鱼类病原菌荧光假单胞菌的致病机制及宿主抗菌免疫因子的作用研究

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	鱼类病原菌荧光假单胞菌的致病机制及宿主抗菌免疫因子的作用研究
	孙园园1,2
学位类型	博士
导师	孙黎
	2017-05-22
学位授予单位	中国科学院大学
学位授予地点	北京
学位专业	海洋生物学
关键词	荧光假单胞菌毒力因子疫苗半滑舌鳎抗感染
摘要	海水养殖鱼类经常被病原微生物所感染，导致产量下降，给水产养殖业带来巨大的经济损失。目前鱼类病害防治仍没有有效的措施，原因之一是病原的致病机制和宿主的免疫防御机制尚未完全阐明。本文从病原和宿主免疫两方面入手，研究了鱼类常见病原菌荧光假单胞菌(Pseudomonas fluorescens)的致病机制和鱼类宿主半滑舌鳎(Cynoglossus semilaevis)免疫因子(杀菌/通透性增强蛋白BPI和B型甘露糖结合凝集素BML)的抗细菌感染功能。本文通过蛋白质组学技术，首先比较了在正常和缺铁培养环境中荧光假单胞菌TSS全菌蛋白的差异表达情况。在蛋白表达谱中发现了22个具有显著性差异表达的蛋白，通过查阅文献和结构分析，选取了其中四个，即Hemo(血红素加氧酶)、PspB(丝氨酸蛋白酶)、Sod(超氧化物歧化酶)和TfeR(TonB依赖型外膜受体)，分别构建了基因突变株，即TSSDhemo、TSSDpspB、TSSDsod和TSSDtfeR并进行了功能研究。实验表明，与野生株TSS相比，TSSDhemo、TSSDpspB和TSSDtfeR在组织侵染、致死率和抗宿主血清杀伤等方面的能力都显著降低，表明pspB、hemO、tfeR和fha是参与感染的毒力基因；对宿主的免疫保护实验结果显示，当重组的TfeR和PspB以亚单位疫苗的形式免疫鱼类宿主后，宿主对于TSS的侵染产生了显著的免疫保护效应；抗体阻断实验表明，抗TfeR的抗体抑制了荧光假单胞菌TSS对宿主细胞的侵染。这些结果表明，环境中铁的含量会影响荧光假单胞菌大量蛋白的表达发生变化，其中包括一些重要的毒力因子。其次同样利用蛋白质组学技术，比较了在正常和缺铁培养条件下荧光假单胞菌TSS外分泌蛋白的差异表达情况。在外分泌蛋白表达谱中，发现了15个具有显著性差异表达的蛋白，经分析预测选取了一个上调蛋白——丝状血凝素(FHA)，并构建了编码该蛋白的基因突变株即TSSfha，比较分析了突变株与野生株在毒力及生理方面的差异，并进一步研究了FHA所发挥的功能。体外实验表明，相比较于野生株TSS，突变株（一）生长速度基本没有改变，但生物膜的形成能力和产生胞外丝状物的能力显著下降；（二）鞭毛受到破坏，运动性下降；（三）粘附宿主细胞的能力和自凝集能力显著下降；（四）凝血能力和抗宿主血清杀伤的能力显著下降。体内感染实验表明，突变株感染宿主组织的能力显著降低，对宿主的致死率下降；抗体阻断天然FHA后，野生株TSS感染能力降低。此外，免疫保护实验表明，重组FHA免疫大菱鲆后可以显著提高宿主抵抗TSS侵染的能力。这些结果首次表明了荧光假单胞菌FHA是一个关键的毒力因子，并且在与致病相关的生理过程中发挥重要作用。以荧光假单胞菌的鱼类宿主半滑舌鳎为研究对象，探究了其两类抗菌因子在抵抗荧光假单胞菌侵染的过程中所发挥的作用。杀菌/通透性增强蛋白(BPI)是先天免疫中一个重要分子，在哺乳动物中可以清除入侵的革兰氏阴性菌。在硬骨鱼中，BPI的功能还未可知。因此研究了半滑舌鳎BPI (CsBPI)在抵抗荧光假单胞菌感染方面所发挥的生物学功能。蛋白定位结果表明，CsBPI可以被半滑舌鳎外周血白细胞(PBL)分泌到细胞外环境中。重组CsBPI（rCsBPI）能够结合荧光假单胞菌等革兰氏阴性菌，且对荧光假单胞菌的结合力最强，但不能结合革兰氏阳性菌；rCsBPI通过与荧光假单胞菌的结合，进而增强其细胞膜的通透性，破坏其结构最终导致细菌死亡，并且与rCsBPI结合的荧光假单胞菌更容易被PBL吞噬。体内实验表明，rCsBPI上调一系列参与抗菌和抗病毒的免疫基因的表达，并且可以增强半滑舌鳎抗荧光假单胞菌以及抗病毒感染的能力。这些结果首次表明，硬骨鱼类的BPI具有显著的免疫调节及抗菌抗病毒功能。凝集素是一类糖结合蛋白，在先天免疫系统中发挥重要作用。因此本研究之后比较了半滑舌鳎三个B型甘露糖结合凝集素(CsBML1、CsBML2和CsBML3)在抗细菌感染方面所发挥的生物学功能。分析发现，这三个凝集素均具有三个保守的甘露糖结合基序QXDXNXVXY，且在肝脏中表达量最高，细菌感染之后表达均上调。重组CsBML1、CsBML2和CsBML3 (rCsBML1、rCsBML2和rCsBML3)以剂量依赖的方式结合荧光假单胞菌等多种病原菌，但对于不同细菌具有不同结合力。进一步研究发现这三个凝集素均具有甘露糖结合特异性和钙离子依赖性，但三者对细菌的凝集能力有所差异。体外实验表明rCsBML1和rCsBML2可以杀死细菌，体内实验表明rCsBML1和rCsBML2能抑制细菌对宿主组织的侵染，但rCsBML3无此功能。这些结果表明，在硬骨鱼类中，B型甘露糖结合凝集素对于抵抗外界病原菌侵染发挥着重要作用，并且此家族的不同成员在抗菌方面可能扮演着不同的角色。
其他摘要	Mariculture fish are often infected by pathogenic microorganisms, resulting in reduced yields and economic losses. However, there is no effective measures at present. One of the reasons is that the pathogenic mechanism of pathogen and the immune defense mechanism of the host are not elucidated clearly. In this study, we have investigated the pathogenic mechanism of Pseudomonas fluorescens, which is a common pathogen of fish. At the same time, we have studied the anti-bacterial function of the immune factors (bactericidal/permeability increasing protein and B-type mannose-specific lectins) in a fish host half-smooth tongue sole (Cynoglossus semilaevis). In this study, we performed proteomic analysis firstly to compare the global protein profiles of P. fluorescens strain TSS cultured under iron-replete and iron-deplete conditions. Twenty two differentially expressed proteins were identified of global proteins. The genes encoding four of the 22 proteins, i.e. HemO (heme oxygenase), PspB (serine protease), Sod (superoxide dismutase), and TfeR (TonB-dependent outermembrane ferric enterobactin receptor), were knocked out, and the pathogenicity of the mutants was examined. The results showed that compared to the wild type, the hemO, pspB, and tfeR knockouts were significantly impaired in the ability to survive in host serum, to invade host tissues, and to cause host mortality. Immunization of turbot with recombinant TfeR (rTfeR) and PspB induced production of specific serum antibodies and significant protections against lethal TSS challenge. Further analysis showed that rTfeR antibodies recognized and bound to TSS, and that treatment of TSS with rTfeR antibodies significantly impaired the infectivity of TSS to fish cells. Taken together, these results indicate for the first time that in