| Streptococcus suis(SS)is an important zoonotic pathogen,which can cause arthritis,septicemia,meningitis,and other diseases in humans and pigs.It brings huge economic losses to the breeding industry and poses a serious threat to food safety and public health safety.SS vaccination is an effective preventive measure to prevent SS related diseases,but there are many SS virulence factors and multiple serotypes,so there is still a lack of commercial vaccines that can provide cross-immunity protection.Therefore,the search for new virulence factors is of great significance for the research of universal SS vaccine and the scientific prevention and control of SS related diseases.In this study,a new SS virulence related factor SUT2 was first screened and identified by transposon random mutagenesis and C.mellonella assay,and then the biological characteristics of its virulence regulation mechanism against SS were studied.Finally,the attenuated vaccine strain was constructed by knockout of SUT2 gene and its immune effect was evaluated.In order to explore new virulence factors,we first used transposon TnYLB-1 to construct a transposon mutation library of SS genome,and then screened virulence altered mutants and isolated virulence factor-related genes using the moth suppressor assay.The results showed that 450 transposon mutant libraries were obtained by screening for kanamycin resistance and spectacamycin resistance,and the transposon efficiency was 83.33%.Subsequently,the library was further screened by toxicity test of C.mellonella,and a total of 18 SS mutant with reduced virulence were obtained.Finally,a total of 8 virulence reduction related genes were identified by reverse PCR and gene sequencing at the insertion site of the mutant.They were diaminoheptanoic acid decarboxylic acid,M24 metallopeptidase,two hypothesis proteins,ABC transporter permease subunit,pyridoxal phosphate dependent aminotransferase,ABC transporter binding substrate,ribonucleic acid G and E.Subsequently,we selected SUT2 gene with the most obvious virulence decline,and constructed SUT2-targeting gene knockout SS strain and its complement strain by homologous recombination technology to study the function of this gene.The results showed that,compared with the wild type,ΔSUT2 did not affect the growth of SS in BHI medium,but could affect the chain length morphology.ΔSUT2 significantly decreased the acid-base tolerance of SS,but significantly increased the biofilm formation ability of SS;The results of in vitro experiments showed that ΔSUT2 significantly decreased the phagocytic ability,whole blood survival,hemolytic activity and other aspects of PAM(porine alveolar macrophages),but enhanced the adhesion and invasion ability of human microvascular endothelial cells(HBMEC/D3)by up-regulating the transcription level of adhesion and invasion related genes.In vivo experiments showed that ΔSUT2 significantly increased the survival rate of mice,indicating that SUT2 could affect the virulence of SS.In conclusion,SUT2 gene is crucial to SS2 pathogenicity,and has a variety of biological effects.Finally,we knocked out SUT2 gene of naturally isolated and weakly pathogenic Streptococcus suis type 2 strain and constructed live vaccine JLY022 ΔSUT2 to explore the cross immune protection against streptococcus type 2 and type 7.The results showed that the protection rate of JLY022 ΔSUT2 against Streptococcus suis type 2was 60% and that of Streptococcus suis type 7 was 40%.In conclusion,this study successfully constructed a library of SS2 transposons,identified 8 virulence related genes not reported in SS,and confirmed that SUT2 is involved in the adhesion and invasion of host cells by SS,biofilm formation,and antiphagocytosis.The live attenuated vaccine JLY022 ΔSUT2 was constructed,the results showed that it had certain cross immune protection between serotypes 2 and 7 of Streptococcus suis.This study provides a scientific basis for the pathogenicity and vaccine research of porcine Streptococcus suis type 2. |
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