本文關(guān)鍵詞：腸炎沙門氏菌毒力蛋白與宿主細(xì)胞蛋白相互作用的篩選和功能研究　出處：《武漢大學(xué)》2012年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 腸炎沙門氏菌 Ⅲ型分泌系統(tǒng)(T3SS) 毒力島(pathogenicity islands) 蛋白組學(xué) E3泛素連接酶 蛋白的泛素化修飾

【摘要】：腸炎沙門氏菌(Salmonella enterica)是一種典型的革蘭氏陰性致病菌。該菌主要引起畜禽的胃腸炎以及人類腸炎和食物中毒,嚴(yán)重威脅著人畜健康。腸炎沙門氏菌表達(dá)兩個(gè)Ⅲ型分泌系統(tǒng)(type Ⅲ secretion systems,T3SS),由毒力島1和毒力島2(pathogenicity islands1and2, SPI-1and SPI-2)分別編碼。細(xì)菌依賴T3SS將毒力蛋白運(yùn)輸至宿主細(xì)胞內(nèi)發(fā)揮作用。通常認(rèn)為,毒力島1編碼的毒力蛋白在細(xì)菌入侵的過程中起到作用,而毒力島2編碼的蛋白則負(fù)責(zé)促進(jìn)細(xì)菌在細(xì)胞中的生存,復(fù)制以及免疫逃逸等的調(diào)節(jié)。目前已經(jīng)鑒定出了30幾個(gè)T3SS毒力蛋白,但是它們中的絕大多數(shù)其生理功能尚未知曉或了解很少。 為探討T3SS兩個(gè)毒力島的分泌蛋白的潛在生物學(xué)功能,并分析它們是怎樣幫助細(xì)菌從入侵上皮細(xì)胞到在細(xì)胞內(nèi)生存、復(fù)制的,鑒定這些毒力蛋白與宿主細(xì)胞蛋白的相互作用關(guān)系成為了必不可少的途徑。本文從以下兩個(gè)方面對T3SS分泌的毒力蛋白與宿主細(xì)胞蛋白的相互作用進(jìn)行了系統(tǒng)分析,分別是： 1.通過酵母雙雜交技術(shù)嚴(yán)格系統(tǒng)地篩選20個(gè)T3SS毒力蛋白(SsPH2, Ssel、 SpiC、SseF、SseG、SlrP、SseJ、SopD2、SifB、SipC、SipB、SipA、SopD、SipD、 SptP、SopA、SopE2、SrfB、SrfA、SrfJ)與宿主細(xì)胞的相互作用蛋白,并最終得到了超過300對的陽性相互作用結(jié)果。我們對其中部分結(jié)果進(jìn)行了免疫共沉淀驗(yàn)證并通過文獻(xiàn)進(jìn)行了初步的功能分類分析,并從蛋白質(zhì)組學(xué)的角度構(gòu)建了相互作用網(wǎng)絡(luò)草圖。這些蛋白功能分布廣泛,涵蓋了能量代謝、細(xì)胞骨架和細(xì)胞運(yùn)動(dòng)、物質(zhì)運(yùn)輸以及細(xì)胞內(nèi)多種信號(hào)通路的調(diào)節(jié)。結(jié)合文獻(xiàn),我們獲得的大量數(shù)據(jù)為揭示細(xì)菌侵染細(xì)胞及之后的生物學(xué)功能和細(xì)菌對宿主細(xì)胞各種生物學(xué)通路系統(tǒng)的影響提供了理論基礎(chǔ)和事實(shí)依據(jù)。 2.選取SPI-2的重要毒力蛋白SsPH2為研究對象,探討其對宿主內(nèi)源泛素通路的影響作用。SsPH2作為一個(gè)最近才知曉的細(xì)菌編碼的E3泛素連接酶,通過影響宿主內(nèi)源泛素通路來促進(jìn)細(xì)菌在宿主內(nèi)的生存、復(fù)制以及調(diào)節(jié)宿主免疫反應(yīng)。我們系統(tǒng)地鑒定了酵母雙雜交得到的SsPH2作用對象,并著重研究了SsPH2與細(xì)胞蛋白LM04的相互作用。我們首先分別用不同方式驗(yàn)證了二者于體內(nèi)、體外的相互作用,并探討了其作用區(qū)域。接著我們通過體內(nèi)、體外實(shí)驗(yàn)證實(shí)了SsPH2可以催化LM04蛋白的泛素化修飾。后面的進(jìn)一步研究闡釋了SsPH2對LM04的表達(dá)調(diào)控作用,我們發(fā)現(xiàn)SsPH2在體內(nèi)穩(wěn)定表達(dá)具有提高LM04蛋白水平的作用,通過深入研究這種調(diào)控的分子機(jī)制,我們發(fā)現(xiàn)SsPH2增強(qiáng)LM04蛋白水平是通過其對LM04的泛素化修飾來實(shí)現(xiàn)的。SsPH2抑制了LM04在細(xì)胞中的降解途徑,從而延長了LM04蛋臼的半衰期�？紤]到LM04與白細(xì)胞介素6(IL-6)途徑的重要關(guān)系,我們猜測這種依賴于細(xì)菌SsPH2的LM04蛋白水平調(diào)節(jié)作用很可能影響了宿主IL-6信號(hào)通路,并調(diào)節(jié)了由IL-6介導(dǎo)的Stat3轉(zhuǎn)錄活性,從而影響宿主的早期炎癥反應(yīng)。以上發(fā)現(xiàn)揭示了SsPH2作為細(xì)菌編碼的E3泛素連接酶通過特異性識(shí)別并泛素化修飾宿主蛋白LM04來實(shí)現(xiàn)細(xì)菌對宿主細(xì)胞生理功能的調(diào)節(jié)作用。 綜上所述,通過本課題的研究我們鑒定出了數(shù)量龐大的腸炎沙門氏菌T3SS毒力蛋白在宿主細(xì)胞中的相互作用對象,為后續(xù)的進(jìn)一步功能分析打下了堅(jiān)實(shí)的理論基礎(chǔ)。此外我們著重研究了毒力蛋白SsPH2與宿主蛋白LM04的相互作用機(jī)制,并發(fā)現(xiàn)了SsPH2對LM04的泛素化修飾以及調(diào)節(jié)作用。我們的研究工作為腸炎沙門氏菌乃至整個(gè)革蘭氏陰性菌界毒力蛋白與宿主蛋白相互關(guān)系的思考提供很多新的研究思路和方向。
[Abstract]:Salmonella enteritidis (Salmonella enterica) is a kind of typical gram negative bacteria. The bacteria are mainly caused by livestock gastroenteritis and human enteritis and food poisoning, a serious threat to human and animal health. Salmonella enteritidis expression of two type III secretion system (type secretion systems, T3SS), and 1 by pathogenicity island pathogenicity island 2 (pathogenicity islands1and2, SPI-1and SPI-2) respectively. T3SS will depend on the encoding of bacterial virulence protein transport to host cells play a role. Generally, the pathogenicity island encoding virulence protein 1 plays a role in the process of the invasion of bacteria, and 2 pathogenicity island encoding protein is responsible for promoting the survival of bacteria in the cells. Replication and immune escape regulation. At present have identified 30 T3SS virulence proteins, but most of them in their physiological function are currently unknown or poorly understood.
