MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.
Jiangsu Academy of Agricultural Sciences, Veterinary Research Institute, Nanjing, China.
Cell Microbiol. 2018 Oct;20(10):e12862. doi: 10.1111/cmi.12862. Epub 2018 Jun 11.
Streptococcus suis serotype 2 (SS2) is a zoonotic agent that causes meningitis in humans and pigs. However, the mechanism whereby SS2 crosses the microvasculature endothelium of the brain is not understood. In this study, transposon (TnYLB-1) mutagenesis was used to identify virulence factors potentially associated with invasive ability in pathogenic SS2. A poorly invasive mutant was identified and was found to contain a TnYLB-1 insertion in the serine/threonine kinase (stk) gene. Transwell chambers containing hBMECs were used to model the blood-brain barrier (BBB). We observed that the SS2 wild-type ZY05719 strain crossed the BBB model more readily than the mutant strain. Hence, we speculated that STK is associated with the ability of crossing blood-brain barrier in SS2. In vitro, compared with ZY05719, the ability of the stk-deficient strain (Δstk) to adhere to and invade both hBMECs and bEnd.3 cells, as well as to cross the BBB, was significantly attenuated. Immunocytochemistry using antibodies against claudin-5 in bEnd.3 cells showed that infection by ZY05719 disrupted BBB tight junction proteins to a greater extent than in infection by Δstk. The studies revealed that SS2 initially binds at or near intercellular junctions and crosses the BBB via paracellular traversal. Claudin-5 mRNA levels were indistinguishable in ZY05719- and Δstk-infected cells. This result indicated that the decrease of claudin-5 was maybe induced by protein degradation. Cells infected by ZY05719 exhibited higher ubiquitination levels than cells infected by Δstk. This result indicated that ubiquitination was involved in the degradation of claudin-5. Differential proteomic analysis showed that E3 ubiquitin protein ligase HECTD1 decreased by 1.5-fold in Δstk-infected bEnd.3 cells relative to ZY05719-infected cells. Together, the results suggested that STK may affect the expression of E3 ubiquitin ligase HECTD1 and subsequently increase the degradation of claudin-5, thus enabling SS2 to traverse the BBB.
猪链球菌 2 型（SS2）是一种人畜共患病原体，可引起人类和猪的脑膜炎。然而，SS2 穿过大脑微血管内皮的机制尚不清楚。在本研究中，转座子（TnYLB-1）诱变用于鉴定与致病性 SS2 侵袭能力相关的潜在毒力因子。鉴定到一个侵袭能力较弱的突变体，发现其丝氨酸/苏氨酸激酶（stk）基因中存在 TnYLB-1 插入。使用含有 hBMECs 的 Transwell 室来模拟血脑屏障（BBB）。我们观察到 SS2 野生型 ZY05719 菌株比突变菌株更容易穿过 BBB 模型。因此，我们推测 STK 与 SS2 穿越血脑屏障的能力有关。在体外，与 ZY05719 相比，stk 缺失菌株（Δstk）黏附和侵袭 hBMECs 和 bEnd.3 细胞以及穿过 BBB 的能力显著减弱。用针对 bEnd.3 细胞中闭合蛋白-5 的抗体进行免疫细胞化学染色显示，与 Δstk 感染相比，ZY05719 感染更能破坏 BBB 紧密连接蛋白。研究表明，SS2 最初结合在细胞间连接附近或附近，并通过细胞旁穿越穿过 BBB。在 ZY05719 和 Δstk 感染的细胞中，闭合蛋白-5 的 mRNA 水平没有区别。这一结果表明，闭合蛋白-5 的减少可能是由蛋白降解引起的。与 Δstk 感染的细胞相比，感染 ZY05719 的细胞显示出更高的泛素化水平。这一结果表明泛素化参与了闭合蛋白-5 的降解。差异蛋白质组学分析显示，E3 泛素蛋白连接酶 HECTD1 在 Δstk 感染的 bEnd.3 细胞中的表达水平比 ZY05719 感染的细胞低 1.5 倍。总的来说，结果表明 STK 可能影响 E3 泛素连接酶 HECTD1 的表达，随后增加闭合蛋白-5 的降解，从而使 SS2 能够穿过 BBB。
