The Effect And The Mechanism Of Enolase From Streptococcus Suis Type2on Promoting Blood Brain Barrier Permeability

Streptococcus suis serotype2(SS2) is an important zoonotic pathogen, and itsmain clinical symptoms is meningitis, with high mortality, high morbidity and highincidence of sequelae. Currently, the pathogenesis of S. suis meningitis is not clear. Ingeneral, Streptococcus suis could develop meningitis through a series of bacteria-hostinteractions. How the Streptococcus suis cross the blood-brain barrier and enter thecentral nervous system is one of the most critical step. Blood-brain barrier is astructural and functional barrier which mainly consist of brain microvascularendothelial cells (BMEC), astrocytes (AC) and peripheral cells. The primary site ofbreakdown of the BBB in most bacterial meningitis appears to be BMEC.In our previous work, the SS2genome phage display libraries were built to screenthe main virulent factors which may be involved in the destruction of the blood-brainbarrier by SS2, and we verified that Enolase was one of the factors which wererelatived with BBB. In this study, Enolase from SS2was prokaryoticly expressed andthe effect of Enolase on BBB permeability in vitro was detected by constructing theBBB model in vitro. Meanwhile, the effect of Enolase on mice BBB permeability invivo was detected by Evans blue method, and the mechanism of Enolase on BBBpermeability were explored. All of that could lay a theoretical foundation for SS2meningitis prevention and therapy.The results shows that Eno could be expressed in E.coil BL21Codon Plus (DE3),that Eno was soluble protein and the purity of Eno was more than95%and that thepolyclonal antibody of Eno was also obtained. Then BBB model constructed byco-culture of procine brain microvascular endothelial cells and astroglia cell wastreated with Eno. The trans epithellal electric resistance (TEER) of BBB in Eno groupwas found to be lower than that of control group and the concentration of horse radishperoxidase (HRP) which was in the lower chamber of the culture chamber in Enogroup was higher than that of control group. Meanwhile, Eno was injected into mice by tail vein injection and the EB content of brain in Eno group increased. It was illustratedthat Eno could increase BBB permeability and damage the integrity of BBB.After PBMECs were treated with Eno, we found that Eno could have cytotoxicityon PBMEC, inhibit cell proliferation, induce cell apoptosis, inhibit the expression oftight junction and promote cell to secrete IL-8、 IL-6、 IL-1α and MCP-1. It wassuggested that Eno may increase the BBB permeability through the above mechanisms.In the Cytokine antibody microarray experiment, we found that Eno could promotePBMECs of BBB model in vitro to secrete cytokine such as IL-1β、IL-6、IL-8、IL-10、TNFa、TGFβ1,that the change folds of IL-8in Eno group was significant comparedwith the control group at6h,12h and that IL-8at24h remained relatively high levels. Itwas suggested that IL-8may play an important role in Eno increasing the BBBpermeability. We also detected the signaling pathway by which Eno promotedPBMECs to secrete IL-8and the effect of IL-8on the BBB permeability in vitro. Theresults showed that Eno could activate PBMEC p38MAPK and ERK signalingpathway to secrete IL-8and IL-8decreased TEER of BBB, increased the concentrationof HRP which was in the lower chamber of the culture chamber, and increased theBBB permeability.In conclution, Eno increased the BBB permeability, and the mechanism was likelythat Eno had cytotoxicity on PBMECs, induceed cell apoptosis, inhibited theexpression of tight junction or Eno could activate PBMEC p38MAPK and ERKsignaling pathway to secrete IL-8, then IL-8increased the BBB permeability. The studyenriches the pathogenesis of meningitis caused by SS2and provides a theoretical basisfor prevention and therapy of meningitis caused by SS2.