Establishment Of Mouse Assessment Model For Blood Brain Barrier Opening Caused By Piscine Streptococcus Agalactiae Infection And The Analysis Of Cas9 Founction

Streptococcus agalactiae,also known as group B streptococcus(GBS),has a broad host range of infection.It can not only cause neonatal meningitis,pneumonia and mastitis,but cause meningitis and septicemia in fish.In recent years,S.agalactiae has become an important pathogen of fish and has caused heavy economic losses to the aquaculture industry in China.The mortality rate in fish caused by S.agalactiae is high and even up to 100%,but its pathogenesis is not cler.Therefore,to study the pathogenesis of S.agalactiae is essential for prevention and control of this bacterium infection.1 Establishment of a mouse assessment model for blood brain barrier caused by S.agalactiae infectionS.agalactiae is one of the main pathogens causing meningitis in animals and human.To explore the pathogenesis of meningitis caused by this bacterium,mouse model was developed.The ?-galactosidase-positive Escherichia coli M5 was screened as an indicator bacterium for blood-brain barrier(BBB)opening.E.coli M5 was inoculated to mice by the intravenous injection after the intraperitoneal injection of S.agalactiae,and 5min later,blood was collectedion from the venae chorioideae oculi.The mice were killed and the brains were sterilely removed.Then S.agalactiae and E.coli M5 in the brain tissues were counted.The results showed that the numbers of M5 and S.agalactiae GD201008-001 in the brain tissues was increased with time of intraperitoneal injection.It suggests this meningitis model in mice with good stability and reproducibility was successfullydeveloped,providing a convenient and reliable means for investigation of the pathogenesis of bacterial meningitis.2 Prokaryotic expression of hyaluroni,dase from S.agalactiae and its pathogenicity in miceTo determine the role of hyaluronidase(Hyl)in the pathogenicity of S.agalactiae,a pair of specific primers was designed according to the hylB gene sequence of piscine S.agalactiae published in GenBank.A 3156bp long whole fragment of hylB was amplified from the genome of S.agalactiae GD201008-001 by PCR.This amplified fragment was inserted into the pET3 2a vector,thus creating a recombinant expression vector pET32a-hylB.The recombinant vector was transformed into E.coli BL21 and induced by IPTG to express.SDS-PAGE and Western blotting analyses demonstrated that the target protein of 13 7.1 kD was obtained.Hyaluronic acid(HA)degradation assays showed that the expressed protein had a good enzymatic activity.Further,the protein was injected into the ICR mice through the tail vein.Histopathological observation showed that both of the lung and the brain were damaged seriously with manifestations of pulmonary interstitial broadening,the infiltration of inflammatory cells and cerebrovascular hemorrhage.It suggests that Hyl plays an important role in the pathogenicity of pneumonia and meningitis caused by piscine S.agalactiae.3 Construction of the cas9 deleted mutant in S.agalactiaeIn order to investigate Cas9 function of S.agalactiae,primers were designed based on S.agalactiae GD201008-001 genome published in Genebank to amplifiy the downstream and upstream homology arms.The two arms were used as a template to generate a PCR fragment comprising the flanking sequences of the cas9 genomic locus.The recombinant plasmid was electrotransformed into GD2010008-001 competent cells,and the strains were selected on spectinomycin resistance plate.The suspected cas9 mutant strains were verified by PCR and qRT-PCR.The target gene was connected to plasmid pSET2 and electroporated into the mutant strain and screened by PCR.PCR,qRT-PCR and sequencing tests proved that the cas9 deletion strain and complemented strain were successfully constructed,which lay the foundation for the subsequent analysis of biological properties.4 Characterization of the cas9 mutant in S.agalactiaeTo further determine the function of cas9 gene in S.agalactiae GD201008-001,the characteristics of wild type strain,cas9 gene deletion strain and complemented strain were investigated in vivo and in vitro.The results showed that there was no significant difference in growth among the wild strain,cas9 deletion strain and complemented strain.LD50 value of cas9 mutant in zebrafish was increased,while.the LD50 value of cas9 mutant in mice was similar to that wild strain,but the death time in mice was delayed as compared to the wild strain.Also,the inactivation of cas9 caused the significantly decreased intracellular survival and adhesion capacity.The capacity to break BBB in mouse model of cas9 mutant was decreased compared to the wild type.This study demonstrated that cas9 gene has a direct or indirect impact on the virulence of S.agalactiae,and plays a key role in the pathogenesis of meningitis.