马链球菌兽疫亚种突破猪脑微血管内皮细胞的分子机制

Streptococcus equi ssp.zooepidemicus(SEZ) is a major pathogen worldwide that causes swine streptococcosis, meningitis is the most striking feature of the infection,although other pathologies, such as septicemia ,arthritis have been described,but the mechanism is unclear.In this study, the transposon mutagenesis technology was employed to construct the SEZ library of transposon mutants and the porcine brain microvascular endothelial cells (PBMEC) was used as the blood brain barrier (BBB)model for SEZ. We tried to screen the virulence factors of SEZ which were related to cross the PBMEC. With the yeast two-hybrid technology, The proteins which could interact with these virulence factors related to cross the BBB from the constructed PBMEC library were screened ,after identification by immunoprecipitation and GST -pull down ,the adhesion receptor was found from these screened proteins . After SEZ infecting, we detected the IFN-γ and the other cytokines in supernatant and the expression level of metalloproteases in PBMEC, invasive assay was used to evaluate the influence to permeability of PBMEC, and the effect of SEZ hemolysin and streptokinase to PBMEC permeability was also evaluated. We employed the Oligo Signal Transduction Pathway Finder Microarray to detect the key factors of several Signal Pathways and tried to find out which signal pathways of PBMEC were related to the treatment of SEZ hemolysin and streptokinase. These results might be helpful to clarify the molecular mechanism of SEZ crossing PBMEC,and contribute to the understanding of the pathogenic mechanism of meningitis caused by SEZ, and also are helpful to design of novel medicines and vaccines to control swine streptococcosis.

马链球菌兽疫亚种（SEZ）旧称兽疫链球菌，是猪链球菌病的主要病原。其可引起猪的脑炎，但机制不明。本研究以SEZ强毒菌株ATCC35246为研究对象，利用转座子诱变技术，建立转座子突变体库，以猪脑微血管内皮细胞（PBMEC）为血脑屏障模型，筛选SEZ与突破血脑屏障模型相关的毒力因子，发现粘附蛋白，并通过小鼠模型评价其功能；构建PBMEC文库，利用酵母双杂交技术，从文库中筛选与SEZ突破血脑屏障相关毒力因子的互作蛋白，并进行鉴定，寻找相关粘附受体；同时通过SEZ对PBMEC的侵袭实验，评价SEZ产生的溶血素、链激酶和PBMEC金属蛋白酶表达量的变化对血脑屏障模型通透性的影响。在此基础上，采用细胞信号通路发现芯片分析蛋白互作等引起PBMEC细胞信号激活的途径。结果通过阐明SEZ突破PBMEC血脑屏障模型的分子机制，能进一步解析SEZ致猪脑炎的机理，并为相应的药物及疫苗设计提供依据。

马链球菌兽疫亚种（SEZ）旧称兽疫链球菌，是猪链球菌病的主要病原。其可引起猪的脑炎，但机制不明。本研究以SEZ 强毒菌株ATCC35246为研究对象，利用转座子诱变技术，建立转座子突变体库；同时建立鼠脑微血管内皮细胞（bEnd.3）、猪脑微血管内皮细胞（pBMEC）和人脑微血管内皮细胞（hBMEC）体外血脑屏障模型，筛选SEZ与突破宿主血脑屏障相关的毒力因子，并进行功能鉴定。利用SILAC技术，从SEZ中筛选到25个有结合宿主脑微血管内皮细胞活性的细菌蛋白，以上毒力因子与该菌突破血脑屏障相关。同时，从SEZ转座子突变体库中筛选到25个与通过血脑屏障相关的抗吞噬毒力因子。利用SCOTS 技术对SEZ生物被膜态的差异表达基因进行了筛选，鉴定出24个差异表达基因，初步阐明了SEZ生物被膜形成的机制。发现SEZ荚膜的缺失极显著的增加了SEZ对细胞的粘附和侵袭，证实arcA是SEZ耐受酸性环境的重要毒力因子，在SEZ通过特洛伊木马突进穿过血脑屏障中起重要作用。发现SEZ感染bEnd.3后，TNF-α、IL-6的分泌量显著增加（p＜0.05），并使MMP9等表达上升，促进了SEZ穿透bEnd.3血脑屏障模型。同时发现SEZ不表达链激酶，该酶与SEZ通过血脑屏障无关。揭示SEZ Fic蛋白很可能影响到宿主细胞内小G蛋白活性，引起细胞骨架变化，细胞形态改变，进而破坏血管内皮细胞形成屏障结构的完整性，促进细菌穿透血脑屏障。初步阐明了SEZ突破脑微血管内皮细胞的分子机制，为解析SEZ致猪脑炎的机理以及相应的药物或疫苗的设计提供了科学的理论依据。