Chemical Biology Tools for Visualization of Bacterial Chemoreceptor Signaling

用于细菌化学感受器信号传导可视化的化学生物学工具

• 批准号: 8836413
• 负责人: heather L hodges
• 金额: $ 3.04万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-01 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8836413
• 关键词: Affect; Aspartate; Bacteria; Behavior; Binding; Biological; Biological Models; Biology; Cells; Chemicals; Chemoreceptors; Chemotaxis; Complex; Cues; Disease; Electron Microscopy; Electrons; Environment; Escherichia coli; Family; Fluorescence; Fluorescence Microscopy; Freezing; Health; Histidine; Image; Imagery; Individual; Infection; Label; Laboratories; Ligand Binding; Ligands; Mammals; Membrane; Molecular; Movement; Noise; Nutrient; Organizational Change; Pathogenicity; Pathway interactions; Plants; Polymers; Preparation; Property; Proteins; Research; Resolution; Role; Route; Sample Size; Sampling; Scheme; Sensory; Signal Transduction; Signaling Molecule; Signaling Protein; Site; Structure; Symbiosis; System; Techniques; Vesicle; Work; appendage; base; biological systems; cell motility; design; extracellular; fluorophore; frontier; improved; information processing; insight; nanometer; nanoparticle; nanoscale; novel strategies; pathogenic bacteria; public health relevance; receptor; receptor binding; reconstitution; reconstruction; response; scaffold; signal processing; success; tool; tool development

DESCRIPTION (provided by applicant): Bacteria are able to sense and respond to their environment through multi-protein signal transduction cascades, the most common of which is the histidine-aspartate sensory pathways (HAPs). The most characterized HAP is a bacterial motility system termed bacterial chemotaxis. The components of the chemotaxis core signaling complex are conserved among motile bacteria and within HAPs. This research will investigate fundamental principles underlying bacterial signal transduction mechanisms using Escherichia coli as a model system. Despite over five decades of research and extensive characterization of the individual E. coli chemotaxis pathway components, a full molecular level understanding of signal transduction has not been elucidated. Signal transduction in chemotaxis is initiated by the binding of extracellular ligands to a specialized family of transmembrane receptors. These transmembrane receptors or chemoreceptors, cluster at distinct regions of the cell and are arranged in an extended lattice. Chemoreceptor organization is conserved across bacteria. However, the importance of this organization has yet to be fully realized. The application of multivalent ligands to the chemotaxis system afforded the first evidence that an extended, membrane associated lattice of chemotaxis signaling proteins is critical for transducing signals. Additionally, our group provided evidence that attractants transduce signals by disrupting organization within the signaling array by demonstrating receptor delocalization upon activation by immuno-fluorescence microscopy. This formulated a hypothesis that changes in the conserved array organization controls signaling. We aim to image changes in chemoreceptor organization upon stimulation. Chemoreceptor imaging will be performed by electron cryotomography (ECT); an electron microscopy technique that allows for 3D reconstructions of nanometer scale biological structures. Rapid freezing of samples permits visualization of preserved protein organizations. Advances within our group developing ligand polymers will provide the tools necessary to probe receptor organization. The synthetic tractability of these polymers will allow the appendage of a fluorophore for imaging by combination fluorescent/ECT providing assurance that images are of actively engaged receptors. The results of this work will be vital to understanding bacterial motility and transmembrane signaling in general. The proposed tools for ECT will be broadly applicable to elucidating features of other important biological systems. Moreover, the chemotaxis system has been implicated in regulating the differentiation of some bacteria to a pathogenic swarmer cell state. Uncovering the chemotaxis signaling mechanisms will have ramifications in understanding the impact of the chemotaxis system on bacterial pathogenicity and provide a new unexplored mode for control and mitigation.

描述(申请人提供)：细菌能够通过多蛋白质信号转导通路感知和响应环境，其中最常见的是组氨酸-天冬氨酸感觉通路(HAPS)。HAP最具特征的是一种细菌运动系统，称为细菌趋化性。趋化性核心信号复合体的组成在可移动细菌之间和HAPS内是保守的。这项研究将以大肠杆菌为模型系统，研究细菌信号转导机制的基本原理。尽管经过了50多年的研究和对单个大肠杆菌趋化途径成分的广泛表征，但对信号转导的全分子水平的理解还没有阐明。趋化作用中的信号转导是由细胞外配体与一个特殊的跨膜受体家族结合而启动的。这些跨膜受体或化学受体聚集在细胞的不同区域，并以扩展的格子排列。化学感受器组织在细菌中是保守的。然而，这一组织的重要性尚未得到充分认识。多价配体在趋化系统中的应用首次证明了一个延伸的膜相关趋化信号蛋白晶格对于信号转导是至关重要的。此外，我们的团队提供了证据，证明了引诱剂通过破坏信号阵列中的组织来传递信号，方法是在免疫荧光显微镜下显示受体激活时的离域。这形成了一个假设，即保守的阵列组织中的变化控制信号。我们的目标是成像化学感受器组织在刺激时的变化。化学感受器成像将通过电子冷冻断层扫描(ECT)进行；这是一种电子显微镜技术，可以对纳米级的生物结构进行3D重建。样品的快速冷冻可以使保存的蛋白质组织可视化。我们团队在开发配体聚合物方面的进展将提供探测受体组织所需的工具。这些聚合物的合成可处理性将允许通过结合荧光/ECT进行成像的荧光团的附属物，从而确保图像是活跃参与的受体。这项工作的结果将是至关重要的了解细菌的运动性和跨膜信号一般。建议的ECT工具将广泛适用于阐明其他重要生物系统的特征。此外，趋化系统还参与调节某些细菌向致病群集性细胞状态的分化。揭示趋化信号机制将对理解趋化系统对细菌致病性的影响具有重要意义，并为控制和缓解提供一种新的尚未探索的模式。