The Mechanism Of Transcriptional Regulator XtgS Affecting Streptococcus Agalactiae Virulence And Screening Of Superantigen In Piscine Streptococcus Agalactiae

Streptococcus agalactiae（S.agalactiae）is an important zoonotic pathogen with a broad spectrum of infection,such as newborn,unpregnant women,the elderly,dairy cows,warm-water fish,and its colonization ability is powerful,which can adapt to the host for the different parts.In recent years,the outbreak of S.agalactiae disease in tilapia has caused serious damage to the fishery and caused huge economic losses.Therefore,the researh on the pathogenic mechanism of piscine S.agalactiae has become a hot issue.There is a transcriptional regulator XtgS related to virulence in the unique sequence of piscine S.agalactiae GD201008-001,which elucidated the mechanism of XtgS to regulate S.agalctiae virulence,further understood the pathogenic mechanism of S.agalactiae,and provided theoretical basis for the prevention and control of the disease pathogen.1 XtgS complementary strain construction and the effect of XtgS on virulence.Previous studies have found that the deletion strain △xtgS virulence increased,but the plasmid complemented strain CΔxtgSpSET2-xtgS could not restore the function of XtgS.In this experiment,a plasmid complemented strain CΔxtgSpSET2-xtgS,a genomic in situ complemented strain CΔxtgSxtgS and two ectopic complemented strains CΔxtgS1-PxtgST and CΔxtgS2-PxtgST were constructed.Real-time PCR results revealed that CΔxtgSxtgS and CΔxtgS2-PxtgST（named CΔxtgS）could fully restore the normal transcription level of xrgS and its upstream and downstream genes.and transcription level of upstream gene A964₁958 inΔxtgS increased significantly.In a zebrafish infection model,the LD50 indicated that ΔxtgS virulence significantly increased（p<0.05）,which was the same as the previous results,and complemented strain CΔxtgS recovered some virulence,the survival curve showed an increase in the survival rate of zebrafish infected with CΔxtgS.The above results confirmed that complemented strain CΔxtgS restored the function of xtgS,and it was speculated that XtgS affected S.agalactiae virulence by regulating the expression of its upstream gene A964₁958.2 Transcriptome analysis and virulence change of XtgS overexpressed strain（A909xtgS）.To further clarify the function of XtgS,human strain A909 without xtgS gene was used as a research object to obtain an overexpressing strain A909xtgS and an empty plasmid control strain A909pSET2.Transcriptome sequencing analysis and Real-time PCR displayed that transcription levels of SAK₁102～1105（iron transfer system 1）and SAK₁425～1428.（iron transfer system 2）in A909xtgS were down-reguLated,and transcription level of SAK₁829－1835（PTS system）was up-regulated.The biological characteristics among A909,A909xtgS and A909 pSET2 showed that there was no difference in the growth rate,and A909xtgS virulence to zebrafish was significantly decreased（p<0.05）,the virulence of A909 pSET2 was closed to A909,and survival rate of zebrafish challenged with A909xtgS apparently increased.Verification of the differential gene transcription levels in GD201008-001,ΔxtgS and CΔxtgS revealed that transcriptional expression of iron transfer systems 1 and 2 was up-regulated inΔxtgS deletion strain which corresponded to the down-regulation effect produced in A909xtgS.Meanwhile,transcription level of the unique 10kb sequence in GD201008-001 was studied,found that A964₁958 and A964₁957 transcription levels raised several times in ΔxtgS,which was consistent with the Real-time PCR results in chapter 3,and the rest of genes had no obvious change.The above results show that XtgS was a transcriptional regulatory repressor,which affected the different S.agalactiae virulence by inhibiting the transcriptional level of iron transfer system 1,2 and A964₁958 and_A964₁957.3 Recombinant expression of XtgS protein and determination of its transcriptional binding site.In order to determine the transcriptional binding site of XtgS protein,we first obtained the soluble haploid protein rXtgS1-75aa and the dimeric protein rXtgSdimer by prokaryotic expression and purification,respectively co-incubated with promoter sequences of three operon that may be regulated（about 250bp）to performed electrophoretic mobility shift assay（EMSA）.The results revealed that rXtgS1-75aa had no DNA binding function.and rXtgSdimer could bind directly to the promoter region of A964₁958,but not directly to the promoter region of iron transfer system 1 and 2.The promoter sequences of different lengths were ligated into pTCV-lac plasmid and respectively transferred into GD201008-001,ΔxtgS and CΔxtgS to perform β-galactosidase activity assay.The results showed that the target sequence of XtgS binding was located at P149-191 bp of the A964₁958 promoter.In conclusion,XtgS protein inhibited the transcriptional expression of the gene by directly binding to the key part of the promoter of A964₁958（P149-191）,and combined with Real-time PCR data,XtgS protein was indirect to the transcriptional regulation of transfer system 1 and 2.4 The screening of superantigen in piscine Streptococcus agalactiae.The lethality rate of tilapia-derived S.agalactiae GD201008-001 in mice was extremely high.After 12 hours of attack on a very low number of bacteria（10 CFU/body）,the mice showed various clinical symptoms,and subsequently all died within 24 hours.This phenomenon was similar to the symptoms of Streptococcal toxic shock syndrome（STSS）produced by humans infected with hemolytic streptococci,and STSS was mostly thought to be caused by streptococcal superantigens.Therefore,we speculated that GD201008-001 infected mice may secrete certain superantigen during the process.To detect whether GD201008-001 released superantigen,we performed a bioinformatic analysis of superantigens against the genome of S.agalactiae,and used the supernatant of bacteria in the medium environment in vitro and the serum isolated from the infected mice stimulated lymphocytes respectively,then measured lymphocyte proliferation by MTS colorimetry to to detect possible superantigen that bacteria may release.The results showed that no known superantigen or superantigen-like protein gene was found in GD201008-001 genome,and that neither S.agalactiae released in the in vitro culture medium nor in vivo mouse infection could release superantigen.These showed that pathogenic mechanism of GD201008-001 still needs further exploration.