| Streptococcus suis(SS)is an emerging zoonotic pathogen that causes septicemia,arthritis,meningitis and endocarditis in swines and humans,making serious threat to public health and safety.Among the 33 known S.suis serotypes,serotype 2(SS2)is the most virulent.Human S.suis outbreaks occurred twices in China and caused serious deaths.Although SS has been researched decades,expecially for its virulence factors,pathogenic mechanisms of SS are still unclear.The virulence of SS2 shows high diversity,however,lacking suitable marker genes has hindered evaluation of virulence in a given SS serotype,especially for SS2.In this study,internally standardized zabrafish model was used to identify SS2 strains with different virulence.Lots of diversities between virulent and avirulent genomes were identified through pan-genome analysis.This contributed to understanding the pathogenicity of SS2.1 Screening avirulent strains of Streptococcus suis serotype 2A total of 105 SS2 strains,covering different isolation regions and dates,were assessed by PCR using genes sbp2’,mrp,epf and sly.Six genotypes including sbp2’-lmrp-/epf-/sly-、sbp2’+/mrp+/epf+/sly+ and so on,were determined.Zebrafish was used as an infection model to evaluate the virulence of strains with different genotypes;avirulent strain T15 and virulent isolate ZY05719 served as references(i.e.,an internal standard).Virulence of a strain was determined by comparing its LD50 with references’ LD50 16 avirulent strains with genotypes sbp2’-/mrp-/epf-/sly-(13)or sbp2’-/mrp+/epf+/sly+(3)were identified,showing that sbp2’ may be a more suitable virulence marker gene than mrp,epf or sly.2 Pan-genome analysis of Streptococcus suis serotype 2Genomes of 7 avirulent strains screened by zebrafish model were sequenced.By means of NCBI datas,10 virulent and 9 avirulent genomes were used to perform pan-genome analysis.Pan-genome shared by 19 SS2 strains was composed of 1,239 core genes and 2,436 variable genes.9 avirulent strains were classified into two groups(Ⅰ and Ⅱ)according to the evolution relationship.Avirulent strains possessed more prophage sequences than virulent ons.A pilus genes cluster specific to virulent strains was identified by comparing the core-genomes shared by virulent and avirulent strains,respectively.And 53 and 58 genes belonging to virulent and avirulent strains were screened by comparing the core-genomes shared by virulent and group Ⅱ avirulent strains.3 Genotype of mrp and its relationship with the virulence of Streptococcus suis serotype 2Core-genome shared by 9 avirulent strains consist of 1,459 genes,including gene mrp,which conflicted to the PCR result that 13 avirulent strains were mrp-.Variable regions may locate in mrp sequence.Conserved and non-conservative regions of mrp were identified by sequences alignment of mrp from different SS2 genomes.Primers(mrp-1、mrp-2)targeting to different regions were designed,then were used to determine the mrp types of SS2 isolates.Zebrafish model was used to detect the virulence of strains with different mrp types;and results showed that type mrp-A(mrp-1+/mrp-2+)strains tend to be virulent than type B(mrp-1+/mrp-2-)strains.Quantitative real-time PCR was used to detect the transcription level of mrp in SS2 strains;and results showed that mrp in mrp-A strains showed higher transcription level than mrp in type B strains.4 Construction of pnuC gene knock-out mutant in Streptococcus suis serotype 2Comparing analysis between core-genomes shared by virulent and group Ⅱ avirulent strains present 53 virulent strains-specific genes,including nadR,pnuC and mutT,which are all locate in the same operon.△pnuC,pnuC knock-out mutant of SS2 virulent strain ZY05719,was constructed successfully,using methods including fusion PCR and homologous recombination,and Streptococcus-Escherichia coli shuttle vector pSET-4s.Compared to wild strain ZY05719,△ pnuC showed significantly attenuated virulence to zebrafish,which suggested that gene pnuC contributes to the pathogenicity of SS2 strains. |
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