细菌的CYCLIC-DI-GMP信号分子与两个新受体蛋白之间的相互作用研究

发布时间：2018-02-20 03:07

  本文关键词： c-di-GMP 结核分枝杆菌 噬铁蛋白 莽草酸激酶 病原-宿主相互作用　出处：《华中农业大学》2016年博士论文　论文类型：学位论文

【摘要】：环二鸟苷单磷酸(cyclic-di-GMP,简称c-di-GMP)是细菌中普遍存在的多功能第二信使分子,广泛参与调控细菌生物膜形成、运动性、细胞周期进程、毒力以及病原-宿主相互作用等过程。分枝杆菌具有独特的c-di-GMP信号系统并能影响其多种生理和病理特性,但是该信号分子如何介导病原性结核分枝杆菌(Mycobacterium tuberculosis)与宿主之间的相互作用还基本不清楚,而且目前在该重要人类病原菌中还没有明确的c-di-GMP信号受体蛋白报道。本研究发现了两个新的c-di-GMP受体蛋白:一个是参与病原-宿主相互作用的人免疫系统噬铁蛋白LCN2,另一个是结核分枝杆菌的莽草酸激酶Aro K。1.人噬铁蛋白LCN2是c-di-GMP分子的直接受体。LCN2是人类先天免疫系统的重要抗菌元件,在营养缺乏条件下它通过截获细菌生长必须的铁载体而抑制细菌在宿主体内的存活。我们首先通过生物信息学方法预测LCN2是c-di-GMP的可能受体,并利用表面等离子共振(Surface Plasmon Resonance,SPR)和等温滴定量热(Isothermal Titration Calorimetry,ITC)实验证实,c-di-GMP也与铁载体Fe-Ent/Fe-CMBs一样,能够与r LCN2直接结合。但是,在相似条件下我们不能清楚探测到r LCN2蛋白与c-di-AMP、GTP和c GAMP等与c-di-GMP类似的其它几个小分子之间的相互作用。这表明,c-di-GMP与r LCN2之间的相互作用具有特异性。进一步,ITC定量测定发现,c-di-GMP滴定r LCN2蛋白的化学计量比是1:1,亲和力Kd为1.63±0.05μM,表明一分子c-di-GMP结合一分子r LCN2单体蛋白。因此,我们的研究发现了噬铁蛋白LCN2是c-di-GMP的直接受体,细菌在感染过程中可能利用自己的这个信号分子阻塞LCN2蛋白与铁载体的结合,从而解除其抗菌效应。2.结核分枝杆菌莽草酸激酶Aro K是c-di-GMP的直接受体。莽草酸激酶Aro K是结核分枝杆菌莽草酸途径和细胞壁合成代谢的关键酶,已经成为抗结核药物设计的潜在靶标蛋白。首先我们选取了大约80个在PDB数据库中有晶体结构信息的结核分枝杆菌H37Rv蛋白,建立了结核分枝杆菌蛋白质文库,筛选文库发现莽草酸激酶Aro K是可能的c-di-GMP受体。然后利用紫外交联实验证明放射性同位素标记的c-di-GMP分子能与Aro K蛋白直接结合。进一步,利用竞争性结合实验证实c-di-GMP与Aro K结合具有特异性。通过ITC定量测定发现,c-di-GMP滴定Aro K蛋白的化学计量比是1:1,亲和力Kd为2.453±0.05μM,表明一分子c-di-GMP结合一分子Aro K单体蛋白。通过设计并成功表达纯化Aro K一系列关键氨基酸残基突变蛋白,发现了位于催化中心的关键氨基酸Ser16也是Aro K与c-di-GMP相互作用的关键氨基酸。最后,通过测定激酶活性表明c-di-GMP对Aro K的酶促反应有明显的抑制作用。因此,Aro K是一个新的c-di-GMP受体,c-di-GMP能够直接靶向Aro K的活性中心从而影响其酶活。这些结果表明c-di-GMP信号分子可能通过影响莽草酸激酶Aro K的活性,调控结核分枝杆菌杆菌的细胞壁分支酸代谢,从而影响病原菌的生长和毒力;同时,该工作也为病原菌莽草酸激酶抑制剂的设计提供了新的线索。
[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�澶栦氦鑱斿疄楠岃瘉鏄庢斁灏勬�у悓浣嶇礌鏍囪�扮殑c-di-GMP鍒嗗瓙鑳戒笌Aro K铔嬬櫧鐩存帴缁撳悎.杩涗竴姝，

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