Genome-wide CRISPR screening identifies RAB8B as a candidate target for improving disease resistance against Glaesserella parasuis in pigs

Glaesserella parasuis ( G. parasuis ) is a major pathogen causing Glässer’s disease in pigs, leading to substantial economic losses in the global swine industry. However, the host genetic factors governing its pathogenesis remain poorly understood. Here, we performed a genome-wide CRISPR-Cas9 knockout screen in porcine LLC-PK1 cells and identified RAB8B as a critical host factor promoting G. parasuis infection. Functional validation showed that RAB8B knockout significantly improved host cell viability, reduced bacterial adhesion and invasion, and preserved epithelial barrier integrity after infection. Transcriptomic and mechanistic analyses revealed that G. parasuis infection triggered pathological and sustained hyperactivation of the TLR5/NF-κB signaling pathway. RAB8B depletion specifically suppressed this sustained activation at the late stage of infection. This effect attenuated the production of key pro-inflammatory cytokines ( IL8 , IL6 , TNF-α ) and promoted cell survival. This study demonstrates that RAB8B exacerbates G. parasuis infection by maintaining pathological sustained activation of the TLR5/NF-κB pathway. It uncovers a novel host-directed regulatory mechanism and identifies RAB8B as an in vitro candidate gene meriting further in vivo evaluation for improving disease resistance in pigs. These in vitro findings provide preliminary insights that may inform future research on pig health management.