c-di-AMP候选受体蛋白TerD的结构功能及其信号途径研究

Cyclic diadenosine monophosphate (c-di-AMP) is a new member of bacterial cyclic nucleotide messengers which is firstly discovered in 2008. For now it is reported that c-di-AMP is involved in two physiological functions in bacteria: i) to regulate the formation of endospore in Bacillus subtilis, and ii) to control the synthesis of peptidoglycan in both Staphyloccocus aureus and Bacillus subtilis. However, the receptors and targets along with these c-di-AMP modulating signaling pathways are is still unknown. ..In our previous research, we have expressed and purified the c-di-AMP synthetase DisA from Bacillus thuringiensis, and obtained a large amount of chromatographically pure c-di-AMP. Then we successfully established an affinity chromatography system concerning c-di-AMP as ligand. As a result, a probably receptor protein named TerD annotated as a stress response protein were gained...To confirm that TerD is a candidate acceptor of c-di-AMP and reveal its signaling pathway, we will focus on the following experiment: .1) To solve the structure of TerD and the complex structure of TerD together with c-di-AMP;.2) To construct a series of bacterial strains including the terD knock-out strain, the complementary terD knock-out strain and the overexpression terD strain, and observe the phenotype and determine the transcriptomic differences under various conditions, including culture conditions and stimulus conditions, together with mother strain Bacillus thuringiensis BMB171;.3) To set up a affinity chromatography system with TerD as the ligand and fish DNA fragments and proteins having specially affinity with TerD, respectively...Based on the foundation of these tests, we try to find out the kind of target genes and reveal the intact signaling pathway from the receptors to the targets mediated by c-di-AMP as well as its physiological function in bacteria.

c-di-AMP 是2008年新发现的细菌第二信使分子。目前，只报道了c-di-AMP参与调控细菌的芽胞形成与肽聚糖的合成两种生理功能。而其信号调节途径相关受体与靶标完全未知。我们利用苏云金芽胞杆菌（Bt）中的DisA合成了c-di-AMP，构建了以c-di-AMP为配基的亲和色谱体系，捕获并鉴定了一种特异结合的蛋白TerD。为了证明TerD是c-di-AMP的受体，并解析c-di-AMP的信号通路，我们拟进行以下几个方面的研究：1）解析TerD的结构及TerD与c-di-AMP的复合结构；2）构建敲除与过表达terD的Bt菌株，并观察不同条件下的表型变化及测定转录表达谱差异；3）构建以TerD为配基的亲和色谱体系，分别垂钓能与TerD特异结合的DNA片段或蛋白质。在这些实验基础上，推测TerD调控的靶基因及调控方式，构建一条从结合受体到作用靶标的完整的c-di-AMP信号通路。

c-di-AMP 是2008年新发现的细菌核苷酸类第二信使分子。在外界信号的刺激下，细菌细胞分别通过合成酶——二腺苷酸环化酶（diadenylate cyclase，DAC）与降解酶——磷酸二酯酶（phosphodiesterase，PDE）来调节胞内c-di-AMP的浓度。c-di-AMP通过特定的受体或效应物，调节靶标基因的表达或蛋白质的构象，从而行使不同的生理功能。项目申请时，其信号调节途径相关受体与靶标完全未知。我们研究了三种DAC即：DisA、CdaA与CdaS以及一种PDE——GdpP的酶学性质及转录特性。特别揭示了CdaS为芽胞形成期特异性DAC，影响芽胞的形成。基因敲除实验表明对数生长期表达的disA与cdaA不能同时被敲除。我们利用苏云金芽胞杆菌中的DAC——DisA合成了c-di-AMP，构建了以c-di-AMP为配基的亲和垂钓体系，捕获并鉴定了一类特异结合的蛋白TerD。同位素实验结合质谱与ITC的结果表明，在某些金属离子（Ca2+）的参与下，不同的TerD既可能是c-di-AMP的生理效应物（如TerD2），也可能是cAMP的效应物（如TerD3）。terD操纵子由三个同源的terD基因（terD1、terD2和terD3）和一个terC基因（TerC为跨膜蛋白）以及yceG、telA组成，采用不同组合方式的基因敲除策略，我们发现TerD不仅与细菌的碲（Te）抗性相关，而且影响细菌对高温的敏感性。进一步我们揭示了c-di-AMP的RNA类受体，即c-di-AMP的核糖开关存在于K+转运蛋白操纵子kdpFABCD的5’-非翻译区，调控K+的输送。同时我们发现，在蜡样芽胞杆菌群中c-di-AMP与另外一种非常重要的第二信使分子c-di-GMP的蛋白类受体或效应物很少，可能主要是通过核糖开关起调控作用，这些结果对于细菌信号途径以及细菌代谢调控的研究具有重要意义。