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

RNA结合蛋白在双组分信号传导中的调节作用及其对细胞生理学和炭疽发病机制的影响

• 批准号: 10436636
• 负责人: Hualiang Pi
• 金额: $ 0.25万
• 依托单位: VANDERBILT UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10436636
• 关键词: Affect; Animal Model; Animals; Anthrax disease; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotics; Bacillus anthracis; Bacteria; Bacterial Physiology; Binding; Biochemical; Cell physiology; Cells; Coupled; Data; Detection; Development; Electrophoretic Mobility Shift Assay; Environment; Formaldehyde; Genes; Genetic; Genetic Transcription; Gram-Positive Bacteria; Growth; High-Throughput Nucleotide Sequencing; Human; Immune; Immune response; Immunoprecipitation; In Vitro; Incidence; Infection; Kinetics; Knowledge; Light; Link; Mediating; Membrane; Messenger RNA; Microscopy; Modeling; Molecular; Mus; Nutrient; Osmotic Pressure; Oxidation-Reduction; Pathogenesis; Pathway interactions; Phagocytes; Phagocytosis; Plants; Play; Post-Transcriptional Regulation; Process; Proteins; Proteomics; RNA; RNA Decay; RNA Degradation; RNA Stability; RNA-Binding Proteins; Regulation; Regulon; Reporter; Resolution; Role; Signal Transduction; Site; Stress; System; Temperature; Testing; Therapeutic; Time; Transcript; Virulence; antibiotic resistant infections; antimicrobial; antimicrobial drug; base; biological adaptation to stress; cell envelope; crosslink; defined contribution; experience; experimental study; fitness; genetic selection; host colonization; human pathogen; insight; live cell imaging; mRNA Decay; mRNA Stability; mRNA Transcript Degradation; macrophage; mouse model; novel; novel therapeutic intervention; pathogen; pathogenic bacteria; preference; quorum sensing; response; small molecule; stressor; transcriptome; transcriptome sequencing; transcriptomics; uptake

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.

概括 细菌病原体的抗生素耐药性正在世界各地迅速蔓延。因此，它是 迫切需要制定策略来发现新型抗菌药物。两组分系统 (TCS) 是理想的选择 开发新型抗菌治疗的目标至少有两个原因：（i）它们通常对于 细菌在宿主体内生长； (ii) 它们在细菌中很常见，但在人类和动物中显然不存在。 TCS 的研究已经有几十年了，信号转导的分子基础众所周知，然而， 关于这些信号系统的监管仍然存在重要问题。在此应用程序中，我将使用 以细胞内人类病原体炭疽杆菌为模型生物并探讨其调控机制 HitRS 信号系统。该 TCS 感知吞噬细胞环境并提供直接的适应性 在与宿主免疫细胞相互作用过程中具有优势。此外，HitRS 系统是由 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 用于激活该系统。事实上，使用公正的遗传选择策略，我们鉴定了 RNA 结合 蛋白质 KreA（炭疽芽孢杆菌中的 ComK 阻遏蛋白）通过调节在 HitRS 激活中发挥关键作用 TCS 转录本的 mRNA 稳定性。此外，我们的初步数据表明 KreA 的功能是 RNA 结合蛋白 (RBP) 在 HitRS 信号传导中发挥重要的转录后调节作用。 近年来，细菌转录后控制的重要性越来越受到人们的重视 尽管人们对细菌中这些调节网络的机制知之甚少。基于我们的 初步数据，我们提出了一个新鉴定的 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 动态相互作用以促进 mRNA 降解，并促进哺乳动物宿主内细菌的存活。在这个应用程序中，我们将结合 许多策略，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) 定义 KreA 的直接 RNA 靶点和结合偏好，(ii) 阐明 KreA 调节 HitRS 信号传导的潜在机制，(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) 剖析 HitRS 信号转导的贡献 吞噬作用和炭疽发病过程中 KreA 介导的 RNA 调节。而且，结果 从这项研究中获得的结果将为 TCS 调节提供新的见解，扩展我们对细菌后的了解。 转录调控网络，并为开发新型抗菌疗法奠定基础。