Proteomic Analysis Of Cell Wall-associated Proteins Of Streptococcus Suis Serotype 2

Streptococcus suis serotype 2(SS2) is an important zoonotic pathogen,causing human or swine suffering from meningitis,arthritis,endocarditis,sepsis.S.suis was first discovered in Denmark.Of the 35 capsular serotypes known,serotype 2 is considered the most virulent.Two large outbreaks due to SS2 emerged in 1998, Jiangsu and 2005,Sichuan in China,resulting in more than 12-thousand swine death and 200 human infections,20%of which were fatal.Attempts to control the disease have been hampered by the lack of effective vaccines and suitable diagnostics.SS2 has been studied by the technologies of genomic or immunity,in the past. Some virulent factors were founded,for example:extracellular factor(EF), muramidase-released protein(MRP),capsular polysaccharide(CPS) and suilysin (SLY),Sao protein,fiber-binding protein and IgG binding protein.But these virulence factors didn't exist stably at all strains of Streptococcus suis type 2.The understanding of this bacteria is still not very clear.With arriving of postgenome times,proteomics has being rising gradually which allows the study by the proteome of organism and opens a new door for the study of SS2.In our study,the technology of proteomics was employed to compare the cell wall-associated proteins which came from the virulent and avirulent SS2.A total of two compared groups was made.In this two groups,the virulent strain 449 and 606 compared to the avirluent strain 1330,respectively.Firstly,a simple,rapid and non-destructive procedure was used to extract cell wall-associated proteins from virulent and avirulent strains.This method was the first used in the research of SS2.Comparing to the traditional methods(for example:crush the bacteria,and extract the proteins),this method is convenient,quickly,stable and low cost.What's more fewer interference things were mixed.The crude sample including cell wall-associated proteins was dealed by a modified method of TCA/acetone.In the first electrophoresis,pH 3-10 IPG stripe was choosed to detect the distribution of the isoelectric point of samples and pH 4-7 IPG stripe was selected as the first electrophoresis gel,finally.Isoelectric focusing electrophoresis reach a total of 80 kVh.After IEF,strips were equilibrated twice.At the beginning of the second electrophoresis,12.5%gel was used to separate the protein spot.But some spots gathered and hindered the following analysis.So,we selected 12%gel to separate the spots.The two dimensional electrophoresis(2DE) of the each sample was repeated 4 times.And coomassie brilliant blue R350 was used to stain the gels,which is more quickly(about 10min) and more clearly.The software ImageMaster was used to analysis the 2DE gels.Finally,a total of 155 alternative proteins were found.The value of changes was at least 2.5 fold.These proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS),and 39 unique proteins were identified,at last. These proteins participate the metabolism,pathopoiesis and regulation of organism.Some of 39 alternative proteins are recognized as virulent factors for instance muramidase-release protein(MRP),which was studied extensively.Some are potential virulent factors.For example:①plasminogen-binding proteins.This is a kind of proteins which can bind with host's plasminogen,including Enolase,putative phosphoglycerate mutase(PGM),triosephosphate isomerase(TPI).The binding and activation of plasminogen can be seen as an important step in the dissemination of pathogens and survived in the host.②Endothelin-converting enzyme 1(ECE-1). ECE-1 can activate endothelin-1(ET-1) and ET-1 contribute to the potent vasoconstrictor.Recently ET-1 has also been shown to affect mitogenesis and apoptosis in various cell lines and tissues.③GroEL,④HSP70.There are all molecular chaperone and the most conserved proteins known.They may emerge the surface of the bacteria,and play a role in attachment to host's extracellular matrix components.Some are the regulator of SS2.For example:⑤tagatose 1,6-diphosphate aldolase(TDP aldolase).In Streptococcus pyogenes,TDP aldolase negatively regulated the expression of cysteine protease SpeB,which was a virulent factor. Interestingly,phosphoglycerate kinase(PGK) is either a plasminogen-binding proteins or a regulatory factor of SS2.When the dose reach to a quantity,it can inhibite group B streptococci invasion.The protein is ideally down-regulated in strain 449' compared with srain 1330'.The identification and study of these 39 proteins provided a basis for selection of proteins that may be useful in detection of SS2 infections,and in vaccine development.