| Streptococcus suis is an important zoonotic pathogen worldwide.Streptococcus suis can be divided into 33 serotypes(1/2,1-31,33).Among them,serotype 2,because of its strongest virulence and most infectious cases,are always reported the most,no matter in China、Southeast Asia or around the world.It can cause severe disease in pigs,for example,sepsis,meningitis,arthritis,pneumonia,leading to the huge economic losses of swine industry.Among diseases caused by Streptococcus suis,meningitis is the most common phenotype,but other systemic complications such as sepsis,pneumonia,are also typically directly related to Streptococcus suis.There used to be two serious outbreaks of swine streptococcus infecting human beings in China,causing heavy casualties.Also in the two outbreaks,toxic shock syndrome(STSS)caused by Streptococcus suis was first observed.At the same time,these infections also had the clinical features of deep tissue infections and high attack rates.However,differing from virus,bacteria have larger genomes and the genome structure is more complex,and the number of cell proteins is huge,which has been bringing more difficulties for its study of pathogenesis,virulence factors.Although there are a great number of studies about the virulence factor of Streptococcus sui,the pathogenesis of it is still unclear.Therefore,exploration of virulence factors of pathogenic strains of S.suis remains to be the hotspot.Our study was also focused on the exploration and verification of virulence factors of Streptococcus suis.It mainly included the following three aspects:1.Identification of mmuM owned only by virulent strains of SS2Identicication of the virulence of S.suis 2 strains was conducted through the internally standardized zabrafish model in previous study.7 avirulent strains were selected to do the whole genome sequencing.Then these whole genome sequences were put together with 10 virulent whole genomes published on the NCBI to direct the pan-genome analysis,in order to find out the genes owned by virulent strains but not by the avirulent strains.Through pan-genome analysis,53 genes which were owned by virulent strains but not owned by the avirulent strains were found,including mmuM gene,which encoded the homocysteine methyltransferase.Furthermore,by PCR test,mmuM was found to only consist in the virulent strains of SS2.Based on this,it could be speculated that mmuM gene was related to the pathogenicity of Streptococcus suis.type 2 and might be a potential virulence factor.2.Adhesion assays of mmuM,OppA and IMPDH recombinant proteins to HEp-2 cellsmmuM was prokaryotic expressed.At the same time,two proteins of the 15 LN-binding proteins and 5 FN-binding proteins idetified by 2D far-western blot,OppA and IMPDH,were prokaryotic expressed,and their ECM-binding activities were verified.Indirect Immunofluorescence Assays suggested that all these three proteins could adhere to HEp-2 cells.This results also proved the feasibility of using 2D far-western blot to identify ECM-binding proteins in order to find out the potential adhesion factors.3.Construction and characterization of mmuM gene-knockout mutant of ZY05719In order to verify this speculation,we chose the virulent strain ZY05719 as the research object.Using the classic method of homologous recombination,and the temperature-sensitive suicide plasmid pSET4s the mmuM gene-deletion strain △mmuM was successfully constructed.Then the complemented strain CAmmuM was also constructed successfully using the shuttle plasmid pSET2.Some basic biological traits of ZY05719、△mmuM and C△mmuM,such as the growth characteristics,hemolytic characteristics,Gram staining characteristics and transmission electron microscopy of them showed no significant changes.After PCR detection of S.suis of other serotypes,the mmuM gene was found not existing in other serotypes,which further suggested that mmuM gene existed only in the virulent strain of Streptococcus suis type 2;After virulence tests with zebrafish,the mutant strain △mmuM was found to have lower virulence than the wild-type strain ZY05719.The adhesion of△mmuM to Hep-2 cells decreased significantly,compared to the wild-type strain.Whole blood survival assay showed that the survival rate of the mmuM-deficient strain in pig anticoagulated whole blood declined significantly compared with the wild-type strain;Real-time PCR indicated that the trancriptional levels of the reported virulence factors sly2C,mrp,sly decreased in△mmuM.According to these experimental results,the mmuM gene might be another virulence factor of S.suis 2.4.Analysis of the differential proteomics of emmuM and ZY05719 with the method of Label-free.Through label-free proteomic analysis of the whole cell protein expression levels of the wild strains ZY05719 and the delection mutant strain △mmuM,91 genes were found down-regulated for 1.5 times and 66 up-regulated for 1.5 times in AmmuM than in ZY05719(P<0.05).Among the down-regulated proteins of △mmuM,several ones were reported to be related to the adhesivity and virulence of S.suis,including the glyceraldehyde-3-phosphate dehydrogenase(GAPDH),6-phosphofructokinase(6-PGD),inosine-5-monophosphate dehydrogenase(IMPDH),and glutamate dehydrogenase(GDH). |
