内肽酶O对马链球菌兽疫亚种突破血脑屏障的影响

Role of endopeptidase O on the breakthrough of the blood-brain barrier of Streptococcus equi ssp.zooepidemicus

运用同源重组法构建马链球菌兽疫亚种(SEZ) ATCC35246株(SEZ WT)pepO基因缺失株(SEZ△pepO)及互补株(SEZ C△pepO),比较分析pepO基因对SEZ形态特征、生长性能、感染小鼠的能力、小鼠全血存活率及小鼠脑组织定殖能力的影响;观察感染后小鼠脑组织的病理变化;选取人脑微血管内皮细胞(hBMEC)为体外血脑屏障模型细胞,测定pepO基因对hBMEC细胞的黏附侵袭能力以及对体外血脑屏障模型的穿透能力的影响。结果显示,pepO基因缺失后不影响SEZ在培养基中的生长速率,也不影响SEZ在小鼠全血中的存活率,但缺失株感染小鼠的能力显著降低(P<0.05),在小鼠脑组织细菌载量显著降低(P<0.05);SEZ WT株和SEZ C△pepO株感染组小鼠脑组织出现明显充血、出血、水肿的脑膜炎症状,组织切片可见大量出血及炎性细胞浸润,SEZ△pepO株感染组小鼠脑组织未见明显病变;SEZ△pepO对hBMEC细胞的黏附侵袭率以及对体外血脑屏障模型的穿透率较SEZ WT和SEZ C△pepO均显著降低(P<0.001)。本研究初步证明内肽酶O与SEZ毒力密切相关,并可促进SEZ穿透血脑屏障,有助于进一步阐明SEZ致脑膜炎的机制。

The purpose of this study is to explore the role of endopeptidase O(pepO) gene of Streptococcus equi subsp.zooepidemics(SEZ) in the process of SEZ breaking through the blood-brain barrier.The homologous recombination method was used to construct pepO gene deletion strain(SEZ △pepO) and complementary strain(SEZ C△pepO) of SEZ ATCC35246(SEZ WT).The effects of pepO gene in SEZ on morphological characteristics, growth performance, the ability to infect mice, the survival rate in whole blood of mice and the ability to colonize mouse brain tissue were analyzed.The pathological changes of brain tissue were observed after infection.Human brain microvascular endothelial cells(hBMEC) were used as in vitro blood-brain barrier model cells to detect the effect of pepO gene on adhesion and invasion of hBMEC cells and penetration of blood-brain barrier model in vitro.The results showed that deletion of the pepO gene did not affect the growth rate of SEZ in the THB medium or the survivability in the whole blood of mice, but significantly reduced the ability to infect mice(P<0.05) and the bacterial load in mouse brain tissue(P<0.05).Brain tissues of mice infected with SEZ WT and SEZ C△pepO strains showed signs of meningitis with obvious congestion, hemorrhage and edema, and the sections showed massive hemorrhage and inflammatory cell infiltration, while brain tissues of mice infected with SEZ △pepO strains showed no obvious lesions.The adhesion and invasion rate of SEZ △pepO to hBMEC cells and the penetration rate of the in vitro blood-brain barrier model were significantly lower(P<0.001)than both SEZ WT and SEZ C△pepO.The present study provides preliminary evidence that endopeptidase O is closely related to SEZ virulence and can promote SEZ to penetrate the blood-brain barrier,which helps to further elucidate the mechanism of SEZ-causing meningitis.