Biological Characteristics Of Streptococcus Suis Serotype 4 Isolates And Transcriptional Regulator GalR

Streptococcus suis is an important zoonotic pathogen that poses a serious threat to human health and hinders the economic development of the global swine industry.According to the difference of bacterial capsular polysaccharide,it can be divided into 33 serotypes?1-31,33 and1/2?,of which Streptococcus suis type 2 is the most pathogenic.Studies have reported that Streptococcus suis serotype 4?SS4?can cause disease and even death in humans and animals,and has become a major epidemic serotype in China.At present,there are few studies on SS4 at home and abroad.This study conducted a systematic analysis of the pathogenic biological characteristics of SS4 in different regions of China,and provided a reference for the prevention and control of SS4.At the same time,the laboratory analyzed the bacterial protein expression profile of SS4 virulent and attenuated strains by comparative proteomics,and screened the transcriptional regulator GalR.We performed bioinformatics analysis and prokaryotic expression on GalR,which laid the foundation for further study of the biological characteristics of GalR protein.We successfully constructed the deletion strain SH1510?GalR,and studied the biological characteristics of GalR,which provided a theoretical basis for further study on the regulation of GalR on the regulation of galactose metabolism and its pathogenic mechanism.The study is divided into three parts:1.Biological characteristics of Streptococcus suis type 4 isolatesWe performed multi-site sequence typing,virulence factor testing,and animal pathogenicity tests on 10 strains of SS4 from different regions of China.The results showed that6 strains were ST850 type,3 strains were ST1006 type,and 1 strain was ST94 type.According to the analysis of the isolated areas of the strains,the strains in Guangdong Province and the strains in Jiangsu Province have higher homology,and the strains in Jianghu area show genetic diversity.Gdh,gapdh and orf2 were detected in 10 strains,sly was detected in 6 strains,and fbps were detected in 4 strains.According to the virulence factor spectrum,there were 3 virulence genotypes,6 strains were gdh+sly+gapdh+orf2+,3 strains were gdh+fbps+gapdh+orf2+,and 1strain was gdh+sly+fbps+Gapdh+orf2+type.Animal pathogenicity tests showed that SH1510strain had the highest mortality rate in mice and could cause death in New Zealand rabbits.2.Bioinformatics analysis and prokaryotic expression of Streptococcus suis type 4 GalR geneWe used the relevant bioinformatics analysis website to perform protein sequence homology search,functional domain search,signal peptide prediction,and transmembrane region prediction of the transcriptional regulator GalR.We used the expression vector pET32a to prokaryotic expression of GalR gene,SDS-PAGE analysis of protein solubility,and Western blotting to identify protein reactogenicity.The results showed that the GalR protein of SH1510strain had some homology with the amino acid sequences of some other bacterial GalR family proteins.Domain search revealed a small DNA-binding domain with an HTH motif at the N-terminus,with a regulatory ligand binding domain at the C-terminus and a linker linking the two functional domains.The GalR protein has no signal peptide and no transmembrane region.SDS-PAGE results showed that the GalR protein was successfully expressed and the soluble expression reached more than 99%,and the GalR protein was successfully purified.Western blotting results showed that the GalR protein has good reactogenicity.3.Construction and biological characteristics of the deletion strain of GalR gene of SH1510 strainWe constructed the deletion strain SH1510?GalR by homologous recombination.After the construction of the deleted strains was successfully identified by PCR and Western blotting,Gram staining and comparison of different sugar utilization rates were performed on the parent strain and the deletion strain.We conducted a mouse pathogenicity test and a competitive infection test using BALB/c mice as test animals,and conducted a whole blood survival test by mimicking the in vivo environment of whole blood of healthy pigs.The results showed that there was no significant change in the bacterial morphology of SH1510 after the loss of GalR gene.Under the condition that glucose and sucrose are glycogen,the growth of bacteria is not affected.Under the condition that D-galactose is glycogen,the growth rate of bacteria and the value of OD₆₀₀ are decreased.The pathogenicity of mice decreased by 1.62 times.The colonization ability in the body was significantly reduced?P<0.01?.Whole blood viability was significantly reduced?P<0.05?.