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: 10488627
• 负责人: Mclaughlin Maeve
• 金额: $ 6.76万
• 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/10488627
• 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 细菌具有不可思议的能力，可以感知和应对细胞内和细胞外波动。 为了维持细胞稳态。在细菌中，环境适应通常是 由两种组分系统（TC）介导的，由传感器组氨酸激酶（HK）组成，该激酶（HK）磷酸化A 响应信号检测的同源响应调节剂（RR）。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。历史上一直认为古典TC 以最小的相互作用或与其他信号通路进行交叉调节，以高度线性的方式发出信号。一个 我们小组和其他人的数据越来越多地提供了证据，表明一类不寻常的组氨酸激酶， 称为HWE激酶，可以形成多蛋白信号传导复合物，在细菌信号中产生新的范式 转导。这些信号系统可以将信息从众多环境输入到 协调一系列生理反应。在Caulobacter Crescentus中，一个这样的信号复合物 已被称为字母摩菌病信号体，已被鉴定为协调调节细胞表面 附件，生物膜形成的关键初步步骤。我们已经证明了字母摩托菌的信号体 由A）HWE激酶SKAH组成，可作为分子轮毂蛋白，b）HWE激酶LOVK和C） 经典香港，spds。 Lovk和SPD在调节一般压力响应中起着至关重要的作用 和固定相适应。有趣的是，可以集成Lovk和SPD的感官信息 通过信号体通过下游转录因子，RTRA和 RTRB和假设蛋白，RTRC。初步数据提供了信号体包含的证据 额外的HWE和经典的HK激酶，表明感觉络合物可以整合更广泛的范围 信号比以前怀疑。这里提出的研究采用多学科的方法 表征HWE信号体的结构和功能。第一个目标将使用生化方法 和质谱法以识别SKAH的分子伴侣并剖析直接相互作用 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 分析信号流的方法通过信号体组件。初步证据表明 假设蛋白RTRC是一个神秘的转录因子，可作为HWE的关键输出 信号体。在第三个目标中，我将通过X射线晶体学和 荧光记者。此外，我将使用基于FRET的生物传感器和运动性测定来检查 RTRC和C-DI-GMP信号传导之间的调节联系。 HWE信号体充当主要模型系统 用于检查多激酶感觉系统如何秩序检测和处理复杂的环境信息 调节生理反应。此外，由于许多细菌病原体存在HWE激酶，因此 从这项工作中获得的见解将有助于开发针对TC的抗菌疗法。