Construction And Characteristic Analysis Of Suilysin Gene Knock-Out Mutant Of Streptococcus Suis Type2Isolated In Jiangsu Province

Streptococcus suis (S. suis) is a major swine pathogen which is responsible for a wide range of diseases, including septicaemia, meningitis, endocarditis, arthritis and even acute death. In addition to causing disease in pigs, it is also an important zoonotic agent that afflicts people in close contact with infected pigs or pork-derived products. Thirty-five types (1-34and1/2) have been described based on capsular polysaccharides. Streptococcus suis serotype2(SS2) is the most frequently isolated and associated with disease.Hemolysin (Suilysin, SLY) is an important virulent factor of SS2. Geographical differences in prevalence of sly+strains have been described. The most striking difference is the low prevalence of suilysin positive strains in North America in comparison to Europe and Asia.This reminds us that SLY may play different role in the pathogenicity of different region SS2.Up to now, the biological function and pathogenic mechanism of SLY is not very clear. It is certain that SLY can lysis many kinds of animal red cells and has toxicity to many cells. It remains to be further elucidated its ability of inducing expression of cytokines and its role of occurrence and development in the disease, especially in meningitis.In this study, the distribution and genetic variance of suilysin gene of streptococcus suis type2strains isolated from diseased pigs in china was identified.The recombinant full length suilysin was expressed in E.coli and its bioactivity and immunogenicity was analyzed. We constructed the suilysin knock-out mutant of streptococcus suis type2strain isolated in Jiangsu provine and studied its biological characteristics and pathogenicity. All these help us to further understand the biological function of suilysin, especially for its molecular pathogenic mechanism. 1PCR detection and sequence analysis of the gene encoding suilysin from streptococcus suis type2strains isolated from diseased pigs in chinaThe genes encoding suilysin (SLY) of Streptococcus suis type2strains isolated from diseased pigs in China were detected and sequence analyzed. The genes encoding SLY from the29strains isolated at different time and areas were detected by polymerase chain reaction (PCR). The sly genes of SS2-1, HA9801, HA0507, JR0507and ZY05719were cloned and sequence analyzed. The results were as following:the genes encoding SLY of all29strains were positive by PCR; the ORFs of sly gene of the5strains were comprised of1494base pairs which encoded497amino acids; nucleotide sequence homology were99.7%-100.0%and the derived protein sequence homology were99.8%-100.0%. The sly genes of SS2-1, HA9801, HA0507and JR0507were identical to that of the foreign referenced strain1/7. From the above, it came to the conclusion that the sly gene is prevalent in the Streptococcus suis type2isolates from diseased pigs in China and the sequence of sly gene is extremely conservative.2Prokaryotic expression, bioactivity and immunogenicity of the full length suilysin of streptococcus suis type2The suilysin full length gene of Streptococcus suis type2was construction by the methods of molecular biology and was cloned into plasmid pET-32a.The recombinatant plamsid was transformed into the expression host strain E.coli BL21(DE3).After4hour induced by1mM IPTG at37℃, the protein which molecular weight was about72K was at the highest level. The content of the protein was about20%of that of total bacterial proteins. The proteins in sonicated supernatant accounted for50%. The purified protein by Ni-NTA His resin was positive by Western blotting. Its hemolytice titer to sheep red cell and chick red cell attained1.707×105HU/mg and4.267×104HU/mg, respectively. The purified protein could cause the Vero cell or HEp-2cell fusion, grain increase and fall. The protection rate of rSLY to BALB/c mice was83.3%(5/6). This established a solid foundation for further research such as pathogenesis and vaccine development.3Construction of suilysin gene mutation of streptococcus suis type2strain isolated in Jiangsu provinceUsing the genomic DNA of JR0507as the template,569bp (upstream sly arm) and559bp (downstream sly arm) fragments were amplified, respectively. The cat gene was amplified according to the pSET3DNA. The homologous arms and the cat gene were linked to pSET4S by turns, and then the linked products were transferred into DH5a competent cells to construction the vectors pSET4sΔSLY. Subsequencely pSET4sΔSLY were introducted into competent cells of JR0507by electrotransformation. And the transformants were screened on the plates containing chloromycetin and detected by PCR and Western blotting. The results showed that JR0507(ΔSLY) was successfully constructed, which laid the foundation of studying function of suilysin.4Analysis of biological characteristics and pathogenicity of suilysin knock-out mutant of Streptococcus suis type2strain isolated in Jiangsu provinceThe biological characteristics and pathogenicity of JR0507(ΔSLY) and wild type JR0507(WT) was compared. The results showed that the hemolytic activity of JR0507(ASLY) were lost completely. Compared with WT, growth and biochemical characteristics and adhesion ability to PK-15, HEp-2and PIEC cell of the mutant had not changed. But the mutant exhibited a significant decrease toxicity to the cells (P<0.01). The percent survival rate of the wild type parent strain was (40.7±4.8)%in whole blood. In contrast, the mutant was much more sensitive, with a percent survival rate of only (15.5±2.8)%. Animal pathogenicity tests showed that the MLD of ΔSLY to BALB/c mice was1.0×109CFU, ten-fold higher than that of the wild type. And the mutant showed no pathogenicity to New Zealand rabbit and weaning piglet. After6hours incubation,3D4porcine alveolar macrophages expressed no inflammation related cytokines of IL-1β, IL-18, IL-8, IL-12and IL-10by the mutant and WT. But after12hours incubation,3D4expressed the four cytokines except IL-12by field strain, but not by the mutant. In short, the virulence of suilysin knock-out mutant is attenuated when compared with the WT, which may be associated with its decreased ability to survive in whole blood, destruct to cell and induce the express of inflammation related cytokines. All are further clarified the biological function and molecular pathogenic mechanism of hemolysin and proved its important role in the occurrence and development of the disease associated with Streptococcus suis.