志贺氏菌Ⅲ型分泌系统效应子蛋白IpaH4.5功能研究

发布时间：2018-01-16 23:12

本文关键词：志贺氏菌Ⅲ型分泌系统效应子蛋白IpaH4.5功能研究　出处：《中国人民解放军军事医学科学院》2010年博士论文　论文类型：学位论文

【摘要】： 志贺氏菌是一类不形成芽孢的革兰氏阴性致病菌,其致病性依赖于Ⅲ型分泌系统(Type III secretion systems, T3SS)。T3SS是一种能分泌细菌毒力因子的多蛋白复合体,是细菌致病的重要毒力武器。许多动植物致病菌利用T3SS对宿主侵袭致病。细菌的T3SS由分泌器装置(apparatus)、转位子(translocators)、效应子(effectors)、分子伴侣(chaperones)等构成。T3SS分泌的效应子具有诱导侵袭、介导吞噬逃逸、促进细菌在宿主细胞内运动和细胞间播散、干扰宿主细胞信号通路等功能。志贺氏菌在侵袭致病过程中,利用T3SS向宿主细胞释放超过25种毒力蛋白,这些毒力蛋白称为效应子(effectors),目前仅对少数效应子的功能有研究报道,大部分效应子功能未知。志贺氏菌IpaH家族蛋白由毒力大质粒和染色体上的基因共同编码,福氏志贺氏菌2a 301株的毒力大质粒上共有5个ipaH拷贝:ipaH1.4、ipaH2.5、ipaH4.5、ipaH7.8和ipaH9.8,染色体上有7个ipaH的拷贝。IpaH家族成员氨基端为6~8个富含亮氨酸蛋白(LRR)的结构域,羧基端为839个碱基构成的保守区。目前已知志贺氏菌IpaH家族蛋白由T3SS分泌,但对其功能研究并不多见。有报道IpaH7.8能够促进志贺氏菌吞噬逃逸、IpaH9.8能与哺乳动物剪接因子U2AF35结合干扰宿主的免疫反应、染色体IpaH能够抑制宿主炎症反应等,但对于毒力大质粒编码的IpaH4.5的功能,尚未见到详细的研究报道。为此,我们利用基因缺失突变技术、细胞侵袭实验、蛋白质相互作用等技术手段对IpaH4.5的功能进行了深入系统的研究。研究IpaH4.5的功能,首先需要构建ipaH4.5缺失突变株和互补株。我们利用λ-red同源重组技术构建了ipaH4.5缺失突变株,利用能够在志贺氏菌中复制且携带卡那抗性基因的pAK载体,构建了ipaH4.5缺失突变株的互补株。经PCR证实突变株中ipaH4.5缺失,互补株中ipaH4.5回复。利用RT-PCR技术对突变株和互补株中ipaH4.5的转录进行验证,结果表明突变株中的ipaH4.5没有转录活性,互补株中ipaH4.5的转录活性得到恢复,表明突变株和互补株构建成功。接下来我们对突变株、野生株和互补株的生存和侵袭相关表型进行了比较,分析ipaH4.5对志贺氏菌生长、代谢、侵袭力和毒力的影响。通过测定突变株、野生株、互补株的生长曲线和生化实验,我们发现ipaH4.5不影响志贺氏菌的生长和代谢。利用体外应激实验,我们又发现ipaH4.5也不影响志贺氏菌的对极端环境如热休克、低pH值、氧压力、渗透压等的抗力。HeLa细胞和鼠J774.A.1细胞侵袭实验结果表明,ipaH4.5缺失突变株、野生株和互补株入侵细胞的能力无明显差异。我们进一步测定了突变株、野生株和互补株感染鼠J774.A.1细胞后,培养上清中炎性因子TNF-α、IL-1β的分泌水平。结果表明,与突变株相比,野生株和互补株能够抑制鼠J774.A.1细胞分泌TNF-α和IL-1β等炎性因子,提示IpaH4.5具有抑制宿主炎症反应的功能。通过小鼠肺感染模型和豚鼠角膜实验,进一步证实IpaH4.5能够抑制宿主炎症反应,志贺氏菌缺失ipaH4.5后引起小鼠肺组织和豚鼠角膜炎症反应增强。为深入探讨IpaH4.5抑制炎症反应的机制,我们利用双荧光素酶报告基因系统检测IpaH4.5对NF-κB信号途径的影响。构建带有Flag标签和GFP标签的IpaH4.5的真核表达载体。Flag-IpaH4.5和Flag空载体分别转染293T细胞后,用相同剂量TNF-α刺激细胞,与空载体相比,IpaH4.5能够抑制NF-κB的转录活性,并且这种抑制作用随着ipaH4.5转染剂量的增加而增强,呈现明显的剂量反应关系,说明IpaH4.5能够抑制宿主细胞的NF-κB信号途径。我们随后利用激光共聚焦技术观察GFP-IpaH4.5在真核细胞中的定位,结果表明,IpaH4.5分布于细胞质和细胞核,提示IpaH4.5在细胞内通过与多种蛋白相互作用来执行功能。为阐明IpaH4.5抑制NF-κB信号途径的机制,我们利用酵母双杂交技术筛选了细胞内与IpaH4.5相互作用的蛋白。我们以ipaH4.5基因全长为诱饵,从人脾cDNA文库中筛选与其相互作用的蛋白质,共得到52个候选基因,通过回筛实验初步验证筛选结果的可靠性,通过测序、序列比对、功能分类,发现筛选到的蛋白基因主要涉及以下一些功能:凋亡、免疫调控、细胞黏附、转录调控等。其中包括NF-κB的p65亚基、PMSB9、HLA-C、UXT、POMP等蛋白基因,我们选择感兴趣的p65蛋白进行深入的研究。通过体外的GST pull-down实验和体内的免疫共沉淀实验(Co-IP)验证IpaH4.5与p65亚基确实存在相互作用,进一步的研究表明p65亚基上与IpaH4.5相互作用的结构域位于p65的第1~190个aa之间。随后我们又利用双荧光素酶报告基因系统验证IpaH4.5与NF-κB的p65亚基相互作用的生物学意义,证实IpaH4.5通过结合p65直接抑制NF-κB信号途径。以上研究结果表明,志贺氏菌IpaH家族蛋白成员之一—IpaH4.5与其他一些已报道的志贺氏菌T3SS效应子蛋白一样,能够干扰宿主的天然免疫、抑制宿主的炎症反应。双荧光素酶报告系统和酵母双杂交实验证明IpaH4.5通过与NF-κB的p65亚基相互作用直接抑制NF-κB信号途径,进而抑制宿主的天然免疫。有关IpaH家族蛋白功能最新的研究表明,IpaH家族蛋白成员具有非常独特的E3泛素连接酶活性,生物化学实验证据表明IpaH选择性地利用宿主中一类特殊的泛素结合酶,通过泛素化途径导致靶蛋白降解,进而在志贺氏菌对宿主的免疫抑制中起重要作用。目前还不清楚IpaH作为一种特殊的E3泛素连接酶,能够泛素化哪些宿主靶蛋白。我们通过研究筛选到与IpaH4.5相互作用的宿主蛋白,这些蛋白很可能是IpaH型E3泛素连接酶作用的底物。因此有必要进一步通过泛素化实验来证实这一猜想。通过本课题的研究,揭示了IpaH4.5抑制宿主炎症反应的分子机制,使我们对IpaH4.5的功能有了比较全面的认识,为阐明研究志贺氏菌致病的分子机制提供了重要线索,也为研制志贺氏菌疫苗提供新的思路。
