蜡状芽胞杆菌群中c-di-AMP新型受体蛋白PrpD的功能及其调控芽胞形成的分子机制研究

c-di-AMP is an important bacterial second messenger in regulating various cellular physiological functions, for example sporulation. However, the molecular mechanisms are largely unknown. Identification and characterization of novel c-di-AMP receptors are critical for uncovering the regulation pathways. In our previous research, we developed an affinity chromatography system connected with c-di-AMP ligand, and successfully fished a potential c-di-AMP receptor protein of Bacillus thuringiensis which was later identified as PrpD, 2-methylcitrate dehydratase. Further, we demonstrated that when prpD gene was deleted based on the markerless gene knock-out method, sporulation of Bacillus thuringiensis was significantly delayed. In this program, to confirm the hypothesis that PrpD is a novel receptor of c-di-AMP and reveal its signal pathway, we plan to perform the following experiments: 1) identification of the interaction between PrpD and c-di-AMP and determination of their binding characteristics; (2) determination the effect of c-di-AMP on the enzymatic activity of PrpD; (3) sequencing the transcriptome and proteome of the start strain BMB171, ΔprpD mutant strain, sporulation-specific diadenylate cyclase gene mutant strain ΔcdaS and ΔcdaSΔprpD double mutant strain, respectively, and identifying the key sporulation-related genes whose expression levels are significantly altered. Based on these design, we try to reveal a novel mechanism of c-di-AMP regulated sporulation through PrpD protein. This project is believed to provide new insights into the pathogenesis of B. cereus group.

c-di-AMP是细菌重要的第二信使分子，能够调控芽胞形成等多种生理功能，但其调节机制仍不清楚。c-di-AMP新型受体的鉴定是揭示其调节路径的关键。本项目前期构建了以c-di-AMP为配基的亲和色谱体系，在苏云金芽胞杆菌中钓取到了一种能与之特异结合的蛋白2-甲基柠檬酸脱水酶（PrpD），并发现prpD的缺失能够显著推迟芽胞形成。基于此，拟进行以下研究：1）证实c-di-AMP能够与PrpD结合，并测定其结合特性参数；2）分析c-di-AMP对PrpD酶活力的影响；3）分别测定出发菌株BMB171、潜在受体基因缺失突变株ΔprpD、芽胞特异性c-di-AMP合成酶基因缺失突变株ΔcdaS、双缺失突变株ΔcdaSΔprpD的转录组和蛋白组，比较和分析芽胞形成关键基因的表达差异，揭示c-di-AMP通过PrpD调控芽胞形成的分子机制。本项目的开展为蜡状芽胞杆菌群的致病机理研究提供新的方向。

c-di-AMP是细菌重要的第二信使分子，参与调节多种重要的生理活动，包括感知DNA损伤、调控钾离子转运、调节芽胞形成等。c-di-AMP通过与受体结合并使受体变构，从而激活或抑制下游效应物的活性，调节相应的生理活动。我们利用苏云金芽胞杆菌中的c-di-AMP合成酶DisA合成了c-di-AMP，并以c-di-AMP配基亲和色谱“钓取”和鉴定了一类特异性结合蛋白PrpD。等温滴定量热法（ITC）结果表明，c-di-AMP与PrpD具有明显的结合作用。利用I-SceI酶介导的基因无痕敲除技术获得了苏云金芽胞杆菌prpD基因缺失突变株ΔprpD，发现该菌株的芽胞形成过程受到明显抑制。质谱结果显示，与出发菌株BMB171相比，ΔprpD菌株中PrpD蛋白的代谢底物2-甲基柠檬酸浓度提高了210倍，该代谢物的积累是导致ΔprpD菌株芽胞形成延迟的关键因素。2-甲基柠檬酸添加等实验进一步证实了2-甲基柠檬酸具备抑制芽胞形成的新颖功能。接着，利用比较转录组学手段明确了2-甲基柠檬酸是通过转录后水平影响sigF的表达进而调控芽胞形成。同时，发现2-甲基柠檬酸的累积不利于细菌的生长，并从转录组水平揭示了该物质诱发细菌代谢紊乱的机理。此外，揭示了细菌中PrpD蛋白所在2-甲基柠檬酸循环的全新转录调控机制，即该循环关键酶基因同时受两个全局性转录调控因子CcpA、AbrB的负调控和LysR家族转录调控因子CcpC的正调控。另外，获得分辨率达1.3 Å的PrpD与柠檬酸的复合物晶体结构，说明柠檬酸可能能够调节PrpD酶活力。本课题的研究结果对细菌信号途径以及代谢调控的研究具有重要意义。