致幼畜脑膜炎粪肠球菌穿越血脑屏障的机制初探

Enterococcus faecalis, as an important opportunistic pathogen in nosocomial environment, has a unique mechanism of drug resistance and opportunistic infection. And virulence relates to drug resistance characteristics. With the increasing of meningitis cases caused by Enterococcus faecalis in young stock year by year, it’s especially important to study on the mechanism of Enterococcus faecalis traversing the blood-brain barrier(BBB) into central nervous system. Therefore, this study constructed the model of the white mice blood-brain barrier in vitro by using Millicell and evaluated it. Extracting the whole genome of meningitis-causing Enterococcus faecalis and build the genome phage display libraries, using the cerebral microvascular endothelial cell in the model as target cells to screen the virulence factors which may be related by phage display libraries. Finally, construct the mutants with one or several virulence factor genes deleted, using the white mice as experimental animal to detect the optimal dose of artificial infection, observe the effect on permeability (MBP and Evans blue) and morphology (Electron microscope) of blood-brain barrier and evaluate the tight junction (Occludin and ZO-1) and integrity of basement membrane (MMP-9). In order to provide a reliable theoretical basis for revealing the pathogenesis of meningitis caused by Enterococcus faecalis in young stock and protect animals’ safety absolutely from Enterococcus faecalis.

粪肠球菌作为医源性感染的重要机会致病菌之一，存在着独特的耐药和机会感染机制。随着粪肠球菌引起幼畜脑膜炎病例的逐年增多，对其如何穿越血脑屏障进入中枢神经系统机制的研究显得尤为重要。为此，本试验采用Millcell的方法构建小白鼠血脑屏障体外模型并对其进行评价；然后提取致脑膜炎粪肠球菌的全基因组，构建其噬菌体随机展示文库，以构建的体外模型中脑微血管内皮细胞作为靶细胞，利用噬菌体随机展示文库筛选出与穿越血脑屏障可能相关的毒力因子；最后构建出一种或几种毒力因子缺失的突变体，以小白鼠作为试验动物，检测该突变株人工感染的最佳剂量，观察对小白鼠血脑屏障通透性（MBP和伊文思蓝）和血脑屏障形态学的影响（电镜），评估紧密连接（Occludin和ZO-1）和基膜的完整性（MMP-9）。以期为揭示粪肠球菌致幼畜致脑膜炎的发病机制提供可靠的理论依据，有望使得粪肠球菌这一机会感染菌“无计可施”。

粪肠球菌作为医源性感染的重要机会致病菌之一，存在着独特的耐药和机会感染机制。随着粪肠球菌引起幼畜脑膜炎病例的逐年增多，对其如何穿越血脑屏障进入中枢神经系统的机制研究显得尤为重要。为此，研究首先建立粪肠球菌感染小鼠穿越血脑屏障和体外血脑屏障损伤的最佳模型，观察对血脑屏障通透性及紧密连接的影响，采用全基因组噬菌体随机展示文库技术筛选粪肠球菌穿越血脑屏障的相关毒力因子并研究其生物学特性，并构建小鼠血脑屏障损伤模型的mRNA水平转录文库并验证。研究结果表明共培养模型是粪肠球菌穿越小鼠血脑屏障的最佳模型，模型中小鼠脑组织EB含量和血清MBP浓度明显升高，透射电镜观察到紧密连接结构松散，结构蛋白ZO-1、Occludin的mRNA和蛋白的表达量明显降低，基质金属蛋白酶MMP-9的mRNA和蛋白的表达量显著升高，表明粪肠球菌可以导致小鼠血脑屏障的通透性增强和紧密连接的结构破坏；粪肠球菌全基因组噬菌体随机展示文库筛选出DNA指导RNA聚合酶、胶原酶样蛋白酶、假定的膜蛋白、透明质酸酶、溶血素、糖原磷酸化酶、烯醇化酶、纤维蛋白原结合蛋白和表面锚定蛋白等9个参与粪肠球菌穿越血脑屏障的毒力因子；携带不同毒力因子的粪肠球菌能造成脑内氧化应激反应，进而加重血脑屏障的损伤，其中Esp、CylA基因在对血脑屏障损伤起着重要的作用。成功构建小鼠脑组织转录文库，并筛选出在致脑膜炎粪肠球菌感染后的差异表达基因，候选基因的低表达对PI3K/AKT信号通路具有影响。另外p38 MAPK信号通路的激活也参与到血脑屏障的损伤过程中。研究结果初步揭示出粪肠球菌致致脑膜炎的发病机制，为粪肠球菌这一机会感染菌“无机可施”奠定了基础。