[Abstract]:Shigella is a kind of non spore forming gram negative pathogens, the pathogenicity depends on type III secretion system (Type III secretion systems, T3SS.T3SS) is a multi protein complex can secrete virulence factors, pathogenic bacteria is an important virulence weapon. Many animal and plant pathogenic bacteria by T3SS invasion of pathogenic host. The bacterial T3SS secreted by the device (apparatus), transposon (translocators), effector (effectors), molecular chaperones (chaperones) constitute the effector secretion of.T3SS induced invasion mediated phagocytosis escape, promote bacterial movement in host cells and cell to cell spread, interfering host cell signaling pathway and other functions.
Shigella invasion in the pathogenic process, using T3SS to host cells to release more than 25 kinds of virulence proteins, these virulence proteins called effectors (effectors), currently only a few studies have reported the effector function, most effector functions unknown. Shigella IpaH family protein encoding by common virulence plasmid and chromosome the genes of Shigella flexneri 2a 301 strain virulence plasmid of a total of 5 copies of ipaH: ipaH1.4, ipaH2.5, ipaH4.5, ipaH7.8 and ipaH9.8, on chromosome 7 copy of ipaH.IpaH family members 6~8 amino terminal leucine containing rich protein (LRR) domain, C-terminal the conserved region of 839 base. Now known Shigella IpaH family proteins secreted by T3SS, but the function of research is rare. It has been reported that IpaH7.8 can promote the phagocytosis of Shigella IpaH9.8 can escape, and mammalian splicing factor U2AF35 According to interfere with the host immune response, chromosome IpaH can suppress host inflammatory reaction, but for the large virulence plasmid encoding IpaH4.5 function has not been reported in detail. Therefore, we use mutation of gene technology, cell invasion assay, functional protein interaction techniques of IpaH4.5 are studied.
Study on the function of IpaH4.5, the first step is to build a ipaH4.5 mutant and complementary strain. We use lambda -red homologous recombination technique to construct ipaH4.5 mutant of Shigella in use can copy and carry the carrier card pAK resistance gene, the ipaH4.5 mutant complementation strains. Confirmed by PCR ipaH4.5 deletion mutation strain, complementary strain ipaH4.5 reply. To verify the transcription mutation strain ipaH4.5 and complementary strain by using RT-PCR technique, results showed that the mutant ipaH4.5 in the transcriptional activity of transcription activity, complementary strain ipaH4.5 was restored, showed that the mutant and complementary strain was successfully constructed.
