本文關(guān)鍵詞：志賀氏菌Ⅲ型分泌系統(tǒng)效應(yīng)子蛋白IpaH4.5功能研究　出處：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2010年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 志賀氏菌 Ⅲ型分泌系統(tǒng) IpaH4.5 炎癥反應(yīng) NF-κB 酵母雙雜交

【摘要】： 志賀氏菌是一類不形成芽孢的革蘭氏陰性致病菌,其致病性依賴于Ⅲ型分泌系統(tǒng)(Type III secretion systems, T3SS)。T3SS是一種能分泌細(xì)菌毒力因子的多蛋白復(fù)合體,是細(xì)菌致病的重要毒力武器。許多動植物致病菌利用T3SS對宿主侵襲致病。細(xì)菌的T3SS由分泌器裝置(apparatus)、轉(zhuǎn)位子(translocators)、效應(yīng)子(effectors)、分子伴侶(chaperones)等構(gòu)成。T3SS分泌的效應(yīng)子具有誘導(dǎo)侵襲、介導(dǎo)吞噬逃逸、促進細(xì)菌在宿主細(xì)胞內(nèi)運動和細(xì)胞間播散、干擾宿主細(xì)胞信號通路等功能。 志賀氏菌在侵襲致病過程中,利用T3SS向宿主細(xì)胞釋放超過25種毒力蛋白,這些毒力蛋白稱為效應(yīng)子(effectors),目前僅對少數(shù)效應(yīng)子的功能有研究報道,大部分效應(yīng)子功能未知。志賀氏菌IpaH家族蛋白由毒力大質(zhì)粒和染色體上的基因共同編碼,福氏志賀氏菌2a 301株的毒力大質(zhì)粒上共有5個ipaH拷貝:ipaH1.4、ipaH2.5、ipaH4.5、ipaH7.8和ipaH9.8,染色體上有7個ipaH的拷貝。IpaH家族成員氨基端為6~8個富含亮氨酸蛋白(LRR)的結(jié)構(gòu)域,羧基端為839個堿基構(gòu)成的保守區(qū)。目前已知志賀氏菌IpaH家族蛋白由T3SS分泌,但對其功能研究并不多見。有報道IpaH7.8能夠促進志賀氏菌吞噬逃逸、IpaH9.8能與哺乳動物剪接因子U2AF35結(jié)合干擾宿主的免疫反應(yīng)、染色體IpaH能夠抑制宿主炎癥反應(yīng)等,但對于毒力大質(zhì)粒編碼的IpaH4.5的功能,尚未見到詳細(xì)的研究報道。為此,我們利用基因缺失突變技術(shù)、細(xì)胞侵襲實驗、蛋白質(zhì)相互作用等技術(shù)手段對IpaH4.5的功能進行了深入系統(tǒng)的研究。 研究IpaH4.5的功能,首先需要構(gòu)建ipaH4.5缺失突變株和互補株。我們利用λ-red同源重組技術(shù)構(gòu)建了ipaH4.5缺失突變株,利用能夠在志賀氏菌中復(fù)制且攜帶卡那抗性基因的pAK載體,構(gòu)建了ipaH4.5缺失突變株的互補株。經(jīng)PCR證實突變株中ipaH4.5缺失,互補株中ipaH4.5回復(fù)。利用RT-PCR技術(shù)對突變株和互補株中ipaH4.5的轉(zhuǎn)錄進行驗證,結(jié)果表明突變株中的ipaH4.5沒有轉(zhuǎn)錄活性,互補株中ipaH4.5的轉(zhuǎn)錄活性得到恢復(fù),表明突變株和互補株構(gòu)建成功。 接下來我們對突變株、野生株和互補株的生存和侵襲相關(guān)表型進行了比較,分析ipaH4.5對志賀氏菌生長、代謝、侵襲力和毒力的影響。通過測定突變株、野生株、互補株的生長曲線和生化實驗,我們發(fā)現(xiàn)ipaH4.5不影響志賀氏菌的生長和代謝。利用體外應(yīng)激實驗,我們又發(fā)現(xiàn)ipaH4.5也不影響志賀氏菌的對極端環(huán)境如熱休克、低pH值、氧壓力、滲透壓等的抗力。HeLa細(xì)胞和鼠J774.A.1細(xì)胞侵襲實驗結(jié)果表明,ipaH4.5缺失突變株、野生株和互補株入侵細(xì)胞的能力無明顯差異。我們進一步測定了突變株、野生株和互補株感染鼠J774.A.1細(xì)胞后,培養(yǎng)上清中炎性因子TNF-α、IL-1β的分泌水平。