A Preliminary Study On The Phosphorylation Mechanism Of The Orphan Regulator CovR Of Streptococcus Suis Type 2 05ZYH33

Streptococcus suis(S.suis)is a Gram-positive facultative anaerobe,and is an important zoonosis pathogen.Among the various characterized Streptococcus suis serotypes,Streptococcus suis serotype 2(S.suis 2)is the most widely distributed and most pathogenic one,which causes meningitis,sepsis,endocarditis and Streptococcal toxic shock syndrome(STSS).It not only brings huge economic losses to the pig industry,but also seriously threatens public health security.Unfortunately,the specific pathogenesis of Streptococcus suis has not yet been fully elucidated.Our previous study found that CovR,as a global negative regulator,plays an important role in the pathogenicity of S.suis 2 Chinese virulent strain 05ZYH33.However,since CovR is an orphan regulation factor in S.suis,how it is phosphorylated and activated,so that the regulation of the expression of downstream genes is still unclear.Therefore,further in-depth research is urgently needed to address it.Recent studies have found that CovR is phosphorylated by serine/threonine kinase(STK)in Streptococcus such as Streptococcus pneumoniae.This study investigated whether CovR is also phosphorylated by the STK protein and explored the potential mechanism.The specific content is as follows:1.Bioinformatics Analysis of CovR Protein Encoding Gene SSU05₁732Bioinformatics analysis of SSU05₁732 found that the gene consists of 687 nucleotides.The encoded protein CovR protein has no signal peptide and transmembrane region.It has a typical CovR protein domain.The tertiary structure of CovR protein is composed of ?-helix,?-sheetand and random line composition.2.Prokaryotic Coexpression and Detection of Phosphorylation of CovR by STKAccording to the covR and stk gene sequences,PCR amplification primers were designed to amplify the target gene.The pCDFDuet-1::covR and pCDFDuet-1::covR/stk expression vectors were constructed.The CovR recombinant protein was obtained by prokaryotic expression and purification and,named CovR1 and CovR2.Phosphorylation detected of the obtained recombinant CovR protein revealed that the CovR2 protein co-expressed with STK had a significant phosphorylation signal,while the phosphorylation signal was not detected in the CovR protein alone,suggesting that co-expression of STK and CovR may cause CovR to occur phosphorylation.3.Identify CovR phosphorylation sitesThe phosphorylation of CovR protein co-expressed with STK was detected by mass spectrometry.The threonine residues at positions 45,148,150,159,168,194 and 219,the serine residue at positions 40,172,and 215 and the 225th tyrosine residue were phosphorylated.The covR expression plasmids that were mutated in the above three types of amino acid residues were further constructed to transform E.coli BL21(DE3),and the mutant protein was induced to express.Phosphorylation detected of threonine revealed that the phosphorylation signal of CovRT(threonine mutation)and CovRA(threonine/serine/tyrosine mutation)disappeared;serine phosphorylation detected results showed CovRS(serine mutation),phosphorylation of CovRA The disappearance of the signal;tyrosine phosphorylation detected results showed that CovRY(tyrosine mutation),CovRA phosphorylation signal disappeared.This experiment confirmed the phosphorylation of STK and CovR,and the phosphorylation sites of CovR were identified.And our work provides theoretical guidance for the effect of CovR phosphorylation on bacterial pathogenicity.To provide theoretical guidance for further study of the effect of CovR phosphorylation on bacterial pathogenicity.Additionally,we firstly reported the tyrosine kinase activity of the S.suis STK.