As a potential biological function of secretory protein two T3SS pathogenicity island, and analyze how they help the bacteria from invading epithelial cells to survive and replicate inside cells, the interaction between the identification of these virulence proteins with host cells has become an indispensable way. This interaction on the secretion of T3SS from the following two aspects the virulence protein and host cell proteins were analyzed, respectively:
1. through the yeast two hybrid system to strictly screened 20 T3SS virulence proteins (SsPH2, Ssel, SpiC, SseF, SseG, SlrP, SseJ, SopD2, SifB, SipC, SipB, SipA, SopD, SipD, SptP, SopA, SopE2, SrfB, SrfA, SrfJ) protein interaction with host cells and finally, got more than 300 of the positive interaction. We some of these results were verified by immunoprecipitation and analyzed preliminary functional classification through the literature, from the perspective of proteomics constructed the interaction network sketch. These proteins function are widely distributed, covering energy metabolism, cytoskeleton and cell movement, substance transport and regulation of multiple signaling pathways in cells. Combining with literature, a large amount of data we obtained provides to reveal the biological function and the bacterial cells and bacterial infection after the host cell biological pathway system On the basis of the foundation and the facts.
The most important virulence protein SsPH2 2. SPI-2 is selected as the research object, to explore the role of endogenous ubiquitin pathway of host.SsPH2 as a recently known bacteria encoding E3 ubiquitin ligase, to promote the survival of bacteria in the host by affecting the host endogenous ubiquitin proteasome pathway, replication and regulation of host immune response. We systematically identified SsPH2 object yeast two hybrid, and focuses on the interaction between SsPH2 and cell protein LM04. We used different ways to verify the two in vivo interaction in vitro, and to explore the role of regional. Then we through in vivo, in vitro experiments confirmed that SsPH2 can catalyze LM04 ubiquitination protein. Further study behind the expression regulation of SsPH2 on LM04, we found that SsPH2 expression is stable in vivo with increased LM04 protein levels for With the further study of molecular mechanism, through this regulation, we found that SsPH2 increased LM04 protein level is achieved through the degradation pathway of LM04 ubiquitination of.SsPH2 inhibited LM04 in the cells, thereby prolonging the half-life of LM04 egg mortar. Considering LM04 and interleukin 6 (IL-6) important relationship way, we guess this depends on the level of LM04 protein in bacterial SsPH2 regulation is likely to affect the host IL-6 signaling pathway, and regulates the transcriptional activity of Stat3 mediated by IL-6, thus affecting the early inflammatory response of the host. The above findings reveal SsPH2 as bacteria encoding E3 ubiquitin ligase by specific recognition and ubiquitination modification of the host protein LM04 to achieve the regulatory role of bacteria on physiological function of host cells.
In summary, through this study we identified the interaction between a large number of Salmonella enteritidis T3SS virulence proteins in host cells, and lay a solid theoretical foundation for the further function of subsequent analysis. In addition we studied the interaction mechanism of virulence protein SsPH2 and host protein LM04, and found SsPH2 of LM04 ubiquitination and regulation. Our research provides many new research ideas and directions for the thinking of the relationship between Salmonella enteritidis and the gram negative bacteria community virulence proteins and host proteins.

【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R378

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