Screening And Identification Of Virulence Factors Involving In The Traversal Of Streptococcus Suis Across The Human Blood-brain Barrier

Streptococcus suis serotype2is an important zoonotic pathogen that can cause severesystemic infection in humans with high mortality and morbidity. Meningitis is the mostimportant clinical symptom of S.suis infection in humans. Similar to other bacterialmeningitis, Streptococcus suis also caused meningitis through multi-step process, andhow Streptococcus suis traverses the blood-brain barrier （BBB） into the central nervoussystem （CNS） is the most critical step.The BBB is an important structural and functional barrier, and the main functions are toprotect CNS by preventing the entry of blood-bourne toxic compounds and maintain theCNS homeostasis. The BBB is composed of brain microvascular endothelial cells（BMEC）, astrocytes and pericytes. Among them, Brain microvascular endothelial cell isthe most important. Therefore, the study of the interaction of Streptococcus suis andbrain microvascular endothelial cells have a great significance to reveal the mechanismof Streptococcus suis across the BBB and meningitis.We used human brain microvascular endothelial cells （hCMEC/D3） as an in vitro BBBmodel in our previous work. And our experiment results had demonstratedStreptococcus suis traversed the BBB by paracellular pathway. In this study, our goal isto screen the virulence factors involved in the traversal of Streptococcus suis across theBBB by in vitro model and explored the molecule mechanism. Severe possiblevirulence genes identified in our previous work such as SSU05₁000, SSU05_MRP,SSU05₁815and SSU05₁664were evaluated their role on the traversal of S.suis across the BBB. Results showed that gene-deficient of SSU05₁000or SSU05_MRPsignificantly decreased the traversal rate of S.suis across the BBB, while other knockoutstrains had no influence. Therefore, we speculated that SSU05₁000, SSU05_MRPmight involve in the traversal of Streptococcus suis across the BBB.SSU05₁000is a new identified cell wall protein with immunogen activity. Ourprevious animal experiments showed that: SSU05₁000contributed to the survival ofS.suis in blood and the entry of bacteria to the brain. However, it is not clear to thedetailed mechanism and there are no any reports on this protein.In order to study the function of SSU05₁000, we prepared the recombinant protein inE.coli. Briefly, SSU05₁000gene was amplified and cloned into pET30a vector to getthe pET30a-SSU05₁000recombinant expression plasmid which was then transformedinto E.coli BL21（DE3）. The recombinant protein was induced by IPTG and identifiedas soluble expression by SDS-PAGE electrophoresis. Then, the recombinant protein waspurified by nickel affinity chromatography and decontaminated the lipopolysaccharide（LPS） by TritonX-114. Finally, we get the recombinant SSU05₁000protein with thepurity of above90%.In order to further verify the role of SSU05₁000in the traversal of Streptococcus suisacross the BBB, we pretreated the hCMEC/D3cells monolayers by the recombinantprotein, then evaluated the traversal ability of the05ZYH33△1000. Results show:SSU05₁000protein can recover the ability of05ZYH33△1000to the level of wildstrain05ZYH33. In order to ascertain what signaling pathways were being activated andwhether they mediated the traversal of S.suis across the BBB, we treated thehCMEC/D3monolayers with specific signaling pathway inhibitors. Results show that:inhibition of p38MAPK or JNK pathway can significantly decrease the traversal rate of S.suis05ZY across the BBB. In addition, SSU05₁000can activating p38, JNKsignaling pathway by Western blot analysis. These data show that activation of p38MAPK or JNK signaling pathway induced by SSU05₁000is sufficient to the traversalof S.suis across human BBB.In summary, our results indicated that SSU05₁000involved in the traversal ofStreptococcus suis across the BBB by activating p38, JNK signaling pathway whichmight contribute to destroy the integrity of the BBB by opening the tight junctions.