Phosphorylation of RpoD upregulates suilysin and enhances Streptococcus suis virulence

Streptococcus suis ( S. suis ) is a prevalent zoonotic pathogen that severely threatens the swine industry and human public health. Suilysin (SLY), a major pore-forming toxin of S. suis , is indispensable for bacterial hemolytic activity and systemic pathogenicity, yet the upstream regulatory mechanism controlling SLY production remains unclear. Serine/threonine kinase (STK) has been characterized to modulate bacterial physiology and virulence by phosphorylating multiple substrate proteins. Our previous phosphoproteomics analysis indicated that the housekeeping sigma factor RpoD was a potential STK substrate. In vitro phosphorylation assays combined with mass spectrometry, co-immunoprecipitation and site-directed mutagenesis confirmed that STK directly phosphorylated RpoD at Thr-208 residue. Phenotypic tests revealed that RpoD phosphorylation had no obvious impact on bacterial growth and biofilm formation but specifically promoted the thermotolerance and hemolytic activity of S. suis . Notably, the rpoD overexpression mutant strain SC19-rpoD significantly upregulated the transcription and protein production of SLY, whereas the phosphorylation-deficient mutant SC19-rpoD T208A abolished this promotion effect, indicating that Thr-208 phosphorylation is required for sly upregulation, although the underlying molecular mechanism remains to be fully defined. Furthermore, mouse infection models demonstrated that the SC19-rpoD T208A mutant exhibited alleviated splenic pathological injury, reduced bacterial loads in multiple organs, and significantly improved host survival, indicating that the loss of RpoD phosphorylation impairs S. suis virulence. Collectively, these findings demonstrate that STK-mediated phosphorylation of RpoD at Thr-208 is required for sly upregulation and contributes to the S. suis pathogenesis.