This approach reveals the host proteins that the bacterium exploits at different stages of infection. The study identifies SORL1 as a novel host factor for invasion and demonstrates that blocking it with an antibody effectively prevents infection, revealing a novel host-directed therapeutic strategy. Abstract Salmonella enterica, a major cause of gastroenteritis and typhoid fever, hijacks host machinery to invade cells, and replicate within a specialized niche. While some host factors are known, a comprehensive, temporally-resolved understanding of the host-pathogen interface has been hindered by a lack of suitable genome-wide methodologies. To address this, a parallel CRISPR screening platform is developed to identify host determinants for distinct infection stages. An invasion screen captured factors for bacterial entry, while a fitness screen identified factors governing long-term survival. The screens reveal a temporal switch in host dependency, from endosomal trafficking in early infection to cell cycle and DNA damage response pathways governing host cell fitness in long-term infection. Notably, the approach uncovers two novel host factors with stage-specific roles, SORL1 as a mediator of bacterial invasion and ZFYVE19 as a factor supporting intracellular proliferation. Genetic disruption of SORL1 or ZFYVE19 validate these roles, leading to impaired invasion or replication, respectively. Importantly, antibody-mediated blockade of SORL1 effectively prevented Salmonella entry, highlighting it as a novel host-directed therapeutic target. Together, the screening strategy provides a powerful framework for the temporal dissection of host-pathogen interactions, revealing novel biology and promising therapeutic targets. Advanced Science, EarlyView.