Construction Of RevS Gene Mutant Strain Of SS2 And The Protein Interaction Between HYL And The Host

Streptococcus suis(S.suis) is a common pathogenic microorganism and can cause a variety of clinical diseases in human and swine.Based on the CPS antigens of S.suis,35 serotypes or capsular types have been characterized.S.suis serotype 2(SS2) is most commonly and most virulent of these serotypes.revS gene is the response regulator gene of SS2,belonging to a two component system,which can affect the virulence gene expression of streptococcus suis.For further research the virulence gene expression regulated by revS,revS gene mutant strain and reversible mutant strain were constructed and characteristic analyzed.Hyaluronidase is a kind of protein coding by Hyl gene of SS2,it is mueopolysaccharide lytic enzyme which can hydrolyze the hyaluronic acid of periplast, result in the permeability of the tissue augmentation.In addition some research report HYL can act as a modulin,density of HYL in the blood is descended which can induce Blood vessel accrementition.In the course of SS2 infection,whether the hyaluronidase secreted by SS2 can result in the permeability of the tissue augmentation,ease to the ingress of virulence factors secreted by SS2 into the host and play effect is not clear.So in the research hyaluronidase was used as a bait protein to search its interacted protein from Mouse brain cDNA library and human lung cDNA library using the yeast two hybrid system to explore its function in SS2 infection.So the following researches were explored.1.Construction and verification of SS2 revS gene mutant strain and complementation strainIn the research two DNA fragments flanking the revS gene were amplified by PCR from the genome of S.suis 2 SC21.The fragments of the N-terminus and the C terminus flanking region of the revS gene were digested with BamH I/SalⅠand Sal I/EcoRⅠ, respectively.The revS-flanking PCR products were mixed in equal amounts and inserted directly into BamH I/EcoRⅠdigested and dephosphorylated thermosensitive suicide vector pSET4s,ligated,and transformed into E.coli DH5α.The resulting plasmid, pST4S-revS,was electroporated into S.suis 2 SC21,and the resultant strains were grown at 28℃in the presence of spectinomycin(100μg/mL) selection and subsequently passaged at 37℃in the absence of spectinomycin selection as described previously. Successful deletion of the gene revS was confirmed by PCR and Southern blotting. Southern blotting analysis confirmed the successful deletion of the revS gene in the chromosome of the S.suis 2 revS gene mutant strain named SC211.In addition complementation strain was constructed too.The revS structural gene,including its own promoter,was amplified from chromosomal DNA S.suis 2 SC21 by PCR.The PCR product was cut with Sal I/BamHⅠand ligated into the Sal I/BamHⅠdigested E. coli/Streptococcus shuttle vector pAT18(Trieu-Cuot P et al.1991).The resultant plasmid was termed pAT18-revS.The plasmid pAT18-revS were transformed by electroporation into the revS gene mutant strain with subsequent erythromycin selection. Complementation strain designed SC212.The mutant strain and complementation strain were confirmed by PCR and Southern blotting.2.Bionomics research of the mutant strain and complementation strainThe results of the growth curve of the mutant strain,complementation strain and wide type strain show that the mutant strain and complementation strain grow faster than that of wide type strain.The hemolytic activity of wild-type strain was three times higher than that of mutant strain.The hemolytic activity of complementation strain was restored, and was about two times higher than mutant strain SC211.but it did not reach the level of wild-type strain.Reduction of 79%in the adherence to the Hep2 cell of mutant strain compared with wild-type.The adherence of complementation strain was about three times that of mutant strain,but only 81%of the wild-type strain.LD50 values were 7.63×10~6 CFU per mouse for wild-type strain,7.78×10~7 CFU per mouse for mutant strain,and 2.44×10~7 CFU per mouse for complementation strain.Compared with parent strain, mutant strain was attenuated at least ten fold.Complementation strain was restored three fold than the mutant strain.3.Immunogenic research of the mutant strain and complementation strainA vaccine dose of 10~7 CFU mutant strain and 10~6 CFU complementation strain was protective against an extremely high challenge dose of 4×10~7 CFU.These results demonstrated that the Streptococcus suis-2 revS gene mutant was highly attenuated in the mouse model,and 100%protective against homologous bacterial challenge at 10~7 CFU vaccine-challenge doses.The complementation strain was attenuated in the mouse model, and was 100%protective against homologous bacterial challenge at 10~6 CFU vaccine-challenge doses.The ELISA antibody level of mutant strain has no difference with wide type strain,but the ELISA antibody level of the complementation is higher than that of mutant strain and wide type strain.4.Relative quantitative PCR assay of virulence associated gene of SS2The characteristic of SC211 and