Chemical Biology Tools for Visualization of Bacterial Chemoreceptor Signaling

描述（由申请人提供）：细菌能够通过多蛋白信号转导级联来感知和响应环境，其中最常见的是组氨酸-天冬氨酸感觉通路（HAPs）。最典型的HAP是一种称为细菌趋化性的细菌运动系统。趋化核心信号复合物的成分在活动细菌和HAPs中是保守的。本研究将以大肠杆菌为模型系统，探讨细菌信号转导机制的基本原理。尽管经过50多年的研究和对大肠杆菌趋化途径成分的广泛表征，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感受器聚集在细胞的不同区域，并排列在一个扩展的晶格中。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。多价配体在趋化性系统中的应用首次证明了趋化性信号蛋白的扩展膜相关晶格对转导信号至关重要。此外，我们的团队提供了证据，证明引诱剂通过破坏信号阵列内的组织来传递信号，通过免疫荧光显微镜显示受体在激活时的脱位。这形成了一个假设，即保守阵列组织的变化控制着信号。我们的目标是成像化学感受器组织在刺激下的变化。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，可以对纳米尺度的生物结构进行三维重建。快速冷冻样品可以使保存的蛋白质组织可视化。我们小组在发展配体聚合物方面的进展将为探索受体组织提供必要的工具。这些聚合物的合成可追溯性将允许通过荧光/ECT组合成像的荧光团附属物，从而确保图像是积极参与的受体。这项工作的结果将是至关重要的理解细菌运动和跨膜信号。提出的ECT工具将广泛适用于阐明其他重要生物系统的特征。此外，趋化系统还涉及调节一些细菌向致病性群集细胞状态的分化。揭示趋化性信号机制将有助于理解趋化系统对细菌致病性的影响，并为控制和减轻细菌致病性提供一种新的未探索的模式。

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.