Regulaton Of Serine/Threonine Kinase On The Growth And Pathogenesis Of Streptococcus Suis

Microorganism survival depend on their rapid adaption to the constantly changing environment,whose foundation derives from extracellular and intracellular signal transduction.The reverse phosphorylation catalyzed by serine/threonine kinase and its partner serine/threonine phosphatase can regulated mutiple cellular physiological activities.This regulation plays crucial roles in bacterial physiological processes.Streptococcus suis is an important zoonotic Gram-positive pathogen causing lethal infections in pigs and humans.Large outbreaks of S.suis infection in the world have not brought largely economical losses in swine industry,but induced meningitis and toxic shock syndrome in human.Study on the mechnism of S.suis growth and pathogenicity may provided foundation to lead this pathogen into control.Different from other Gram-positive bacteria with multiple STKs,the STK from S.suis is encoded by a single-copy stk gene,suggesting that STK plays crucial regulatory function in S.suis.Previous study have reported that STK played an important role in S.suis growth and pathogenicity(Zhu et al.,2014;孙雯,2014).In this study,the further researches of STK in cell growth,virulence and metabolism were carried on S.suis to explore the intensive mechnism.1.STK regulates the growth and metabolism of zoonotic S.suisIn the previous study of our lab,phenotype assays conducted by the wild-type strain SC-19 and the mutant strain(?)stk showed that STK is relevant in growth and virulence(孙雯,2014).In this study,we have constructed the complementary strain of(?)stk(C(?)stk).Using SC-19,(?)stk and C(?)stk as the experimental materials,we have compared the growth and morphology among these three strains.The results showed that(?)stk displayed impaired growth and longer chain length.To deeply investigated the reasons behind the phenotypes,we have applying whole transcriptome RNA sequencing(RNA-Seq).Numerous genes were differentially expressed in(?)stk compared with the wild-type parental strain SC-19,including 320 up-regulated and 219 down-regulated genes.Particularly,32 virulence-associated genes(VAGs)were significantly down-regulated in(?)stk.These genes involved in adherence and immune evasion,metal ion uptake,multidrug transport systems.Seven metabolic pathways relevant to bacterial central metabolism and translation are significantly repressed in(?)stk.To investigate the Ser/Thr protein kinase activity and the substrates of STK in (?)stk and SC-19,we conducted phosphoproteomic analysis.Of these 12 were differentially expressed phosphoproteins,including 9 down-regulated phosphoproteins and 3up-regulated phosphoproteins.Among 12 differentially expressed phosphoproteins detected,all of the cell division associated proteins,including Fts A,Gps B,Div IVA,and Map Z were down-regulated.Another protein Dna K,which is a classical molecular chaperone,was down-regulated,as well as the predicted RNA-binding protein Jag.Moreover,down-regulated phosphorylation was detected in the predicted periplasmic solute-binding protein Yce G,an uncharacterized protein(SSU05＿0066),and a hypothetical protein(SSU05＿0636).Phosphorylation levels of three proteins,namely translation elongation factor(EF-Tu)involved in protein synthesis,fructose-bisphosphate aldolase(FBA)and GAPDH involved in glycolysis were up-regulated.Moreover,all of these three proteins were also involved in bacteria evasion of host defense,adhesion and invasion.2.STK regulates cell division of zoonotic S.suisBased on the research of the first part,we found that STK could regulated S suis growth.Bacterial cell division is the vital step for mataining cell growth.The study of cell division mechanism has important theoretical and practical significance.To further study the mechnism of STK in cell division,we have identified the protein-protein interaction network in S.suis focused on stk with the bacterial two-hybrid system,and the result showed that the cell division protein FtsZ could interact with STK.In the present study,we using the method of ELISA analysis to verify the interaction domain between STK and FtsZ.The results revealed its N terminal(kinase domain)to N terminal(GTP binding domain)interaction between STK and FtsZ.In vitro super centrifugation showed that STK inhibits the amount of polymerized FtsZ,suggesting that the kinase domain of STK would inhibit Z ring formation through interaction with GTP binding domain of FtsZ.Both in vivo and ex vivo experiments showed that STK can phosphorylated FtsZ and the phosphorylation site located at C terminal domain of FtsZ.Moreover,we identified four phosphorylation sites of FtsZ using the method of mass spectrometry and western blot.These phosphorylation sites were T217、T233、T349 and T383,respectively.The results provide a basis for further study on what roles of phosphoablative form of FtsZ plays in cell division.This study use dye of peptidoglycan synthesis(FDAAs)for SC-19,(?)stk and C(?)stk staining,under the super resolution microscope observation,we found in the absence of stk,bacterial cells displayed abnormal chain length and morphology.These results suggested that STK is involved in maintaining correct Z-ring contraction.But the precise role of STK participating in cell division still need further explore.