The Mechanism Of Brain Microvascular Endothelial Cell Apoptosis That Enhances Blood Brain Barrier Permeability Induced By Enolase Of Streptococcus Suis Type 2

Streptococcus suis Type 2(SS2)is an important pathogen which can cause several symptoms such as meningitis,arthritis,endocarditis and sepsis both in pig and human being.And meningitis is the most severe disease.SS2 infection not only causing tremendous loss in economy but also seriously threatening public health.Enolase(Eno)is a crucial enzyme which can catalyze diphosphoglycerate transforming into phosphoenolpyruvic acid during glycolysis process.Besides function as a catalyst,more and more recent research finds that procaryotic Eno plays an important role in pathogen infection.Eno has already been screened as a critical interaction molecule by our research group through the interaction of genomic random display library and co-culture porcine brain microvascular endothelial cells(PBMEC)/Astrocytes(AC)model.Yet there is no relevant reports demonstrating the function of Eno in the process of SS2 breaking through Blood-Brain Barrier(BBB)and causing meningitis.Thus,the following work was focused on the functions and mechanisms of Eno causing meningitis in this study:1.In order to illuminate the function of Eno,prokaryotic expression of recombinant Eno was performed and BBB co-culture model was established.After the interaction between Eno and BBB model,we find that Eno can increase the permeability of BBB model based on the measurement of model transendothelial cell resistance(TEER).Further more,anti-Eno can also suppress the ability of SS2 adhering to and invading into PBMEC and breaking into BBB.Intravenous anti-Eno significantly promotes the survival rate of ICR mice infected by SS2.Besides,anti-Eno can reduce the permeability of SS2 infected mice brain for Evans Blue.The above results showed that Eno mediated SS2 infection and SS2 damage to BBB.2.In order to explore the mechanisms of Eno damage to BBB,several methodsuch as Annexin-V/PI double staining and Western blot(WB)were performed to analyze the apoptosis of Eno treated PBMEC.The results showed that Eno can induce PBMEC apoptosis through the Caspase-8/Caspase-3 pathway.Anti-Eno can decrease PBMEC apoptosis induced by SS2.These two results confirmed crucial function of Eno during cell apoptosis.In order to investigate the effect of apoptosis on SS2 invasion of BBB,apoptosis inhibitor was used to inhibit the apoptosis of PBMEC in BBB model,and the results showed that inhibition of apoptosis could significantly inhibit SS2 bireaking into of BBB model.Besides apoptosis,the expression of tight junction proteins(TJs)of Eno treated PBMEC was detected in by RT-PCR,WB and immunofluorescence.The result showed that the expression of TJs ZO-1 and Occludin were prominently decreased.Mouse model infected with BL21-Eno strain was established and TJs expression of brain was detected by WB.The results showed that the expression levels of zo-1 and Occludin in BL21-Eno infected mice were significantly lower than that of BL21-23 a infected group.The above results showed that Eno could increase BBB permeability by inducing apoptosis and inhibiting the expression of ZO-1 and Occludin of PBMECs.3.In order to find out the receptor of Eno on PBMEC,Pull down,Mass spectrometry,Co-immunoprecipitation were performed in this study.finally that RPSA is discovered to interact with Eno on the cell surface.And the immunofluorescence analysis find the co-location of Eno and RPSA on the cell surface.4.To explore the role of RPSA and HSPD1 in Eno induced PBMEC apoptosis and in Eno inhibited TJs expression,the relationship of Eno,RPSA and HSPD1 was analyzed.WB results showed that RPSA and HSPD1 were highly expressed in Eno treated cells.Knockdown or knockout RPSA could significantly inhibit HSPD1 expression in Eno treated cells.while knockdown HSPD1 could not inhibit RPSA expression.To sum up,Eno-RPSA interaction induced highly HSPD1 expression in PBMEC.Further research results showed that knockdown or knockout RPSA and knockdown HSPD1 could significantly inhibit Eno induced apoptosis.knockdown or knockout of RPSA could attenuate Eno induced inhibition of TJs expression,but knockdown HSPD1 has no effect on inhibition of TJs expression.Cytoplasmic phosphorylated proteomics and nuclear proteomics analysis were performed to further explore the mechanism of Eno-RPSA interaction induced high HSPD1 expression.After verification and analysis by WB,p38 and ERK were found to be activated,and the downstream nuclear transcription factor e IF4 E was activated.HSPD1 expression was enhanced consequently.5.In order to validate expression of related signaling molecules in vivo,AAV9 with sh RPSA or sh HSPD1 were injected to mice brain to establish brain model of knockdown RPSA and or HSPD1.Various indicators of blood and brain samples were analyzed after Eno injection or SS2 infection.The results showed that the gene knockdown RPSA and HSPD1 significantly improved the survival rate of SS2 infected mice,and weakened the damage of SS2 to the brain.Immunohistochemical method was performed to analyze the expression of signaling molecules(RPSA,HSPD1,p38,ERK,e IF4 E,Caspase-3,ZO-1,Occludin,etc.)in brain.The in vivo results were consistent with the results in vitro test.SS2 meningitis and non-meningitis models were established and signaling molecules of brain were analyzed by immunohistochemical method.The in vivo test results were consistent with the results in vitro test.In summary,this study found that Eno activates the downstream p38/ERK-e IF4 E signal by binding to the cell surface RPSA,which leads to increased expression and extracellular secretion of HSPD1 and consequently cell apoptosis.Eno-RPSA interaction activates cellular Rho A signal,thereby inhibiting the expression of ZO-1and Occludin,resulting in increased BBB permeability.The discovery and mechanism clarification of the above phenomena provide an important theoretical basis for the mechanism research of SS2 breakthrough BBB,which is of great significance to the prevention and treatment of meningitis.