Identification Of Blood Survival-related Genes In Streptococcus Suis

Streptococcus suis(S.suis)is an important zoonotic pathogen.It can cause the host of sepsis,meningitis and endocarditis and other symptoms,It can bring significant economic losses not only to the pig industry,but also a serious threat to human health.According to the antigenicity of polysaccharides,S.suis is divided into 33 serotypes,of which Streptococcus suis type 2 is recognized as the world's most widely distributed and virulent serotype.The main virulence factors of S.suis include capsular polysaccharide(CPS),hemolysin(SLY),extracellular factor(EF),lysozyme release protein(MRP)and so on.Some of the factors involved in the adaptation of the host environment are also involved in S.suis colonization and infection.Survival in the blood and lead to sepsis is one of the important mechanisms of S.suis pathogenesis.In this study,we constructed the transposon mutant library of S.suis,and screened the SS gene associated with pathogen survival in the host blood by high-throughput method.The main results were as follows:1.Construction of mutant library of S.suisThe mutant library of S.suis transposon containing 1918 mutants was constructed by using the p TV408 thermo-sensitive shuttle plasmid containing Tn917 transposon.The sequencing of the mutant library and the COG function classification of the inserted gene were completed.The library contains 393 mutants of different genes,which are relatively random and can be used for subsequent functional genomics studies.2.Whole blood killing experiment screening gene of S.suis blood survivalThe 1918 mutants in the mutant were screened by the whole blood of the pig by establishing a high-throughput whole blood killing test on a 96-well plate.The killing rate of each mutant was calculated and compared with that of the parent strain,A total of 1151 mutants with varying viability in the blood were obtained,including 377 mutants with enhanced survivability in the blood,and 774 mutants with decreased viability in the blood.Functional analysis and classification of the mutated genes in these mutants revealed that 149 genes were reported throughout the bacterium in terms of blood survival or virulence genes and were associated with carbohydrate metabolism,transcription factors,transporters and Enzymes,and so on,in addition to 244 genes in the entire bacteria has not been reported with the blood survival or virulence genes related to the study.3.Validation of S.suis blood survival related genesFour non-reported genes were selected from the selected genes in the blood survival-related genes.Further validation and analysis were performed.The results showed that the viability of No.314(Locus_tag: SSUSC84₀942)and No.1760(Locus_tag: SSUSC84₀561),Each of which encodes a gene product which is a 6-phosphate-galactose isomerase subunit and an aminoglucoside.No.27(Locus_tag:SSUSC84₁385)and No.1688(Locus_tag: SSUSC84₀966)have enhanced blood viability,and their gene products are bifunctional enzymes and glutamine transferases of 5 'nucleotides and metallophosphates.