| Streptococcus suis serotype 2 is a worldwide causative agent of many forms of swine infection and is also recognized as a zoonotic agent causing human disease,including meningitis.Two large-scale outbreaks of severe SS2 epidemics occurred in China in 1998 and 2005 that posed serious concerns to public health and challenged the conventional conception.Since the discovery,that an infection of SS2 could cause human meningitis,more than 1500 human cases worldwide have been recorded.In particular,people with low immunity are seriously threatened.In Vietnam,Thailand,SS became the main pathogen casued meningitis.It is widely known that disruption of the BBB occurs in various meningitis pathogens.The blood-brain barrier(BBB)is composed of brain capillary endothelial cells and has an important role in maintaining homeostasis of the brain separating the blood from the parenchyma of the central nervous system(CNS).The mutant library of transposon and SS could be used for virulence factor screening.and studying the mechanism of SS 2 through BBB,1 A novel suicide shuttle plasmidfor Streptococcus suis serotype2 gene mutationTo provide a forward-genetics technology for finding virulent phenotype-related genes in SS,we constructed a novel temperature-sensitive suicide shuttle plasmid,pMar4s,which contains the Himar1 system transposon,TnYLB-1,and the Himar1 C9 transposase from pMarA and the repTAstemperature-sensitive fragment from pSET4s.The kanamycin(Kan)resistance gene was in the TnYLB-1 transposon.Temperature sensitivity and Kan resistance allowed the selection of mutant strains and construction of the mutant library.The SS2 mutant libraries were successfully constructed using the pMar4s plasmid.Southern bolt results revealed large variability in transposon insertion sites and that the library could be used for phenotype alteration screening.2 New putative virulence factors of Streptococcus suis serotype 2 involved in penetration into the blood-brain barrierSS have the ability to adhere to and invade human brain microvascular endothelial cells(hBMEC)forming the blood-brain barrier.In this study we describe the screening of a mutant library,produced by insertion of transposon TnYLB-1 into the chromosome of SS strain ZY05719,for mutants that are less able to interact with hBMEC.Both qualitative and quantitative screening assays were used to identify poorly invasive mutants.TnYLB-1 insertion sites from 10 poorly invasive mutants were sequenced and characterized.The mutants were selected to interact with hBMEC in the vitro BBB model,ZY05719 as a positive control,the results show that decreased the transcytosis of these mutants.We speculate that the insertion of the transposon TnYLB-1 inactivates the insertion site,that is,the 10 genes may be virulent factors that affect SS2-induced meningitis.3 Regulation of serine/threonine protein kinase expression in Streptococcus suis serotype 2 impacts blood-brain barrier penetrationWe have produced a poorly invision and transcytosis mutants of wild-type pathogenic strain ZY05719 and the gene is serine/threonine protein kinase(stk).Therefore,we hypothesized that the stk gene is the meningitis related virulence fator of SS2.We have described that the pathogen SSencodes a single membrane-associated,serine/threonine kinase(STK)that is important for virulence of this bacteria.In this study,in order to avoid the influence of TnYLB-1,we constructed the gene deletion strain Astk and the complement strains CAstk.Adhesion of and transcytosis of hBMEC by ?stk was more poorly than ZY05719.Astk and ZY05719 induced marked morphological changes in hBMEC.These findings suggest that ?stk and ZY05719 induced actin cytoskeletal reorganization of the host cells.In addition,it was observed that ZY05719 and Astk altered the level of claudin-5 protein in bEnd.3 cells,and these changes were accompanied by a decrease in the transendothelial electrical resistance.Immunocytochemistry with antibodies against claudin-5 showed fragmented patterns of claudin-5 in SS-treated bEnd.3 cells compared to the control and more than in ?stk-treated bEnd.3 cells.Infection in BALB/c mice by ZY05719 can cause bacteremia and it was detected bacteria in CSF.The colonization capacity of Astk in the mouse was significantly reduced.Astk was quickly cleared in the blood and there was no bacteria in CSF.In this study,we show that infection with STK-deficient SS strains resulted in decreased bacteremia.Also STK-deficient SS2 showed decreased ability to invade the brain endothelium,but SS2 itself had a low invasion rate.It did not affect the ability of SS2 to alter the cytoskeleton morphology in hBMEC.However,the ability of SS2 destroyed claudin-5 became weaker,so we speculate that SS2 penetrated into BBB by the paracellular pathway.4 Identification of serine/threonine kinase substrates in Streptococcus suis serotype 2Protein phosphorylation is essential for the regulation of cell growth,division,and differentiation in both prokaryotes and eukaryotes.We have described that the pathogen SSencodes a single membrane-associated,serine/threonine kinase(STK)that is important for virulence of this bacterium.In this study,we used a combination of phosphopeptide enrichment and mass spectrometry to enrich and identify serine(Ser),threonine(Thr)and tyrosine(Tyr)phosphopeptides of SS2.We identified that 41 serine/threonine phosphopeptides,corresponding to 31 proteins.A comparison of Ser/Thr/Tyr phosphopeptides identified from the STK expressing strains to the isogenic stk mutant indicates that 14 proteins are potential substrates of the SS2 STK enzyme.Collectively,these studies provide a novel approach to identify serine/threonine kinase substrates for insight into their signaling in human pathogens like SS2. |
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