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质信号转导级联来感知和响应其环境，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组分在运动细菌和HAP内是保守的。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。尽管经过五十多年的研究和对个体E.大肠杆菌趋化性途径的组成部分，一个完整的分子水平的理解信号转导尚未阐明。趋化性中的信号转导是通过细胞外配体与一个专门的跨膜受体家族的结合而启动的。这些跨膜受体或化学感受器聚集在细胞的不同区域，并排列在扩展的晶格中。化学受体组织在细菌中是保守的。然而，该组织的重要性尚未得到充分认识。应用多价配体的趋化性系统提供了第一个证据表明，一个扩展的，膜相关的晶格的趋化性信号蛋白是至关重要的转导信号。此外，我们的小组提供的证据表明，引诱剂通过破坏信号阵列内的组织来抑制信号，通过免疫荧光显微镜证实激活后受体的离域。这形成了一个假设，即保守阵列组织的变化控制信号传导。我们的目标是成像刺激后化学感受器组织的变化。化学感受器成像将通过电子冷冻断层扫描（ECT）进行;电子显微镜技术，允许纳米级生物结构的3D重建。样品的快速冷冻允许保存的蛋白质组织的可视化。在我们小组开发配体聚合物的进展将提供探测受体组织所需的工具。这些聚合物的合成易处理性将允许荧光团的附属物用于通过组合荧光/ECT进行成像，从而确保图像是主动接合的受体。这项工作的结果将是至关重要的了解细菌运动和跨膜信号一般。建议的ECT工具将广泛适用于阐明其他重要的生物系统的功能。此外，趋化性系统已涉及调节某些细菌分化为致病性群集细胞状态。揭示趋化性信号机制将对理解趋化性系统对细菌致病性的影响产生影响，并提供一种新的未探索的控制和缓解模式。

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.