本文關(guān)鍵詞： c-di-GMP 結(jié)核分枝桿菌 噬鐵蛋白 莽草酸激酶 病原-宿主相互作用　出處：《華中農(nóng)業(yè)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：環(huán)二鳥苷單磷酸(cyclic-di-GMP,簡稱c-di-GMP)是細(xì)菌中普遍存在的多功能第二信使分子,廣泛參與調(diào)控細(xì)菌生物膜形成、運動性、細(xì)胞周期進(jìn)程、毒力以及病原-宿主相互作用等過程。分枝桿菌具有獨特的c-di-GMP信號系統(tǒng)并能影響其多種生理和病理特性,但是該信號分子如何介導(dǎo)病原性結(jié)核分枝桿菌(Mycobacterium tuberculosis)與宿主之間的相互作用還基本不清楚,而且目前在該重要人類病原菌中還沒有明確的c-di-GMP信號受體蛋白報道。本研究發(fā)現(xiàn)了兩個新的c-di-GMP受體蛋白:一個是參與病原-宿主相互作用的人免疫系統(tǒng)噬鐵蛋白LCN2,另一個是結(jié)核分枝桿菌的莽草酸激酶Aro K。1.人噬鐵蛋白LCN2是c-di-GMP分子的直接受體。LCN2是人類先天免疫系統(tǒng)的重要抗菌元件,在營養(yǎng)缺乏條件下它通過截獲細(xì)菌生長必須的鐵載體而抑制細(xì)菌在宿主體內(nèi)的存活。我們首先通過生物信息學(xué)方法預(yù)測LCN2是c-di-GMP的可能受體,并利用表面等離子共振(Surface Plasmon Resonance,SPR)和等溫滴定量熱(Isothermal Titration Calorimetry,ITC)實驗證實,c-di-GMP也與鐵載體Fe-Ent/Fe-CMBs一樣,能夠與r LCN2直接結(jié)合。但是,在相似條件下我們不能清楚探測到r LCN2蛋白與c-di-AMP、GTP和c GAMP等與c-di-GMP類似的其它幾個小分子之間的相互作用。這表明,c-di-GMP與r LCN2之間的相互作用具有特異性。進(jìn)一步,ITC定量測定發(fā)現(xiàn),c-di-GMP滴定r LCN2蛋白的化學(xué)計量比是1:1,親和力Kd為1.63±0.05μM,表明一分子c-di-GMP結(jié)合一分子r LCN2單體蛋白。因此,我們的研究發(fā)現(xiàn)了噬鐵蛋白LCN2是c-di-GMP的直接受體,細(xì)菌在感染過程中可能利用自己的這個信號分子阻塞LCN2蛋白與鐵載體的結(jié)合,從而解除其抗菌效應(yīng)。2.結(jié)核分枝桿菌莽草酸激酶Aro K是c-di-GMP的直接受體。莽草酸激酶Aro K是結(jié)核分枝桿菌莽草酸途徑和細(xì)胞壁合成代謝的關(guān)鍵酶,已經(jīng)成為抗結(jié)核藥物設(shè)計的潛在靶標(biāo)蛋白。首先我們選取了大約80個在PDB數(shù)據(jù)庫中有晶體結(jié)構(gòu)信息的結(jié)核分枝桿菌H37Rv蛋白,建立了結(jié)核分枝桿菌蛋白質(zhì)文庫,篩選文庫發(fā)現(xiàn)莽草酸激酶Aro K是可能的c-di-GMP受體。然后利用紫外交聯(lián)實驗證明放射性同位素標(biāo)記的c-di-GMP分子能與Aro K蛋白直接結(jié)合。進(jìn)一步,利用競爭性結(jié)合實驗證實c-di-GMP與Aro K結(jié)合具有特異性。通過ITC定量測定發(fā)現(xiàn),c-di-GMP滴定Aro K蛋白的化學(xué)計量比是1:1,親和力Kd為2.453±0.05μM,表明一分子c-di-GMP結(jié)合一分子Aro K單體蛋白。通過設(shè)計并成功表達(dá)純化Aro K一系列關(guān)鍵氨基酸殘基突變蛋白,發(fā)現(xiàn)了位于催化中心的關(guān)鍵氨基酸Ser16也是Aro K與c-di-GMP相互作用的關(guān)鍵氨基酸。最后,通過測定激酶活性表明c-di-GMP對Aro K的酶促反應(yīng)有明顯的抑制作用。因此,Aro K是一個新的c-di-GMP受體,c-di-GMP能夠直接靶向Aro K的活性中心從而影響其酶活。這些結(jié)果表明c-di-GMP信號分子可能通過影響莽草酸激酶Aro K的活性,調(diào)控結(jié)核分枝桿菌桿菌的細(xì)胞壁分支酸代謝,從而影響病原菌的生長和毒力;同時,該工作也為病原菌莽草酸激酶抑制劑的設(shè)計提供了新的線索。
[Abstract]:The cyclic Diguanylic monophosphate (cyclic-di-GMP, c-di-GMP) is a multifunctional second messenger molecule exists in bacteria, widely involved in the regulation of bacterial biofilm formation and motility, cell cycle progression, toxicity and pathogen host interactions. In the process of Mycobacterium c-di-GMP signal with unique system and can affect the physiological and pathological the characteristics, but how the signal molecule mediated pathogenic Mycobacterium tuberculosis (Mycobacterium tuberculosis) and the interaction between the host is not clear, but at present in this important human pathogen there is no clear c-di-GMP receptor protein is reported. The study found that two new c-di-GMP receptor protein: a bite ferritin LCN2 human immune system involved in pathogen host interactions, another is Mycobacterium tuberculosis shikimate kinase Aro K.1. LCN2 C-D is the white iron eating eggs I-GMP.LCN2 is an important receptor molecule directly antibacterial components of human innate immune system, siderophore in nutrient deficient conditions it can capture the bacterial growth and to inhibit bacterial survival in vivo. We first through the bioinformatics prediction of LCN2 receptor c-di-GMP is possible, and using surface plasmon resonance (Surface Plasmon Resonance. SPR) and isothermal titration calorimetry (Isothermal Titration, Calorimetry, ITC) experiments confirmed that c-di-GMP is the same with the iron carrier Fe-Ent/Fe-CMBs, can be directly combined with R LCN2. However, under similar conditions we can not clearly detect R protein LCN2 and c-di-AMP, the interaction between GTP and C GAMP c-di-GMP and several other similar small molecules. This indicates that the interaction between c-di-GMP and R LCN2 with specificity. Further, the quantitative determination of ITC, c-di-GMP R LCN2 protein titration The stoichiometric ratio is 1:1, the affinity of Kd was 1.63 + 0.05 M, indicated that one molecule of c-di-GMP combined with LCN2 r a molecular monomer protein. Therefore, our study found that macrophage ferritin LCN2 receptor c-di-GMP is direct, with bacteria may use their blocking the signal molecules of LCN2 protein and iron carrier in the process of infection in, and remove the antibacterial effect of.2. of Mycobacterium tuberculosis shikimate kinase Aro receptor c-di-GMP. K is a direct shikimate kinase Aro K is a key enzyme of Mycobacterium tuberculosis shikimate pathway and cell wall metabolism, anti tuberculosis drug design has become a potential target protein. First we selected about 80 crystal the structure of information in the PDB database of the H37Rv protein of Mycobacterium tuberculosis, established Mycobacterium tuberculosis protein library screening library found shikimate kinase Aro K is possible and then use the c-di-GMP receptor. 绱浜よ仈瀹為獙璇佹槑鏀懼皠鎬у悓浣嶇礌鏍囪鐨刢-di-GMP鍒嗗瓙鑳戒笌Aro K铔嬬櫧鐩存帴緇撳悎.榪涗竴姝，

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