β-毒素调控Claudin-5在金黄色葡萄球菌感染中枢神经系统中的作用机制研究

通过血脑屏障是细菌感染中枢神经系统的第一步。金葡菌是脑膜炎和脑脓肿的常见病原菌，该菌破坏血脑屏障感染中枢的机制至今不清。我们前期发现，金葡菌β-毒素（Hlb）在细胞及动物模型中均有损伤血脑屏障的作用；Hlb可导致靶细胞紧密连接中Claudin-5表达下调，而后者受转录因子FoxO1调控。故提出科学假设：金葡菌Hlb发挥鞘磷脂酶活性，分解内皮细胞的鞘磷脂产生神经酰胺，激活JNK通路，通过FoxO1抑制Claudin-5表达，导致血脑屏障破坏，促进金葡菌入侵。为验证该假设，本项目拟构建hlb敲除/回补及标记菌株，用活体荧光示踪，免疫荧光等技术，结合动物及细胞模型明确Hlb对血脑屏障的破坏作用；再通过色谱法检测神经酰胺，RT-qPCR、Western blot检测及抑制剂阻断等实验，探讨Hlb通过FoxO1调控Claudin-5破坏血脑屏障的机制。研究结果对阐明金葡菌感染中枢的机制具有重要意义。

Penetrating the blood-brain barrier is the first step in the central nervous system (CNS) infection by bacteria. Staphylococcus aureus (S. aureus) is a common pathogen of meningitis and brain abscess. The mechanism by which the bacteria destroys the blood-brain barrier and infects the CNS is still unclear. We have previously found that S. aureus β-toxin (Hlb) can damage the blood-brain barrier in both cell and animal models; Hlb down-regulates the expression of Claudin-5 in tight junctions of target cells, which is regulated by the transcription factor FoxO1. Therefore, a scientific hypothesis is proposed: S. aureus Hlb exerts sphingomyelinase activity, which can decompose sphingomyelin of endothelial cells to produce ceramide, activate the JNK pathway, and inhibit the expression of Claudin-5 through FoxO1, resulting in the destruction of the blood-brain barrier and promoting the invasion of S. aureus. In order to verify this hypothesis, this project intends to construct hlb knockout/complement and labeling strains, applying in vivo fluorescence tracing, immunofluorescence and other techniques, combined with animal and cell models to clarify the destructive effect of Hlb on the blood-brain barrier; and then detect ceramide by chromatography, perform RT-qPCR, Western blot and inhibitor blocking experiments to explore the mechanism by which Hlb regulates Claudin-5 through FoxO1 in destruction of the blood-brain barrier. The research results are of great significance to elucidate the mechanism of S. aureus infection of the CNS.