Genomic Sequence Determination Of Streptococcus Suis Type 9 Virulent And Attenuated Strains And Construction Of Gene 01891,01892 And 00110 Knockout Strains

Streptococcus suis(S.suis)is an important zoonotic pathogen which can infect pigs in different ages and cause meningitis,arthritis,endocarditis and other symptoms.Severe symptoms can be caused when human infected by Streptococcus suis,such as septic arthritis,meningitis,gangrene,sepsis,and it may eventually lead to death.In our country in recent years,Streptococcus suis type 9(SS9)has been isolated from sick swine,and its virulent strains can cause meningitis and death in swine,causing economic losses and harm to the pork industry and threaten to our public health safety.To reveal the pathogenic mechanism of SS9 in more detail and constructe a more complete prevention system for streptococcus suis disease,carrying out in-depth research on the key virulence sites of SS9 has great significance.There are two strains of Streptococcus suis type 9 isolated by our laboratory,the SS9-29 strain was isolated from the brain tissue of a sick swine that eventually died of meningitis due to infection with SS9,it is a virulence strain;SS9-258 was isolated from the sick swine's lungs which is a attenuated strain.In order to explain the reasons for the difference in virulence of SS9,we carried out the genome sequence determination and comparison and genome analysis of the two strains,and further carried out the construction and pathogenicity study of three different genes' knockout strains.The main results are as follows:(1)The biological characteristics of SS9-29 and SS9-258 strains and their pathogenicity to mice were evaluated in vitro and in vivo.SS9-29 and SS9-258 have no significant differences in colony morphology,cell morphology and growth rate,but SS9-29 is significantly more pathogenic to mice than SS9-258.(2)After the whole-genome sequencing and comparison of SS9-29 and SS9-258,we found a series of genes in different virulence strains,among which genes 01891,01892 and00110 only exist in the SS9 virulent strains' genome.(3)Using SS9-29 as a parental strain,homologous recombination methods were used to construct gene-deleted strains of genes 01891,01892 and 00110 respectively.The biological characteristics of the three gene-deleted strains and their pathogenicity to mice were evaluated.We found that the strains with deletions of genes 01891,01892 and 00110 were not significantly different from the wild SS9-29 strain in cell morphology and growth rate.Compared with the wild SS9-29 strain,the murine pathogenicity was not significantly weakened,indicating that none of the three genes are the key genes for SS9-29 to cause the symptoms of porcine meningitis.To sum up,in this experiment,we first performed whole-genome sequencing and comparison of SS9 strains with different virulence,and found hundreds of virulent strainspecific genes,and then select three of them which are 01891,01892,and 00110 to perform a further study.It is found that they are not the key factors affecting the virulence changes of SS9.However,the results of this study provide a basis for further exploration of new SS9 virulence factors,and is convenient for subsequent verification of the remaining specific genes and further exploration of the key virulence factors of SS9 and their mechanism of action more deeply.