马链球菌兽疫亚种（SEZ）是引起猪链球菌病的主要病原菌之一，可引发动物或人类败血症，脑膜脑炎等疾病，其中穿透血脑屏障是SEZ引起脑膜脑炎的重要环节。研究发现敲除bifA基因后，SEZ穿透宿主血脑屏障能力显著降低；BifA蛋白可由细菌释放至环境中并进入宿主脑微血管内皮细胞，破坏血脑屏障完整性，但其分泌及侵入脑微血管内皮细胞机制尚不清晰。本项目从外泌体分泌途径及BifA自身功能结构域研究出发，利用荧光示踪技术，分析BifA的分泌及入胞途径，同时阐明其功能性结构域在此过程中发挥的作用；构建人脑微血管内皮细胞CRISPR突变文库，在此基础上利用流式细胞仪分选以及Miseq测序，获得参与BifA入胞的靶蛋白及相关信号通路。之后采用CRISPR基因敲除，信号通路抑制剂和猪原代脑微血管内皮细胞进行后续验证，最终揭示BifA蛋白分泌及进入猪脑微血管内皮细胞的分子机制，进一步阐明SEZ突破血脑屏障机制。

Streptococcus equi ssp. zooepidemicus (SEZ) is an important zoonotic pathogen which can cause septicemia and meningoencephalitis in both human and animals. The vital step for SEZ to invade brain is penetrating across blood brain barrier (BBB). In previous research, we found that the BBB penetration capacity of SEZ, which contains defective bifA gene, will decrease significantly. BifA protein can be released to environment by SEZ and enter into host brain microvascular endothelial cells (BMECs), BBB integrity collapsed consequently. The mechanism of BifA secretion and entry to host cells are still unknown. In this research, we focus on bacterial extracellular vesicles and BifA functional domain, employing GFP tracking technology to analyze BifA secretion and the role of each functional domains during its interaction to BMECs; CRISPR mutant library based on BMECs will be constructed, combine with flow cytometry sorting and Miseq to identify target protein and signal pathways correlate BifA entry to BMECs. CRISPR knock-out, signal pathway inhibitors and porcine primary BMECs model will be used for further validation research. The purpose of this study is illustrating the mechanism of BifA secretion by SEZ and its invasion to BMECs, filling gaps in research of BBB penetration mechanism of SEZ.