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 细菌病原体之间的抗生素耐药性正在世界各地迅速传播。因此，是 迫切需要制定发现新型抗菌剂的策略。两个组件系统（TCS）是理想的 至少有两个原因，用于开发新型抗菌治疗的靶标：（i）它们通常是必不可少的 宿主内的细菌生长； （ii）它们在细菌中很常见，但在人和动物中显然不存在。 已经研究了TCS数十年了，信号转导的分子基础是众所周知的 关于这些信号系统的调节仍然存在重要问题。在此应用程序中，我将使用 细胞内人类病原体炭疽杆菌作为模型生物，并研究调节机制 HITRS信号系统的。该TCS感应吞噬细胞环境并提供直接健身 与宿主免疫细胞相互作用的优势。此外，HITRS系统被A激活 各种分子不同细胞包膜应激源，表明必须需要其他细胞因子 用于激活该系统。实际上，使用公正的遗传选择策略，我们确定了RNA结合 蛋白质krea（炭疽芽孢杆菌中的Comk抑制剂）通过调节在HITRS激活中起着至关重要的作用 TCS转录本的mRNA稳定性。此外，我们的初步数据表明，krea起作用 RNA结合蛋白（RBP）在HITRS信号传导中起重要的转录后调节作用。 近年来，细菌后转录后控制的重要性越来越多 尽管这些调节网络的机制在细菌中尚未理解。基于我们 初步数据，我们提出了一个模型，即新鉴定的RBP Krea在特定目标位点结合mRNA， 影响功能协调的mRNA集合的表达，并动态与其他RBP相互作用以促进 mRNA衰变，并促进哺乳动物宿主内的细菌生存。在此应用程序中，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（i）定义了直接RNA靶标和Krea的结合偏好，（ii）阐明 KREA在调节HITRS信号传导中的基本机制，（iii）确定了KREA-的功能分析 调节细菌生理的RNA稳定性，（iv）剖析HITRS信号转导的贡献 在吞噬作用和炭疽发病机理期间，Krea介导的RNA调节。而且，结果 从这项研究中获得的将为TCS调节提供新的见解，扩展我们对细菌后的了解 转录调节网络，并为开发新型抗菌治疗剂奠定了基础。