Abstract
Streptococcus suis (S. suis) causes meningitis in humans and pigs. Elongation factor P (EF-P) is an important translation factor that facilitates protein synthesis with polyproline motifs. Posttranslational modification of EF-P is required for its activity and bacterial virulence. However, the regulatory mechanism of EF-P phosphorylation in S. suis-induced blood-brain barrier (BBB) disruption remains unclear. Our phosphoproteomic analysis revealed that EF-P was the key substrate of serine/threonine protein kinase (STK) in the SC19 strain. In vitro and in vivo phosphorylation assays revealed that EF-P was phosphorylated by STK at Ser-148 and Thr-176. Compared with the wild-type SC19 strain, the efp overexpression mutant strain SC19-(pSET2-EF-P) increased ZO-1 degradation in human brain endothelial cells (hCMEC/D3), the bacterial load, and the mouse survival rate; moreover, blue diffusion in the mouse brain; and brain damage, whereas the efp point mutant strain SC19-(pSET2-EF-P-T176A) abolished this enhancement effect. Furthermore, we determined that EF-P regulated the expression of the B9H01_03990 protein, which is defined as a serine protease (SP). Recombinant SP induced ZO-1 degradation in hCMEC/D3. In addition, the B9H01_03990-deficient strain (ΔB9H01_03990) alleviated ZO-1 degradation in hCMEC/D3 cells and BBB disruption in the mouse brain and transwell infection models, whereas the complementary strain CΔB9H01_03990 reversed this reduction in BBB disruption. Our study reveals that S. suis-induced BBB disruption is mediated through a novel mechanism involving EF-P phosphorylation and the proposed STK/EF-P/SP signalling axis, providing new insight into the pathogenesis of S. suis meningitis.