<i>Glaesserella parasuis ClpX</i> participates in stress tolerance and contributes to bacterial pathogenicity

The <i>ClpX</i> 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 <i>Glaesserella parasuis</i> remains unclear. In this study, we used the clinically isolated serotype 5 CF7066 strain (wild type) to investigate the effects of <i>ClpX</i> on the external environmental stress response of <i>G. parasuis</i> by constructing <i>ClpX</i> mutant (<i>Δ-ClpX</i>) and complemented strains. Our data indicated that the viable bacterial count of <i>Δ-ClpX</i> was significantly lower than that of the wild type under high temperature, osmotic pressure, and oxidative stress conditions. Moreover, the <i>Δ-ClpX</i> strain exhibited extreme sensitivity to complement-mediated killing compared with the wild-type strain, whereas its serum-resistance ability was largely restored after <i>ClpX</i> gene complementation. Additionally, <i>ClpX</i> 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 <i>ClpX</i> as a novel virulence gene in <i>G. parasuis</i>, contributing to a deeper understanding of the pathogenic mechanisms employed by this bacterium.<b>IMPORTANCE</b>The <i>ClpX</i> gene in bacteria is crucial for adapting to environmental stresses and is linked to pathogenicity. However, its role in conferring stress resistance in <i>Glaesserella parasuis</i> is unclear. This study investigates the impacts of <i>ClpX</i> on <i>G. parasuis</i>' stress response by creating a <i>ClpX</i> mutant (<i>Δ-ClpX</i>) and complemented strain. The <i>Δ-ClpX</i> 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 <i>ClpX</i> complementation. <i>ClpX</i> knockdown reduced the wild-type strain's pathogenicity in mice and pigs, which was largely restored by gene complementation. Thus, <i>ClpX</i> is identified as a novel virulence factor in <i>G. parasuis</i>, enhancing our understanding of its pathogenic mechanisms.