pathogenic P. fluorescens, iron affects the expression of a large number of proteins including those that are involved in host infection. Then we compared the secreted protein profiles of TSS cultured under iron-replete and iron-deplete conditions by proteomic analysis. Fifteen differentially expressed proteins were identified. In this study, we identified filamentous hemagglutinin (FHA) as an iron-responsive protein secreted by TSS, furthermore, the fha mutant TSSfha was constructed. In vitro study, compared to the wild type, the fha mutant (i) exhibited a largely similar vegetative growth profile but significantly retarded in the ability of biofilm growth and producing extracellular matrix, (ii) displayed no apparent flagella and motility, (iii) was defective in the attachment to host cells and unable to form self-aggregation, (iv) displayed markedly reduced capacity of hemagglutination and surviving in host serum. In vivo infection analysis revealed that mutant was significantly attenuated in the ability of dissemination in fish tissues and inducing host mortality, and that antibody blocking of the natural FHA produced by the wild type TSS impaired the infectivity of the pathogen. Furthermore, when introduced into turbot as a subunit vaccine, recombinant FHA elicited a significant protection against lethal TSS challenge. Taken together, these results indicate for the first time that P. fluorescens FHA is a key virulence factor essential to multiple biological processes associated with pathogenicity. We investigated the role of two antibacterial factors of tongue sole in the process of resistancing to TSS. Bactericidal/permeability-increasing protein (BPI) is an important factor of innate immunity that in mammals is known to take part in the clearance of invading Gram-negative bacteria. In teleost, the function of BPI is unknown. In the present work, we studied the function of tongue sole BPI, CsBPI. We found that CsBPI was produced extracellularly by peripheral blood leukocytes (PBL). Recombinant CsBPI (rCsBPI) was able to bind to a number of Gram-negative bacteria but not Gram-positive bacteria. Binding to bacteria led to bacterial death through membrane permeabilization and structural destruction, and the bound bacteria were more readily taken up by PBL. In vivo, rCsBPI augmented the expression of a wide arrange of genes involved in antibacterial and antiviral immunity. Furthermore, rCsBPI enhanced the resistance of tongue sole against bacterial as well as viral infection. These results indicate for the first time that a teleost BPI possesses immunoregulatory effect and plays a significant role in antibacterial and antiviral defense. Lectins are a group of sugar-binding proteins that are important factors of the innate immune system. In this study, we examined, in a comparative manner, the expression and function of three Bulb-type (B-type) mannose-specific lectins (named CsBML1, CsBML2, and CsBML3) from tongue sole. All three lectins possess three repeats of the conserved mannose binding motif QXDXNXVXY. Expression of CsBML1, CsBML2, and CsBML3 was most abundant in liver and upregulated by bacterial infection. Recombinant (r) CsBML1, CsBML2, and CsBML3 bound to a wide arrange of bacteria in a dose-dependent manner and with different affinities. All three lectins displayed mannose-specific and calcium-dependent agglutinating capacities but differed in agglutinating profiles. rCsBML1 and rCsBML2, but not rCsBML3, killed target bacteria in vitro and inhibited bacterial dissemination in fish tissues in vivo. These results indicate for the first time that in teleost, different members of B-type mannose-specific lectins likely play different roles in antibacterial immunity.
学科领域	微生物免疫学 ; 水产生物学
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/136643
专题	实验海洋生物学重点实验室
作者单位	1.中国科学院海洋研究所 2.中国科学院大学
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	孙园园. 鱼类病原菌荧光假单胞菌的致病机制及宿主抗菌免疫因子的作用研究[D]. 北京. 中国科学院大学,2017.

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