SC212 were researched further.The virulence of different strains were determined by animal infection,the virulence is decreased clearly when the revS gene knocked out,the virulence is reverted when the revS gene reverted, but which can not get to the level of the wide type.Many virulence associated genes, including mrp,ef,sly,fbps,sod,rpob,gyra and hyl,are shown to be effeeted by revS gene, the expression level decreased obviously when revS gene knocked out,but the cps and gapdh is not effected by revS,the virulence gene expression of the reversible mutant were exceeded the wide type.In this study,the differential expression of the key S.suis serotype 2 virulence-associated genes of different strains was further investigated in bacteria harvested from the blood,lungs and brains of mice 24,48,and 72h post intraperitoneal infection with different strain S.suis.Bacterial RNA was extracted and gene expression was monitored using real-time reverse transcriptase-PCR at various time points.In all organs the expression level of wide type strain is higher than mutant strain at all time,the reversible mutant strain were higher than the other two strains,the virulence gene expression were similar to that observed in vitro,while the expression level in vivo were higher than their in vitro levels.But at different time the difference of same virulence gene expression between in vivo and in vitro is changed.With the time of infection delay the difference of same virulence gene expression between reversible mutant strain and wide type strain or mutant strain is contracted.In vivo the cps expression was changed obviously.At all the time postinfection,all genes examined were higher than their in vitro levels in the blood,lungs and brains,revS gene can affect the virulence associated gene expression and contribute to S.suis pathogenesis.5.Screen of the interaction protein of HYL of SS2The full length Hyl gene was amplified from chromosomal DNA of SS2 strain SC21 by PCR,Hly gene was directional cloned into pSos vector after the amplified PCR product and pSos vector were digested with BamHⅠand SalⅠ,yielding the bait plasmid pSos-Hyl.The recombinant expression plasmid pET-28c-Hyl was constructed by the same way at the same time.The library was screened by co-transformation of the pSos-Hyl bait constructed into a temperature- sensitive cdc25H yeast strain that can not grow at 37℃. If the bait protein physically interacts with the target protein,the hSos protein is recruited to the membrane activating the Ras signaling pathway and allowing the cdc25H yeast strain to grow at 37℃.The clones growing at 37℃on SD/galactose(-UL) plates but not on SD/glucose(-UL) plates were selected as "putative positive" clones and were analyzed further.The Target plasmid DNA was isolated from pMyr-cDNA putative positive clones and was transformed into E.coli cells for plasmid amplification.Colonies contains cDNA fragment insertion identified by restriction digest analysis.The positive Target plasmid was sequenced on a 3730 Sequencing system.The sequencing results were sent to NCBI for BLAST searches on line.In this study two positive target plasmid received from the Mouse brain cDNA library.One of these genes is identical to the widely-interspaced zinc finger motifs gene of Mus museulus(WIZF).The other showed homology to ribonuclease/angiogenin inhibitor 1 gene of Mus musculus(AI1).At the same time two positive target plasmid received from the human lung cDNA library.One of these genes is identical to Homo sapiens adducin 3 gene(ADD3).The other showed homology to assembly ion transporter protein gene of homosapiens(AITP).6.Confirmation of the protein-protein interaction between HYL and targets receivedThe protein-protein interaction between HYL and targets received was confirmed by ProFound Co-immunoprecipation assay.Recombinant plasmid pET-28c-Hyl was transformed into E.coli BL21 cells for protein expression.The coding sequence of WIZF,AI1,ADD3 and AITP obtained by PCR from the target vectors respectively,then they were subcloned into pcDNA3.1/myc-His,yielding four recombinant expression plasmids.The recombinant expression plasmids were transfected into 293T cells for protein expression.Anti-myc probe antibody was used to immunoprecipitate the expressed protein(WIZF,AI1,ADD3 or AITP) from the whole cell lysate and supernatant of BL21 transformed with pET-28c-Hyl culture.The complex was then resolved on a 10%SDS polyacrylamide gel.The separated proteins were transferred to a nitrocellulose membrane.Pig anti-sera against SS2 were used for western-blotting.But the interaction between HYL and WIZF were not confirmed by this way.The relation between the function of AI1,ADD3 or AITP and the pathopoiesis was analyzed.AI1 can inhibit the growth of blood vessel cellula epithelialis,which result in the permeability of the blood vessel changed ease to the diffuse of SS2.ADD3 is a kind of adducing,cell membrane skeleton protein.AITP is ion transporter protein of the cell membrane.These two protein belongs to the ion channel of the cell membrane,which is associated with the permeability of the cell membrane.