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

总结 细菌病原体的抗生素耐药性正在世界各地迅速蔓延。因此有 迫切需要开发新的抗微生物药物的策略。双组分体系（TCS）是理想的 开发新型抗微生物治疗的目标至少有两个原因：（i）它们通常是必需的， （ii）它们在细菌中常见，但在人类和动物中明显缺乏。 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调节仍然存在重要的问题。在这个应用程序中，我将使用 细胞内人类病原体炭疽杆菌作为模式生物，并探讨其调控机制 HitRS信号系统这种TCS感觉吞噬细胞的细胞环境，并提供了一个直接的健身 在与宿主免疫细胞的相互作用过程中具有优势。此外，HitRS系统是由一个 多种分子不同的细胞包膜应激源，表明必须需要额外的细胞因子 启动这个系统。事实上，使用无偏见的遗传选择策略，我们确定了一种RNA结合， 蛋白KreA（B.炭疽菌），其通过调节 TCS转录物的mRNA稳定性。此外，我们的初步数据表明，KreA的功能， RNA结合蛋白（RBP）在HitRS信号转导中起着重要的转录后调节作用。 近年来，细菌转录后调控的重要性越来越受到重视 尽管在细菌中对这些调节网络的机制知之甚少。基于我们 初步数据，我们提出了一个新发现的RBP KreA在特定靶位点结合mRNA的模型， 影响功能协调的mRNA组的表达，与其他RBP动态相互作用， mRNA衰变，并促进细菌在哺乳动物宿主内的存活。在此应用程序中，我们将联合收割机 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靶点和结合偏好，（ii）阐明KreA的作用机制。 KreA在调节HitRS信号传导中的潜在机制，（iii）确定KreA的功能分支- 调节RNA稳定性对细菌生理学的影响，以及（iv）分析HitRS信号转导的贡献 和KreA介导的RNA调节过程中的吞噬作用和炭疽发病机制。此外，结果 从这项研究中获得的信息将为TCS调控提供新的见解，扩大我们对细菌后 转录调控网络，并为开发新的抗微生物疗法奠定基础。

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.