| Streptococcus suis(S.suis,SS)is an important zoonotic pathogen causing meningitis,septicemia,and endocarditis,threatening the global pig industry and public health.Of the 35 serotypes,S.suis serotype 2(S.suis 2)is the most prevalent and virulent.In China,two major SS2 infection outbreaks occurred in Haian of Jiangsu Province,1998 and Ziyang of Sichuan Province,2005.Many virulence factors have been found to play important roles in SS2 infection,but it is still far from elucidating the pathogenesis of SS2.Exploration of novel virulence factors and investigation of new functions of already discovered virulence factors are still important for better understanding of SS2 pathogenesis.Eukaryotic-like Ser/Thr protein kinase(eSTK)and phosphatase(eSTP)are related to pathogenicity of some bacterial species,and both have wide substrate profiles.eSTK(Stk)and eSTP(Stpl)of SS2 were reported to play important roles in pathogenesis.However,substrate profiles of Stk and Stpl remain unknown.SS2 foldase(PrsA)suggested as a putative substrate of Stk needs further confirmation.PrsA can regulate secretion of virulence factors and participate in pathogenesis of Gram-positive bacteria.Our previous studies show that PrsA was pro-inflammatory and cytotoxic.But the specific roles of PrsA in SS2 pathogenesis and virulence factor secretion require further investigation.This study aimed to:(1)explore the effect of stk and stpl deletion on pathogenicity of a virulent Streptococcus suis 2 strain;(2)identify the substrates of Stk and Stpl through combination of gene knockout and phosphoproteomic methods,and analyze the function of the substrates using bioinformatic methods;(3)investigate whether PrsA is phosphorylated by Stk,and involved in pathogenicity and secretion of virulence factors of SS2.1.Deletion of stk and stpl attenuated S.suis type 2 virulenceThe stk and stp1 deletion mutants ?stk and ?stpl were constructed by homologous recombination.Both ?stk and ?stp1 strains had longer chains.Deletion of stk did not significantly influence growth of S.suis type 2,but deletion of stp1 affected growth.Adhesion to epithelial cells of both ?stk and ?stpl were significantly higher than their parent strain,while their invasion and survival in macrophages and pig whole blood were significantly attenuated.?stk had significantly enhanced hemolytic activity compared to the parent strain,while ?stpl did not.Biofilm formation of stk and stp1 deletion strains was significantly enhanced.Both deletion mutants showed significantly reduced virulence to mice compared to their parent strain.Western blotting showed that the adhesin glyceraldehyde-3-phosphate dehydrogenase(GAPDH)in secreted and surface-associated fractions was increased in ?stk and ?stpl strains which might be responsible for their enhanced adhesion.There was increased secretion of suilysin(SLY)in the secreted fraction of ?stk which might be related to its enhanced hemolytic ability.Transmission electron microscopy showed that ?stk had thinner capsular layers(CPS),while ?stpl had thicker CPS than that of the parent strain,and these might be responsible for decreased survival of ?stk and increased survival of ?stpl in RAW264.7 macrophages.We discovered for the first time that Stk and Stp1 are involved in the secretory regulation of extracellular proteins and CPS synthesis in S.suis type 2.2.Comparative phosphoproteomic analysis of ?stk or ?stpl with the parent strainEnzymatic digestion,TiO2 phosphorylated peptide enrichment and LC-MS/MS massspectrometry analysis were conducted using the whole bacterial proteins of stk or stp1 deletion mutants and their parental strain.A total of 3 15 phosphorylated peptides were identified,corresponding to 206 phosphorylated proteins and containing 453 phosphorylation sites.Phosphorylation in S.suis type 2 was mostly on serine and threonine,and less on tyrosine with the ratio of Phos-Thr at 48.3%,Phos-Ser at 47%,and Phos-Tyr at 4.64%.?stk had 47 significantly changed phosphorylation sites compared to the parent strain,of which 1 was significantly up-regulated and 46 were significantly down-regulated.There were also 68 sites only modified in the parent strain.?stp1 had 34 significantly different phosphorylation sites compared to the parent strain,with 16 upregulated and 18 downregulated.Bioinformatics analysis indicated that phosphor-modifications by Stk and Stpl influenced various biological processes in SS2,such as biofilm formation,protein secretion and cell division.Eight proteins were identified as potential co-regulated substrate candidates of both Stk and Stp1.Among them,modification of 199Thr of a cell-division protein DivIVA and 116Thr of an RN A-binding protein Jag were significantly down-regulated in ?stk,but significantly up-regulated in ?stp1.The N-terminal kinase domain of Stk,Stp1,DivIVA,Jag and a mutated Jag with its 116 threonine replaced by alanine(designated as Jag-116A)were expressed.In vitro phosphorylation analysis showed that both DivIVA and Jag could be phosphorylated by Stk and dephosphorylated by Stp1.But the Jag-116A could not be phosphorylated by Stk,confirming that the 116 threonine was the phosphorylation site of Jag.3.PrsA participated in S.suis type 2 pathogenesis by regulating translocation of selected virulence factors.PrsA was previously reported as a substrate candidate of Stk.In the present study,we found that PrsA could not be phosphorylated by Stk.A prsA deletion mutant(?prsA)and a complemented strain(C?prsA)were constructed to further explore the roles of PrsA in SS2 infection.Deletion of prsA caused increased bacterial chain length and decreased growth ability.?prsA had enhanced bacterial adhesion to epithelial cells,but its invasion ability to epithelial cells,survival ability in macrophages and whole blood,and virulence to mice were significantly attenuated.All these phenotypes of ?prsA could be largely reversed to the levels of the parent strain by gene complementation.Western blotting analysis revealed that suilysin was markedly reduced both in surface-associated and secreted fractions of ?prsA,which might be responsible for reduced hemolytic activity.Two important adhesins GAPDH]and enolase were significantly increased in both surface-associated and secreted fractions of ?prsA.Adhesion of?prsA to epithelial cells was inhibited by polyclonal antibodies against GAPDH and enolase.These results show that PrsA could regulate secretion or translocation of virulence factors,thereby affecting the microbe-host interaction and participating in the pathogenesis of SS2.In summary,Stk and Stpl are involved in S.suis type 2 adhesion and invasion,survival in macrophages,and secretion of virulence factors,thus contributing to virulence.We provided modification substrate profiles of Stk and Stp1 for further research.DivIVA and Jag proteins were verified as co-regulated substrates of Stk and Stp1.S.suis 2 can regulate translocation of virulence factors through PrsA,thus affecting its interaction with the host. |
PDF Full Text Request