The Influence Of Phosphorylation Modification On The Regulatory Function Of LacI Family Transcription Factor In Streptococcus Suis Serotype 2

Streptococcus suis serotype 2（SS2）is an important zoonotic pathogen.People infected with SS2 caused streptococcal toxic shock syndrome（STSS）and meningoencephalitis,pigs infected with SS showed sepsis,arthritis,endocarditis,pneumonia,and meningitis.The worldwide epidemic of SS2 infection disease has brought a huge threat to public health and pig industry.SS2 causes meningitis in humans and pigs because of its strong ability to cross the blood-brain barrier（BBB）,but the mechanism is still unclear.The preliminary research of our group found that the serine-threonine kinase gene（stk）deletion strainΔstk showed a weaker ability to cross the BBB and decreased bacterial virulence than the SS2 wild-type strain.Phosphorylated proteomics analysis found that the phosphorylation modification of a Lac I family transcription factor（Lac）inΔstk disappeared,compared with the SS2 wild strain.Mass spectrometry results showed that the phosphorylation modification site is threonine 29.Can Lac be directly phosphorylated by STK?What are the biological function and regulatory mechanisms of Lac?How does the phosphorylation modification affect the regulatory function of Lac?This article has launched a series of researches on the above scientific issues.1 The phosphorylation experiment of SS2 Lac I family transcription factor in vitroIn this study,the expression vectors of the STK kinase domain truncated protein,the wild-type Lac full-length protein,and the mutant Lac T29A full-length protein with the 29^ththreonine mutated to alanine were reconstructed,based on bioinformatics analysis.The recombinant STK kinase domain truncated protein,the wild-type Lac full-length protein,and the point mutant Lac T29A protein was expressed and purified in a prokaryotic system.The above recombinant protein was used for phosphorylation experiments in vitro.The results showed that Lac can be used as a direct substrate protein to be phosphorylated by STK in vitro,and the phosphorylation modification site is the 29^th threonine.2.The biological function of LacIn this study,the SS2 gene deletion strainΔlac and complementary strain CΔlac were constructed by using the wild-type SS2 virulent strain ZY05719 as the parent strain.Compared with ZY05719,the related phenotypes ofΔlac and CΔlac were tested to explore how Lac affects the biological characteristics of SS2.The experimental results showed that the ability ofΔlac to invade h BMEC was significantly enhanced,while the ability to resist phagocytosis of RAW264.7,adapt to an acidic environment and antioxidant capacity were significantly reduced,and the function of CΔlac was partially restored to the level of wild strain.The differentially expressed genes ofΔlac were analyzed,and the results showed that the deletion of Lac caused significant changes in the transcription levels of a large number of metabolism-related genes,which suggested that Lac could enhance the virulence and resilience of SS2 by regulating metabolism.3.The transcription regulation mechanism of Lac in vitroIn this study,the expression vector of the mutant Lac T29E full-length protein with the29^th threonine mutated to glutamic was reconstructed by using site-directed mutagenesis.The simulated phosphorylated recombinant protein Lac T29E was obtained through prokaryotic expression and purification.The recombinant proteins Lac and Lac T29E were incubated with the Lac promoter region DNA fragments for the EMSA experiment.The results showed that Lac could regulate the transcription level of its gene by binding to its promoter region directly,and the phosphorylation modification of Lac prevented its binding to the promoter region of the target gene.In summary,this study found that Lac could be phosphorylated by STK as a direct substrate in vitro,Lac could enhance the stress resistance and bacterial virulence of SS2 by regulating the transcription of metabolism-related genes,Lac could self-regulate the transcription level of its gene by binding to its promoter region DNA directly,the phosphorylation modification could prevent Lac form directly binding to the promoter region of its target gene.This study preliminarily clarified the biological function of Lac and the effect of its transcriptional regulation function by phosphorylation modification,which laid a foundation for further analysis of the mechanism of SS2 virulence regulation.