Characterizing the structure and function of a bacterial multi-kinase sensory complex

表征细菌多激酶感觉复合物的结构和功能

• 批准号: 10314187
• 负责人: Mclaughlin Maeve
• 金额: $ 6.6万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-13 至 2023-09-12
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10314187
• 关键词: Adhesions; Affect; Anti-Bacterial Agents; Bacteria; Binding; Biochemical; Bioinformatics; Biological Assay; Biological Models; Biosensor; Caulobacter crescentus; Cell Adhesion; Cell physiology; Cellular biology; ChIP-seq; Complement; Complex; Cues; DNA; DNA Binding; Data; Decision Making; Detection; Development; Environment; Eukaryota; Fluorescence Resonance Energy Transfer; Gene Expression; Genes; Genetic Transcription; Goals; Gram-Negative Bacteria; Homeostasis; In Vitro; Individual; Link; Mass Spectrum Analysis; Mediating; Microbial Biofilms; Modeling; Molecular; Names; Output; Oxidation-Reduction; PAWR protein; Phase; Phosphorylation; Phosphotransferases; Physiological; Physiological Processes; Play; Process; Proteins; Proteobacteria; Reaction; Regulation; Reporter; Research; Role; Sensory; Signal Pathway; Signal Transduction; Structure; Surface; System; Testing; Variant; Work; X-Ray Crystallography; antimicrobial; base; biological adaptation to stress; cell motility; environmental adaptation; extracellular; information processing; insight; interdisciplinary approach; novel; pathogenic bacteria; programs; protein-histidine kinase; reconstitution; response; sensor histidine kinase; sensory system; targeted treatment; transcription factor; yeast two hybrid system

Abstract Bacteria have an incredible capacity to sense and respond to intra- and extracellular fluctuations in the environment in order to maintain cellular homeostasis. In bacteria, environmental adaptation is commonly mediated by two-component systems (TCS) that consist of a sensor histidine kinase (HK) that phosphorylates a cognate response regulator (RR) in response to signal detection. Upon phosphorylation, the RR can bind to DNA and alter gene expression to facilitate environmental adaptation. Classical TCS have historically been thought to signal in a highly linear manner with minimal interaction or cross-regulation with other signaling pathways. A growing body of data from our group and others provide evidence that an unusual class of histidine kinases, known as HWE kinases, can form multi-protein signaling complexes, creating a new paradigm in bacterial signal transduction. These signaling systems can integrate information from numerous environmental inputs to coordinate an array of physiological responses. In Caulobacter crescentus, one such signaling complex, hereby referred to as the Alphaproteobacterial signalosome, has been identified to coordinately regulate cellular surface attachment, a critical initial step in biofilm formation. We have shown that the Alphaproteobacterial signalosome consists of a) the HWE kinase SkaH that functions as a molecular hub protein, b) the HWE kinase LovK, and c) the classical HK, SpdS. Individually, LovK and SpdS play critical roles in modulating the general stress response and stationary phase adaptation. Interestingly, sensory information from LovK and SpdS can be integrated through the signalosome to modulate cellular adhesion through the downstream transcription factors, RtrA and RtrB, and the hypothetical protein, RtrC. Preliminary data provides evidence that the signalosome is comprised of additional HWE and classical HK kinases, suggesting that the sensory complex can integrate a broader range of signals than previously suspected. The research proposed here takes a multidisciplinary approach to characterize the structure and function of the HWE signalosome. The first aim will use biochemical approaches and mass spectrometry to identify molecular partners of SkaH and dissect direct interactions within the signalosome. The second aim will complement the structural analysis of the signalosome by using biochemical approaches to analyze the signal flow through the signalosome components. Preliminary evidence suggests that the hypothetical protein, RtrC, is a cryptic transcription factor that functions as a critical output for the HWE signalosome. In the third aim, I will characterize the structure and function of RtrC with X-ray crystallography and fluorescent reporters. Additionally, I will use FRET-based biosensors and motility assays to examine the regulatory link between RtrC and c-di-GMP signaling. The HWE signalosome serves as a prime model system for examining how multi-kinase sensory systems detect and process complex environmental information in order to regulate physiological responses. Additionally, as HWE kinases are present in many bacterial pathogens, insights gained from this work will aid in the development of antibacterial therapies that target TCS.

抽象的 细菌具有令人难以置信的能力来感知和响应细胞内和细胞外的波动 环境以维持细胞稳态。在细菌中，环境适应通常是 由双组分系统 (TCS) 介导，该系统由传感器组氨酸激酶 (HK) 组成，可磷酸化 同源响应调节器（RR）响应信号检测。磷酸化后，RR 可以与 DNA 结合 并改变基因表达以促进环境适应。传统的 TCS 历史上被认为 以高度线性的方式发出信号，与其他信号传导途径的相互作用或交叉调节最小化。一个 我们小组和其他人的越来越多的数据提供了证据表明一类不寻常的组氨酸激酶， 被称为 HWE 激酶，可以形成多蛋白信号复合物，创造细菌信号的新范例 转导。这些信号系统可以将来自众多环境输入的信息集成到 协调一系列生理反应。在新月柄杆菌中，一种这样的信号复合物，特此 被称为 Alphaproteobacterial 信号体，已被确定可以协调调节细胞表面 附着，生物膜形成的关键初始步骤。我们已经证明，Alphaproteobacterial 信号体 由 a) 作为分子中心蛋白的 HWE 激酶 SkaH、b) HWE 激酶 LovK 和 c) 组成 经典的 HK，SpdS。单独而言，LovK 和 SpdS 在调节一般应激反应中发挥着关键作用 和固定相适应。有趣的是，来自 LovK 和 SpdS 的感官信息可以集成 通过信号体通过下游转录因子 RtrA 和 RtrB 和假设的蛋白质 RtrC。初步数据证明信号体是由 额外的 HWE 和经典 HK 激酶，表明感觉复合体可以整合更广泛的范围 信号数量比之前怀疑的要多。这里提出的研究采用多学科方法 表征 HWE 信号体的结构和功能。第一个目标将使用生化方法 和质谱法来识别 SkaH 的分子伴侣并剖析 SkaH 内的直接相互作用 信号体。第二个目标将通过使用生化来补充信号体的结构分析 分析通过信号体成分的信号流的方法。初步证据表明 假设的蛋白质 RtrC 是一种神秘的转录因子，充当 HWE 的关键输出 信号体。在第三个目标中，我将通过 X 射线晶体学来表征 RtrC 的结构和功能，并 荧光记者。此外，我将使用基于 FRET 的生物传感器和运动测定来检查 RtrC 和 c-di-GMP 信号之间的调节链接。 HWE 信号体作为主要模型系统 用于研究多激酶感觉系统如何按顺序检测和处理复杂的环境信息 来调节生理反应。此外，由于 HWE 激酶存在于许多细菌病原体中， 从这项工作中获得的见解将有助于开发针对 TCS 的抗菌疗法。