We next to the mutant and wild strains and complementary strains survival and compared the invasive phenotype of ipaH4.5, analysis of Shigella growth, metabolism, invasion and virulence. Through the determination of mutant and wild strains, the growth curve of complementary strains and biochemical experiments, we found that ipaH4.5 did not affect the growth and metabolism Shigella. Using in vitro stress test, we also found that ipaH4.5 does not affect Shigella in extreme environments such as heat shock, low pH value, oxygen pressure, the resistance of.HeLa cells and rat J774.A.1 cell invasion assay results of osmotic pressure. It is showed that the ipaH4.5 mutant had no obvious difference between the wild strain and complementary strains the invasion of the cells. We further examined mutant and wild strains and complementary strains infected mice after J774.A.1 cell culture supernatant of inflammatory factor TNF- alpha, IL-1 beta secretion. The results showed that compared with the mutant and wild Strain and complementary strains can inhibit rat J774.A.1 cells secrete TNF- alpha and IL-1 beta and other inflammatory cytokines, suggesting that IpaH4.5 can inhibit the host inflammatory response function. Through the model and experimental guinea pig corneal mouse lung infection, further confirmed that IpaH4.5 can inhibit the host inflammatory response, Shigella ipaH4.5 deletion caused the enhancement of lung tissue in mice and guinea pigs. In response.
In order to further study the mechanism of IpaH4.5 inhibiting the inflammatory reaction, we use dual luciferase reporter assay system for detection of IpaH4.5 effect on NF- kappa B signaling pathway. The construction with Flag tag and GFP tag IpaH4.5.Flag-IpaH4.5 eukaryotic expression vector and Flag vector were transfected into 293T cells, TNF- cells were stimulated with the same dose, compared with no, IpaH4.5 can inhibit the transcriptional activity of NF- K B, and this inhibition increased with ipaH4.5 transfection dose, showing a clear dose-response relationship, indicating that NF- kappa B signaling pathway can inhibit the IpaH4.5 cells of the host. We then use the positioning, laser scanning confocal microscope GFP-IpaH4.5 in eukaryotic cells results show that IpaH4.5 distributed in the cytoplasm and nucleus, suggesting that IpaH4.5 in the cell by interacting with many proteins to perform its function.
To elucidate the mechanism of IpaH4.5 inhibiting NF- kappa B signaling pathway, we use yeast two hybrid technology to screen the proteins interacting with IpaH4.5 cells. We used ipaH4.5 gene as bait, screening and interaction in human spleen cDNA Library of protein, there are 52 candidate genes, through the sieve reliability experiment the preliminary screening results verified by sequencing, sequence alignment, functional classification, found that the protein gene screened mainly involves the following several functions: apoptosis, immune regulation, cell adhesion, regulation of transcription. Including p65 subunit, NF- kappa B PMSB9, HLA-C, UXT, POMP protein gene, we choose interest p65 protein was further investigated. The immune experiments and in vivo GST pull-down in vitro co precipitation experiment (Co-IP) to verify the IpaH4.5 and p65 subunits do exist interaction, further study showed that subunit p65 and Ipa The domain of H4.5 interaction is located between the 1~190 AA of p65. Then we used the dual luciferase reporter gene system to verify the biological significance of the interaction between IpaH4.5 and NF- B B p65, and confirmed that IpaH4.5 directly inhibited the NF- kappa signaling pathway by combining p65.
The above results showed that Shigella IpaH protein family member, IpaH4.5 and some other sub Shigella T3SS effect has been reported to the same protein, natural host immune interference, inhibit the inflammatory response of the host. Dual luciferase reporter system and yeast two hybrid experiments showed that IpaH4.5 with NF- kappa B subunit p65 interaction the role of direct inhibition of NF- kappa B signaling pathway, thereby inhibiting the host innate immunity. Research on IpaH family proteins function in the IpaH protein family members has a very unique E3 ubiquitin ligase activity and biological chemistry experimental evidence that IpaH selectively using a special host ubiquitin conjugating enzyme, by way of ubiquitin lead to degradation of target protein, which play an important role in the host immune to Shigella inhibition. It is unclear IpaH as a special kind of E3 ubiquitin ligase, Which host target proteins can be ubiquitin? We have screened the host proteins interacting with IpaH4.5 by studying. These proteins are likely to be substrates of IpaH E3 ubiquitin ligase. Therefore, it is necessary to further confirm this conjecture through ubiquitination experiments.
Through this study, to reveal the molecular mechanism of IpaH4.5 inhibiting the host inflammatory response, we make the function of IpaH4.5 have a more comprehensive understanding, provide important clues for elucidating the molecular mechanism of Shigella virulence, to provide new ideas for the development of Shigella vaccine.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R378

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