Role of ClpX and ClpP in Streptococcus suis serotype 2 stress tolerance and virulence

Streptococcus suis has received increasing attention for its involvement in severe infections in pigs and humans; however, their pathogenesis remains unclear. ClpX and ClpP, two subunits of the ATP-dependent caseinolytic protease Clp, play key roles in bacterial adaptation to various environmental stresses. In this study, a virulent S. suis serotype 2 strain, ZY05719, was employed to construct clpX and clpP deletion mutants (ΔclpX and ΔclpP, respectively) and their complementation strains. Both ΔclpX and ΔclpP displayed significantly reduced adaptability compared with the wild-type strain, evident through several altered phenotypes: formation of long cell chains, tendency to aggregate in culture, and reduced growth under acidic pH and H₂O₂-induced oxidative stress. ClpP and ClpX were required for the optimal growth during heat and cold stress, respectively. An in vitro experiment on RAW264.7 macrophage cells showed significantly increased sensitivity of ΔclpX and ΔclpP to phagocytosis compared with the wild-type strain. Mouse infection assays verified the deletion of clpX and clpP led to not only fewer clinical symptoms and lower mortality but also to a marked attenuation in bacterial colonization. These virulence-related phenotypes were restored by genetic complementation. Furthermore, the deletion of clpX or clpP caused a significant decrease in the expression of sodA, tpx, and apuA compared with the wild-type strain, suggesting that these genes may be regulated by ClpX and ClpP as downstream response factors to facilitate the bacterial tolerance against various environmental stresses. Taken together, these results suggest that ClpX and ClpP play important roles in stress tolerance for achieving the full virulence of S. suis serotype 2 during infection.