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迟缓爱德华氏菌胞内侵染途径及与宿主补体调节因子CD59的互作研究
隋智海
学位类型博士
导师孙黎
2018-05-11
学位授予单位中国科学院大学
学位授予地点中国科学院海洋研究所
关键词迟缓爱德华菌, 巨噬细胞, 内吞途径, 补体系统, Cd59
摘要

   迟缓爱德华氏菌(Edwardsiella tarda)是一种革兰氏阴性致病菌,可感染多种宿主,尤其是鱼类,长期以来是最为严重的鱼类病原菌之一。研究表明,E. tarda能在宿主吞噬细胞如巨噬细胞中增殖,也能够抵抗宿主血清的免疫杀伤,然而这些现象的内在机制不清楚。本论文的目的是研究E. tarda巨噬细胞内的侵染途径,探索E. tarda与宿主(半滑舌鳎,Cynoglossus semilaevis)补体调节因子CD59CsCD59)的相互作用,从而促进我们对E. tarda逃逸宿主细胞免疫杀伤和补体免疫杀伤机制的理解。

    为了研究E. tarda的胞内侵染途径,我们首先验证了本实验室分离的鱼类致病菌E. tarda TX1可侵染小鼠巨噬细胞系RAW264.7并在其中增殖。利用不同内吞途径的抑制剂处理和RNA干扰技术(RNAi),发现抑制网格蛋白(Clathrin)和小窝蛋白(Caveolin)介导的内吞途径能显著地降低E. tarda的入侵,而抑制巨胞饮途径却对E. tarda入侵没有影响;通过免疫荧光实验,发现E. tarda能与ClathrinCaveolin发生共定位,其比例分别为23.6%20.4%;抑制ClathrinCaveolin介导的内吞途径后,RAW264.7对灭活和非灭活E. tarda相对摄入率都会明显降低,说明这两条内吞途径对E. tarda进入巨噬细胞并非是特异的。内吞体酸化抑制剂处理细胞后,E. tarda的入侵明显降低;免疫荧光显微镜观察发现活E. tarda都可与前期内体(Early endosome)、晚期内体(Late endosome)及内体溶酶体(Endolysosomes)的标志蛋白Rab5Lamp1Cathepsin D共定位,其比例分比为29.6%28.7%36.7%Lyso-tracker染色显示E. tarda存在于酸性细胞器中。灭活E. tarda也具有相似的共定位现象。这些结果说明E. tarda进入胞内后会经历前期内体、后期内体及内体溶酶体途径。此外,抑制微丝(Microfilament)和微管(Microtubule)会显著降低E. tarda的入侵,并且E. tarda能够与微丝和微管共定位,说明E. tarda进入巨噬细胞的过程是依赖于细胞骨架的。总而言之,本论文结果首次揭示了E. tarda极有可能是以一种被动和不依赖毒力的方式进入细胞,这种方式依赖于Clathrin-Caveolin-介导的内吞途径及细胞骨架。

    补体系统是免疫系统中不可缺少的一部分,由多种蛋白组成。其中,CD59是补体系统的一种调节因子,可抑制补体系统膜攻击复合物的形成,保护自身细胞不被补体伤害。为研究E. tarda是否可以利用CD59来逃逸鱼类补体系统杀伤,我们首先对CsCD59进行基因克隆和蛋白表达纯化。序列比对发现CsCD59107个氨基酸组成,具有保守的CD59结构特征,与已知鱼类CD59氨基酸序列相同度介于33%-46%之间qRT-PCR检测发现在正常生理条件下,CsCD59在多个组织中表达,在肝脏中表达水平最高,在血中表达量最低;E. tarda等病原菌刺激后,肝脏、脾脏和肾脏中CsCD59的表达水平显著上调。兔血红细胞溶解实验证实重组CsCD59rCsCD59)能显著地抑制半滑舌鳎补体系统的激活。ELISA和免疫荧光显微镜观察发现rCsCD59能结合E. tarda等革兰氏阴性菌以及Bacillus subtilis等革兰氏阳性菌。这种结合作用对细菌本身存活没有影响,但能显著地提高E. tarda细菌在半滑舌鳎血清中的存活率。这些结果显示鱼类CD59能促进E. tarda等细菌逃逸补体介导的杀伤作用,E. tarda很可能通过上调CD59的表达并与CD59相互作用而得以在血清中存活

其他摘要

Edwardsiella tarda is a Gram-negative bacterial pathogen that can infect a variety of hosts, especially fish, and has been one of the most serious fish pathogens. Previous studies have shown that E. tarda can reproduce in host phagocytic cells such as macrophages and can also resist immune killing of host serum. However, the mechanism of these phenomena is unclear. The purpose of this paper is to study the infection pathway of E. tarda in macrophages and to explore the interaction between E. tarda and complement regulatory factor CD59 (CsCD59) of Cynoglossus semilaevis, which will promotes us to comprehend the mechanism of immune evasion of E. tarda.

To explore the intracellular trafficking pathway of E. tarda, we found that E. tarda TX1 entered RAW264.7 and multiplied intracellularly in a robust manner. Cellular invasion of E. tarda was significantly impaired by inhibition of clathrin- and caveolin-mediated endocytic pathways and by inhibition of endosome acidification, but not by inhibition of macropinocytosis. Consistently, RAW264.7-infecting E. tarda was co-localized with clathrin, caveolin, and hallmarks of early and late endosomes, and intracellular E. tarda was found to exist in acid organelles. In addition, E. tarda in RAW264.7 was associated with microfilament and microtubule, and blocking of the functions of these cytoskeletons by inhibitors significantly decreased E. tarda infection. Furthermore, formaldehyde-killed E. tarda exhibited routes of cellular uptake and intracellular trafficking similar to that of live E. tarda. Together these results provide the first evidence that entry of live E. tarda into macrophages is probably a passive, virulence-independent process of phagocytosis effected by clathrin- and caveolin- mediated endocytosis and cytoskeletons, and that the intracellular traffic of E. tarda involves endosomes and endolysosomes.

The complement system is an integral part of immunity system and consists of multiple proteins. CD59 is a regulator of the complement system that inhibits the formation of membrane attack complexes in the complement system and protects endogenous cells from lysis. To investigate whether E. tarda can utilize CD59 to escape the killing of the fish complement system, we firstly performed molecular cloning, protein expression and purification of CsCD59. CsCD59 possesses the conserved structural features of CD59 and shares 33%-46% sequence identities with other fish CD59. Expression of CsCD59 was high in liver, spleen, and muscle, and was stimulated by infection of bacterial pathogens. Recombinant CsCD59 (rCsCD59) exhibited an apparent inhibition effect on the activation of tongue sole serum complement. ELISA and microscopy detected binding of rCsCD59 to a number of Gram-negative and Gram-positive bacteria. Interaction with rCsCD59 did not affect bacterial viability but significantly enhanced bacterial resistance against the killing effect of fish serum. Together these results indicate that fish CD59 may to some degrees facilitate a general escape of bacteria from complement-mediated immunity.

语种中文
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/154502
专题中国科学院海洋研究所
第一作者单位中国科学院海洋研究所
推荐引用方式
GB/T 7714
隋智海. 迟缓爱德华氏菌胞内侵染途径及与宿主补体调节因子CD59的互作研究[D]. 中国科学院海洋研究所. 中国科学院大学,2018.
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