Blood-brain Barrier Penetration Mechanism Of Streptococcus Equi Subsp.zooepidemicus ATCC35246

Streptococcus equi subsp.zooepidemicus(SEZ)belongs to the group C streptococci beta-haemolytic streptococcus,of which ATCC 35246 strain was isolated from the dead pigs in the outbreak of swine streptococcal disease in Sichuan,which is highly pathogenic to pigs.The main clinical symptoms after infection are sepsis,meningitis and arthritis.Serious symptoms can lead to death.This disease severely affects the development of the pig industry and public health safety,and cause huge economic losses to the pig industry in China.Previous studies have found that BifA(Brain invasion factor A)gene plays an important role in the process of SEZ ATCC35246 penetrating the blood-brain barrier,but the functional domain of this gene is not clear,which affects the analysis of its pathogenic mechanism.This experiment preliminarily clarified that SEZ penetration through the blood-brain barrier is related to the Fic domain of the Bif protein,indicating that the Fic domain is a key domain for the function of this protein.Although it has been proven that SEZ ATCC35246 has the ability to penetrate the host's blood-brain barrier,the details of its penetration into the blood-brain barrier to cause brain infections have not been clarified.In this study,the indirect immunofluorescence technique was used to observe that SEZ mainly penetrated the blood-brain barrier.Distributed in the third and fourth ventricles,preliminary analysis of the colonization site of the early stage of brain infection after SEZ broke through the blood-brain barrier.There is a large amount of cerebrospinal fluid in the ventricle and the nutritional environment is infertile.In order to further screen the genes related to the cerebrospinal fluid inferior nutritional environment in SEZ,this study tried to construct a SEZ Tn-seq library.The above studies have laid the foundation for further elucidating the mechanism of SEZ subspecies breaking through the blood-brain barrier and causing brain infections.1.The role of the BifA gene Fic domain in SEZ's penetration of the blood-brain barrier.The mechanism of SEZ breaking through the blood-brain barrier is related to its BifA gene.In order to further explore its correlation with the specific domain of the BifA gene,in this study,we constructed the BifA gene deletion strain ? Bif,the wildtype BifA gene replacement strain CBif,strain that BifA Fic domain mutation backfilling in genome,and Fic domain knockout backfilling strains in BifA gene.?Bif+pCBif(? Fic)and Bif A gene 247 histidine was mutated to alanine(this site is Fic structure Functional site of the domain)point mutation backfill strain ? Bif+pCBif(H247A).The survival curve of mice showed that CBif had no significant change in virulence compared with wild strains,while ? Bif,? Bif+pCBif(? Fic),? Bif+pCBif(H247A)had lower virulence than wild strains.This indicates that bacterial virulence is related to the Fic domain of the BifA gene.According to the measurement of the bacterial load in the brain tissue showed that the amount of bacteria isolated from the brain tissue.It was found that the amount of bacteria isolated from the brain tissue of the ? Bif,? Bif+pCBif(? Fic),? Bif+pCBif(H247A)treatment group was significantly lower than that of the WT group,and the difference of CBif treatment group and the WT group was not significant,indicating that the Fic domain is related to the function of BifA to assist SEZ to penetrate the blood-brain barrier.2.Indirect immunofluorescence localization of SEZ in brain tissue after breaking through the blood-brain barrierIn this study,indirect immunofluorescence technology was used to label the Mlike protein(SzM)on the surface of SEZ,and fluorescent markers were successfully used to indicate the location of SEZ in brain tissuetissue sections.The results showed that position of SEZ infecting host brains were mainly in the third and fourth ventricles.In order to further study its dynamic process of SEZ brain infection,we construct an autofluorescent SEZ strain.The GFP gene was integrated into the temperaturesensitive suicide plasmid pSET4S by homologous recombination,and the pSET4SGFP plasmid was electrotransformed into the Streptococcus equi subsp.zooepidemicus ATCC 35246 strain,and the GFP gene insertion strain(SEZ-GFP)was successfully obtained.It was found that GFP inserting into the SEZ genome can be detected by western blotting.The green fluorescence intensity of SEZ-GFP detected by flow cytometry is 10 times higher than that of wild strains,but the observation effect under a fluorescent microscope is not ideal.The growth curve showed no significant difference in the growth rate between WT and SEZ-GFP.The SEZ-GFP fluorescent strain was successfully constructed,but the fluorescence intensity of the strain needs to be further optimized for dynamic observation based on fluorescence tracing in the later stage.3.Construction of SEZ mutant library using transposon mutagenesis technology.This study aims to construct a SEZ Tn-seq library and a condition-induced transposition system,and to explore its value in the construction of SEZ Tn-seq library.First,the pMar4S plasmid stored in the laboratory was electrotransformed into SEZ competent cells,and seed strains were selected under different temperature and resistance conditions to construct a SEZ Tn-seq library.In order to optimize the library construction method,we tried to construct a condition-induced transposition system pMAR-LacI.This plasmid contains the thermosensitive fragments repTAs from the pSET4S plasmid,the TnYLB-1 transposon,Himarl C9 transposase from pMar4S,the LacI manipulation system from pJWV102-PL-dCas9,and the LacI inducible promoter PLacI,repressor from pPEPY-PF6-LacI plasmid.However,the results showed that the LacI system in this plasmid did not work normally in SEZ,and the transposase could still function without the presence of an inducer.The system needs further optimization.