Regulation Of The Tyrosine Kinase Gene Cps2C In Streptococcus Suis Type 2 On Capsule Synthesis And Bacterial Virulence

Streptococcus suis(S.suis)is an important zoonotic pathogen that can be transmitted to humans by contact with diseased animals and contaminated raw pork products.Capsular polysaccharide(CPS)is recognized as the most important virulence factors of S.suis.It can not only mediate bacterial adhesion and invasion but also inhibit inflammation and complement-mediated killing and phagocytosis.Studies have found that most genes encoding bacterial protein-tyrosine kinases(BY-kinases)are located on the CPS synthesis operon and participate in biosynthesis and transport of CPS.In Firmicutes,BY-kinases are composed of transmembrane activator and tyrosine kinase protein.Only when the intracellular segment of the transmembrane activator interacts with the catalytic domain of the tyrosine kinase,BY-kinases can have complete kinase activity.In Streptococcus suis serotype 2(S.suis 2),transmembrane activator and tyrosine kinase are encoded by the cps2 B and cps2 C,respectively.Preliminary research in our lab showed that the protein tyrosine kinase Cps2 C expressed alone in E.coli BL21 could not be phosphorylated,but Cps2 Bct C co-expressed Cps2 C and the 23 amino acids at the C-terminal of Cps2 B could be phosphorylated on serine,threonine and tyrosine residues.However,the specific biological function of the tyrosine kinase gene cps2 C in S.suis 2 is still unclear.In the present study,the cps2 C gene knockout strain was constructed to reveal the biological function of cps2 C.The main research contents and results are as follows:1.Construction and identification of cps2 C gene knockout strainThe gene knockout plasmid p UC19::cps2C containing three exogenous fragments,L,S and R,was constructed and electro-transformed into 05ZYH33 competent cells to construct ?cps2C.Ultimately,the knockout strain ?cps2C was successfully obtained by preliminary inspection with the internal primer Check In-F/R of cps2 C and further verification with combined PCR,RT-PCR,Western blot and DNA sequencing.2.Analysis of basic biological characteristics and pathogenicity of ?cps2CBy the Gram staining experiment that was constructed to observe the bacterial morphology and count the cellular chain length,it was found that the cellular chain length of ?cps2C(7.24±3.38)is significantly shorter than that of the 05ZYH33 strain(13.01±10.24),which suggests that the cps2 C maybe involve in cellular chain regulation in S.suis 2.The transmission electron microscopy observation revealed?cps2C lose the typical capsule structure,which indicated the cps2 C participates in the biosynthesis or regulation of capsular polysaccharide.The virulence of ?cps2C was detected by the bacterial challenge experiment,and it was found that the survival rate of mice injected the ?cps2C stain(10/10)is significantly higher than that of the05ZYH33(0/10),which demonstrated the virulence of the ?cps2C strain is obviously decreased.The mouse protective experiment displayed that the survival rate of immunized mice increased,which exhibited that the ?cps2C has the potential as an attenuated vaccine.3.The effect of cps2 C deletion on protein phosphorylation and transcription levels of CPS operonRT-PCR was performed to identify co-transcribed genes in cps gene cluster.The results demonstrated that the CPS operon consists of 12 consecutively transcribed genes,orf2 X and cps2A-cps2 K.The transcription level of each gene in the CPS operon of the ?cps2C strain was measured by q RT-PCR,and it was found that the cps2 B m RNA level is up-regulated approximate 2.6-fold relative to that in wild-type05ZYH33,but other genes have no significant difference with control.The results indicated that the deletion of the tyrosine kinase gene cps2 C affects the transcription level of the transmembrane activator-encoding gene cps2 B,but Cps2 C does not participate in the transcriptional regulation of other CPS synthesis-related genes in the CPS operon,which suggested that Cps2 C regulates the synthesis of CPS in a post-transcriptional manner.Western blot was used to detect the phosphorylation at serine,threonine and tyrosine of whole protein in ?cps2C,?cps2B and 05ZYH33 stain.It was found that the 30 k Da protein of 05ZYH33 rather than ?cps2C and?cps2B undergoes phosphorylation at serine,threonine and tyrosine residues,which provided experimental evidence that Cps2 C may not only have tyrosine kinase properties but also have serine and threonine kinase properties.In addition,it suggests Cps2 B is an essential activator of Cps2 C in S.suis 2.In summary,the ?cps2C knockout strain was constructed successfully.The deletion of the cps2 C contributes to loss of typical capsule structure,the shorter cellular chain length and the decreasing pathogenicity.CPS operon is composed of 12 capsular synthesis-related genes such as orf2 X and cps2A-cps2 K,and Cps2 C does not participate in the transcriptional regulation of CPS synthesis-related genes in the CPS operon except the transmembrane activator-encoding gene cps2 B.Last but not least,The tyrosine kinase Cps2 C is involved in regulation of phosphorylation at serine,threonine and tyrosine of whole protein in S.suis 2,and the Cps2 B is an essential activator of Cps2C kinase activity.