participates in stress tolerance and contributes to bacterial pathogenicity.

The ClpX gene in bacteria, which plays a key role in adaptation to various environmental stresses, is associated with pathogenicity. However, its role in conferring external environmental stress resistance in Glaesserella parasuis remains unclear. In this study, we used the clinically isolated serotype 5 CF7066 strain (wild type) to investigate the effects of ClpX on the external environmental stress response of G. parasuis by constructing ClpX mutant (Δ-ClpX) and complemented strains. Our data indicated that the viable bacterial count of Δ-ClpX was significantly lower than that of the wild type under high temperature, osmotic pressure, and oxidative stress conditions. Moreover, the Δ-ClpX strain exhibited extreme sensitivity to complement-mediated killing compared with the wild-type strain, whereas its serum-resistance ability was largely restored after ClpX gene complementation. Additionally, ClpX knockdown significantly attenuated the pathogenicity of the wild-type strain, and gene complementation largely recovered its phenotype in mouse and pig models. Taken together, our findings reveal ClpX as a novel virulence gene in G. parasuis, contributing to a deeper understanding of the pathogenic mechanisms employed by this bacterium.IMPORTANCEThe ClpX gene in bacteria is crucial for adapting to environmental stresses and is linked to pathogenicity. However, its role in conferring stress resistance in Glaesserella parasuis is unclear. This study investigates the impacts of ClpX on G. parasuis' stress response by creating a ClpX mutant (Δ-ClpX) and complemented strain. The Δ-ClpX had reduced viability under heat, osmotic pressure, and oxidative stress conditions. It was also more sensitive to complement-mediated killing but regained serum resistance with ClpX complementation. ClpX knockdown reduced the wild-type strain's pathogenicity in mice and pigs, which was largely restored by gene complementation. Thus, ClpX is identified as a novel virulence factor in G. parasuis, enhancing our understanding of its pathogenic mechanisms.