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

摘要 细菌有一种不可思议的能力，可以感知和响应细胞内和细胞外的波动， 环境，以维持细胞内稳态。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， 同源反应调节子（RR）响应于信号检测。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一直被认为 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。一 来自我们小组和其他人的越来越多的数据提供了证据，证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复合物，创造了细菌信号传导的新范例 转导这些信号系统可以整合来自众多环境输入的信息， 协调一系列生理反应。在新月柄杆菌（Caulobacter crescentus）中，一种这样的信号传导复合物，由此 被称为α-变形菌信号体，已被鉴定为协调调节细胞表面 附着，生物膜形成的关键初始步骤。我们已经证明，α-变形菌信号体 由a）HWE激酶SkaH，其作为分子中枢蛋白发挥作用，B）HWE激酶LovK，和c） 经典的HK，SpdS。LovK和SpdS分别在调节一般应激反应中起关键作用 和固定相位适应。有趣的是，来自LovK和SpdS的感觉信息可以被整合 通过信号体调节细胞粘附，通过下游转录因子RtrA和 RtrB和假设的蛋白质RtrC。初步数据提供的证据表明，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 比以前怀疑的信号。这里提出的研究采取了多学科的方法， 表征HWE信号体的结构和功能。第一个目标将使用生物化学方法 和质谱法来鉴定SkaH的分子伴侣，并分析SkaH内的直接相互作用。 信号体第二个目标将补充信号体的结构分析， 方法来分析通过信号体组件的信号流。初步证据显示， 假设的蛋白质RtrC是一种隐蔽的转录因子，作为HWE的关键输出发挥作用 信号体第三个目标是用X射线晶体学方法研究RtrC的结构和功能， 荧光记者。此外，我将使用基于FRET的生物传感器和运动分析来检查 RtrC和c-di-GMP信号传导之间的调节联系。HWE信号体作为主要模型系统 研究多激酶感觉系统如何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。

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.