The regulatory role of an RNA binding protein in two-component signaling and its impact on cellular physiology and anthrax pathogenesis

摘要 细菌病原体对抗生素的耐药性正在世界各地迅速传播。因此，它是 迫切需要制定战略，以发现新的抗菌剂。两组分系统(TCS)是理想的 开发新的抗菌疗法的目标至少有两个原因：(1)它们往往对 细菌在宿主内生长；(2)它们在细菌中很常见，但在人类和动物中明显不存在。 TCS已经被研究了几十年，信号转导的分子基础是众所周知的，然而， 关于这些信号系统的监管，仍然存在重要的问题。在此应用程序中，我将使用 细胞内炭疽芽孢杆菌作为模式生物及其调控机制的研究 HITRS信号系统的。这种TCS感知吞噬细胞环境，并提供直接的健康 在与宿主免疫细胞的相互作用过程中具有优势。此外，HITRS系统由一个 各种分子上不同的细胞被膜应激源，表明必须需要额外的细胞因子 来激活这个系统。事实上，使用一种无偏见的基因选择策略，我们确定了一种RNA结合 蛋白Krea(炭疽杆菌中的ComK抑制因子)，通过调节在hITRS激活中起关键作用 TCS转录本的mRNA稳定性。此外，我们的初步数据表明，KREA作为一种 RNA结合蛋白(RBP)，在hTRS信号转导中起重要的转录后调控作用。 近年来，细菌转录后调控的重要性日益受到重视。 尽管这些调控网络的机制在细菌中知之甚少。基于我们的 初步数据，我们提出了一个新发现的RBP Krea与特定靶点的mRNA结合的模型， 影响功能协调的mRNAs集的表达，与其他限制性商业惯例动态相互作用，以促进 信使核糖核酸腐烂，并促进哺乳动物宿主内的细菌存活。在此应用程序中，我们将结合 包括生化分析、遗传学、转录组学、蛋白质组学、活细胞成像、 和小鼠感染模型，以(I)确定Krea的直接RNA靶标和结合偏好，(Ii)阐明 KREA调节HITRS信号的潜在机制，(Iii)确定KREA- 调节RNA的稳定性对细菌生理的影响；(Iv)剖析HitRS信号转导的作用 Krea介导的RNA在吞噬和炭疽发病过程中的调节。而且，结果是， 本研究将为TCS的调控提供新的见解，扩大我们对细菌后感染的认识 转录调控网络，并为开发新的抗菌疗法奠定基础。

SUMMARY Antibiotic resistance among bacterial pathogens is spreading rapidly around the world. Therefore, it is urgent to develop strategies to discover novel antimicrobial agents. Two component system (TCSs) are ideal targets for developing novel antimicrobial treatments for at least two reasons: (i) they are often essential for bacterial growth within the host; (ii) they are common in bacteria but evidently absent in human and animals. TCSs have been studied for decades and the molecular basis of signal transduction is well known, however, important questions remain regarding regulation of these signaling systems. In this application, I will use the intracellular human pathogen Bacillus anthracis as a model organism and investigate the regulatory mechanism of the HitRS signaling system. This TCS senses the phagocyte cell environment and provides a direct fitness advantage during the interactions with the host immune cells. Furthermore, the HitRS system is activated by a variety of molecular distinct cell envelope stressors, suggesting that additional cellular factors must be required for activation of this system. Indeed, using an unbiased genetic selection strategy, we identified an RNA binding protein KreA (ComK repressor in B. anthracis) that plays a critical role in HitRS activation through modulating mRNA stability of the TCS transcripts. In addition, our preliminary data demonstrate that KreA functions as an RNA binding protein (RBP) and plays an important post-transcriptional regulatory role in HitRS signaling. The importance of bacterial post-transcriptional control has been increasingly appreciated in recent years although the mechanisms of these regulatory networks are poorly understood in bacteria. Based on our preliminary data, we propose a model that the newly identified RBP KreA binds mRNA at specific target sites, impacts expression of functionally coordinated sets of mRNAs, interacts with other RBPs dynamically to facilitate mRNA decay, and promotes bacterial survival within the mammalian hosts. In this application, we will combine a number of strategies including biochemical analysis, genetics, transcriptomics, proteomics, live cell imaging, and mouse infection models to (i) define the direct RNA targets and binding preference of KreA, (ii) elucidate the underlying mechanism of KreA in regulating HitRS signaling, (iii) determine the functional ramifications of KreA- modulated RNA stability on bacterial physiology, and (iv) dissect the contribution of HitRS signal transduction and KreA-mediated RNA regulation during phagocytosis and anthrax pathogenesis. Moreover, the results obtained from this study will provide new insights into TCS regulation, expand our knowledge of bacterial post- transcriptional regulatory networks, and lay the groundwork for developing novel antimicrobial therapeutics.