The Mechanism Of STK/STP Phosphorylation Affecting Biological Characteristics And Virulence Of Streptococcus Suis Serotype 2

Streptococcus suis serotype 2(SS2)is an important zoonotic pathogen,which causes streptococcal disease in pigs.The main characteristics of diseased pigs are sepsis,meningitis and arthritis.This bacterium usually causes streptococcal toxic shock-like syndrome(STSS),meningoencephalitis,and endometritis in humans.In 1998 and 2005,two outbreaks of swine streptococcus occurred in Nantong,Jiangsu and Ziyang,Sichuan,causing significant economic losses and casualties.The regulation of phosphorylation modification is an important regulation mechanism in prokaryotic and eukaryotic organisms.There are eukaryotic-like serine/threonine kinase(STK)in prokaryotes,which can affect the growth and metabolism of bacteria,anti-stress ability,and bacterial division.Previous research in our lab found that STK also exists in SS2 and 22 potential phosphorylation substrates of STK were identified by phosphoproteomic analysis.But the effect of STK phosphorylation regulation on SS2 biological characteristics and virulence have not been reported.In this study,the phosphorylation substrate of SS2 STK,phosphoglucosamine mutase(GlmM)and the PTS system beta-glucoside-specific EIIBCA(BglF)were the research objects.And the mechanism by which GlmM and BglF phosphorylation modification regulate the growth metabolism and virulence of SS2 were clarified.The research results will help to further clarify SS2 pathogenesis by deciphering the regulation mechanism of SS2 STK phosphorylation.1.The mechanism of Streptococcus suis serotype 2 GlmM phosphorylation modification affecting its biological characteristics and virulenceIn this study,in vitro phosphorylation experiments and IP phosphorylation experiments verified the results of earlier SS2 phosphoproteomics,confirmed GlmM is the phosphorylation substrate of STK and we found that GlmM Ser-101 is its phosphorylation site and GlmM phosphorylation level can be affected.Compared with the wild-type strain ZY05719,the deficient strain ΔglmM and the point mutant strain CΔglmM S101A showed abnormal cell morphology,such as increased cell size and loss of capsule.The anti-oxidative stress ability,anti-lysozyme killing ability,whole blood survival rate,anti-neutrophil killing ability,and the pathogenicity in mouse infection model of ΔglmM and CΔglmM S101A significantly decreased.GlmM enzyme activity was determined by coupling GlmU acetyltransferase chemical colorimetric method.Compared with the wild-type strain ZY05719,GlmM enzyme activity in Δstk and CΔglmM S101A decreased by 3.2-fold and 6.80-fold respectively,which indicates that STK can phosphorylate GlmM to regulate GlmM enzyme activity.The Morgan-Elson reaction method was used to determine the N-acetylhexosamine content at the reducing terminus of the peptidoglycan chain.Compared with the wild-type strain ZY05719,the peptidoglycan content of ΔglmM,Δstk and CΔglmM S101A reduced by 2.2-fold,1.6-fold and 1.7-fold,respectively.It indicates that the phosphorylation modification of GlmM can affect SS2 cell wall peptidoglycan.The decreased phosphorylation level of GlmM induces the reduction of GlmM enzyme activity,which inhibits the cell wall peptidoglycan synthesis and changes the above mentioned biological characteristics of ΔglmM and CΔglmM S101A,in order to decrease the pathogenicity.2.The mechanism of Streptococcus suis serotype 2 BglF phosphorylation modification affecting its biological characteristics and virulenceIn this study,in vitro phosphorylation experiments confirmed that BglF is the phosphorylation substrate of STK,and its phosphorylation site is Ser-462.Compared with the wild-type strain ZY05719 and the complementary strain CΔbglF,the growth metabolism rate of the deficient strain ΔbglF decreased significantly.The reason of this phenomenon was that the utilization rate of β-glucoside and other sugars in ΔbglF reduced.In vitro biofilm formation ability,blood survival rate,anti-phagocytosis ability,adhesion and invasion ability,and mice infection model pathogenicity of ΔbglF all significantly reduced.BglF is an important component of PTS.In addition to affecting the growth and metabolism of bacteria,BglF can also affect the transcription level of some TCSs in bacteria,thereby regulating the virulence.Based on this,the transcription levels of TCS in the the wild-type strain ZY05719,the deficient strain ΔbglF and the complementary strain CΔbglF were compared.CovR mRNA levels were significantly up-regulated;CiaR/CiaH,SalK/SalR,Ihk/Irr,and VirR/VirS mRNA levels were down-regulated,of which the transcription levels of CiaR/CiaH,Ihk/Irr,and VirR were significantly down-regulated;While the mRNA levels of the above mentioned genes in the complementary strain CΔbglF recovered to the level of the wild-type strain.It indicated that BglF can affect the transcription levels of TCS,thereby further affecting virulence.The above studies show that BglF phosphorylation by STK at Ser-462 can affect the growth and metabolism of SS2 and TCS transcription levels to further regulate SS2 virulence.3.The effect of Streptococcus suis type 2 STK on protein phosphorylation modification and function of bEnd.3 cellsIn this study,bEnd.3 cells were used as the blood-brain barrier model,and SILAC comparative quantitative proteomics were performed to determine the differentially expressed proteins between SS2 ZY05719 and Δstk infected bEnd.3 cells,in order to explore the role of STK in penetrating the blood-brain barrier.Compared with the cells infected with the wild-type strain ZY05719,241 proteins were significantly up-regulated and 81 proteins were significantly down-regulated in cells infected with the deficient strainΔstk.Proteomics data revealed that significant changes in related proteins involved in the RNA process,host cytoskeleton,tight junction destruction,and immune response.Further analyses found that many differential proteins significantly down-regulated in Δstk-infected cells are related to phosphorylation,indicating that SS2 STK can affect the cellular protein phosphorylation process of the host during the process of penetrating the blood-brain barrier.At the same time,it was found that SS2 STK can activate the MAPK,PI3K-Akt,and NFκB signaling pathways that are closely related to the development of meningitis by affecting the expression levels of STK4,PKA,and Akt proteins in host cells,and may promote SS2 to penetrate the blood-brain barrier.The protein level and transcription levels of some differentially expressed proteins were verified by Westernblot and q-RT PCR experiments,confirming the reliability of the proteomics results.The results of this study are helpful to further decipher the mechanism of SS2 STK affecting penetrating the blood-brain barrier.In summary,GlmM phosphorylation modification by SS2 STK can affect the phosphorylation level of GlmM and GlmM enzyme activity,thereby regulating the cell wall synthesis,in order to control SS2 biological characteristics and virulence.BglF in the PTS system is also a phosphorylation substrate of SS2 STK.The phosphorylation modification of this substrate mainly affects growth and metabolism of SS2 and the transcription levels of some TCSs,so that regulate biological characteristics and SS2 virulence.SS2 STK can activate the MAPK,PI3K-Akt,and NFκB signaling pathways that are closely related to the development of meningitis by affecting the expression levels of STK4,PKA,and Akt proteins in host cells,and may promote SS2 to penetrate the blood-brain barrier.