結(jié)果表明,與突變株相比,野生株和互補株能夠抑制鼠J774.A.1細(xì)胞分泌TNF-α和IL-1β等炎性因子,提示IpaH4.5具有抑制宿主炎癥反應(yīng)的功能。通過小鼠肺感染模型和豚鼠角膜實驗,進一步證實IpaH4.5能夠抑制宿主炎癥反應(yīng),志賀氏菌缺失ipaH4.5后引起小鼠肺組織和豚鼠角膜炎癥反應(yīng)增強。 為深入探討IpaH4.5抑制炎癥反應(yīng)的機制,我們利用雙熒光素酶報告基因系統(tǒng)檢測IpaH4.5對NF-κB信號途徑的影響。構(gòu)建帶有Flag標(biāo)簽和GFP標(biāo)簽的IpaH4.5的真核表達(dá)載體。Flag-IpaH4.5和Flag空載體分別轉(zhuǎn)染293T細(xì)胞后,用相同劑量TNF-α刺激細(xì)胞,與空載體相比,IpaH4.5能夠抑制NF-κB的轉(zhuǎn)錄活性,并且這種抑制作用隨著ipaH4.5轉(zhuǎn)染劑量的增加而增強,呈現(xiàn)明顯的劑量反應(yīng)關(guān)系,說明IpaH4.5能夠抑制宿主細(xì)胞的NF-κB信號途徑。我們隨后利用激光共聚焦技術(shù)觀察GFP-IpaH4.5在真核細(xì)胞中的定位,結(jié)果表明,IpaH4.5分布于細(xì)胞質(zhì)和細(xì)胞核,提示IpaH4.5在細(xì)胞內(nèi)通過與多種蛋白相互作用來執(zhí)行功能。 為闡明IpaH4.5抑制NF-κB信號途徑的機制,我們利用酵母雙雜交技術(shù)篩選了細(xì)胞內(nèi)與IpaH4.5相互作用的蛋白。我們以ipaH4.5基因全長為誘餌,從人脾cDNA文庫中篩選與其相互作用的蛋白質(zhì),共得到52個候選基因,通過回篩實驗初步驗證篩選結(jié)果的可靠性,通過測序、序列比對、功能分類,發(fā)現(xiàn)篩選到的蛋白基因主要涉及以下一些功能:凋亡、免疫調(diào)控、細(xì)胞黏附、轉(zhuǎn)錄調(diào)控等。其中包括NF-κB的p65亞基、PMSB9、HLA-C、UXT、POMP等蛋白基因,我們選擇感興趣的p65蛋白進行深入的研究。通過體外的GST pull-down實驗和體內(nèi)的免疫共沉淀實驗(Co-IP)驗證IpaH4.5與p65亞基確實存在相互作用,進一步的研究表明p65亞基上與IpaH4.5相互作用的結(jié)構(gòu)域位于p65的第1~190個aa之間。隨后我們又利用雙熒光素酶報告基因系統(tǒng)驗證IpaH4.5與NF-κB的p65亞基相互作用的生物學(xué)意義,證實IpaH4.5通過結(jié)合p65直接抑制NF-κB信號途徑。 以上研究結(jié)果表明,志賀氏菌IpaH家族蛋白成員之一—IpaH4.5與其他一些已報道的志賀氏菌T3SS效應(yīng)子蛋白一樣,能夠干擾宿主的天然免疫、抑制宿主的炎癥反應(yīng)。雙熒光素酶報告系統(tǒng)和酵母雙雜交實驗證明IpaH4.5通過與NF-κB的p65亞基相互作用直接抑制NF-κB信號途徑,進而抑制宿主的天然免疫。有關(guān)IpaH家族蛋白功能最新的研究表明,IpaH家族蛋白成員具有非常獨特的E3泛素連接酶活性,生物化學(xué)實驗證據(jù)表明IpaH選擇性地利用宿主中一類特殊的泛素結(jié)合酶,通過泛素化途徑導(dǎo)致靶蛋白降解,進而在志賀氏菌對宿主的免疫抑制中起重要作用。目前還不清楚IpaH作為一種特殊的E3泛素連接酶,能夠泛素化哪些宿主靶蛋白。我們通過研究篩選到與IpaH4.5相互作用的宿主蛋白,這些蛋白很可能是IpaH型E3泛素連接酶作用的底物。因此有必要進一步通過泛素化實驗來證實這一猜想。 通過本課題的研究,揭示了IpaH4.5抑制宿主炎癥反應(yīng)的分子機制,使我們對IpaH4.5的功能有了比較全面的認(rèn)識,為闡明研究志賀氏菌致病的分子機制提供了重要線索,也為研制志賀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.

【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R378

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