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

基本信息

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
项目类别：
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

项目摘要

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（由申请人提供）：细菌能够通过多蛋白信号转导级联感知并响应其环境，其中最常见的是组氨酸-天冬氨酸感觉通路（HAP）。最具特征的 HAP 是称为细菌趋化性的细菌运动系统。趋化性核心信号复合物的成分在运动细菌和 HAP 内是保守的。这项研究将使用大肠杆菌作为模型系统来研究细菌信号转导机制的基本原理。尽管对单个大肠杆菌趋化途径成分进行了五十多年的研究和广泛的表征，但对信号转导的完整分子水平理解尚未阐明。趋化性中的信号转导是通过细胞外配体与专门的跨膜受体家族的结合来启动的。这些跨膜受体或化学感受器聚集在细胞的不同区域，并排列成扩展的网格。化学感受器组织在细菌中是保守的。然而，这个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提供了第一个证据，证明趋化信号蛋白的延伸的、膜相关的晶格对于转导信号至关重要。此外，我们的小组提供了证据，证明引诱剂通过免疫荧光显微镜激活后受体离域破坏信号阵列内的组织来转导信号。这提出了一个假设，即保守阵列组织的变化控制着信号传导。我们的目标是对刺激后化学感受器组织的变化进行成像。化学感受器成像将通过电子冷冻断层扫描（ECT）进行；一种电子显微镜技术，可对纳米级生物结构进行 3D 重建。快速冷冻样品可以使保存的蛋白质组织可视化。我们小组开发配体聚合物的进展将为探测受体组织提供必要的工具。这些聚合物的合成易处理性将允许附加荧光团，通过组合荧光/ECT 进行成像，从而确保图像是积极参与的受体的。这项工作的结果对于理解细菌运动和跨膜信号传导至关重要。拟议的 ECT 工具将广泛适用于阐明其他重要生物系统的特征。此外，趋化系统参与调节一些细菌向致病性群细胞状态的分化。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产生影响，并为控制和缓解提供一种新的、未经探索的模式。