Abstract

ST6GALNAC5 is a novel host factor essential for Glaesserella parasuis infection. • Its knockout reduces bacterial adhesion by 50% and invasion by 90%. • Host cell viability is rescued from 18% to 55% without ST6GALNAC5 . • Transcriptome reprogramming blunts inflammatory responses after knockout. • Proposes a host-directed therapeutic strategy against Glässer's disease. Glaesserella parasuis ( G. parasuis ), the causative agent of Glässer’s disease in pigs, relies on adhesion to host epithelial cells and induction of inflammation for pathogenesis. However, the potential role of ST6GALNAC5 , a host sialyltransferase involved in glycan modification, in G. parasuis infection remains unclear. Using the porcine kidney epithelial cell line LLC-PK1, we investigated the function of the host sialyltransferase gene ST6GALNAC5 during G. parasuis infection. Infection significantly upregulated ST6GALNAC5 mRNA expression. CRISPR/Cas9-mediated knockout of ST6GALNAC5 reduced bacterial adhesion to and invasion of host cells. Compared to wild-type cells, ST6GALNAC5 -KO cells exhibited higher proliferation and survival rates post-infection. Transcriptomic analysis revealed that ST6GALNAC5 knockout alone altered host cell pathways: DNA replication and cell cycle pathways were activated, while antigen processing and presentation and Toll-like receptor signaling pathways were suppressed. Consistently, upon infection, knockout cells showed significantly reduced expression of pro-inflammatory cytokines TNF-α , IL-8 , IL-6 , and IL-11 . These findings demonstrate that ST6GALNAC5 promotes G. parasuis infection by enhancing bacterial adhesion, invasion and potentiating host inflammatory responses. Therefore, ST6GALNAC5 may serve as a potential host-directed target for controlling G. parasuis infection.