Molecular Mechanisms of Phosphate Signaling in E. coli

描述（由申请人提供）：拟议研究的目的是了解有关环境磷酸盐水平的信息如何通过磷酸盐信号转导途径传递以控制大肠杆菌中的基因表达。该通路的核心是反应调节因子PhoB和组氨酸激酶PhoR。大量的工作已经完成阐明这两种蛋白质的功能机制。本研究的重点是了解PstSCAB2和PhoU这两个辅助蛋白是如何参与磷酸盐的感知和信号通路的控制的。考虑到磷酸盐调控研究的历史，关于细胞如何感知磷酸盐以及该信息如何在细胞内传递的基本问题仍然存在，这多少有些令人惊讶。PstSCAB2是一种磷酸盐转运蛋白，也可作为该系统的传感器。PhoU控制PstSCAB2的活性，是PstSCAB2向PhoR传递信号所必需的。我们的第一个假设是，PstSCAB2转运体通过转运过程中固有的构象变化传递有关环境磷酸盐水平的信息。我们的第二个假设是，PhoU通过与特定的PstSCAB2构象特异性相互作用来传递这一信息，并介导与PhoR的蛋白/蛋白相互作用。我们计划通过遗传和生化方法来解决这些假设。转运蛋白的突变版本将被隔离，“锁定”成各种构象。我们将使用这些转运体的版本来捕获与其他蛋白质的复合物，使用共洗脱和共免疫沉淀实验以及细菌双杂交分析。这些方法应该被证明是相互补充的，并将提供最大的机会来观察所提议的信号复合体内的蛋白质/蛋白质相互作用。在分析PhoU的作用时，将在缺乏转运体的情况下分离组成型信号突变体，并研究其与PhoR的相互作用。此外，PhoU高度保守的氨基酸残基对其功能至关重要，这些残基将被突变并研究其功能和细胞定位。PhoU-GFP融合也将被构建并用于研究定位。这项工作很重要，因为这些信号蛋白是细菌在不断变化的环境中生存所必需的——包括人类免疫系统。这一特征与它们在高等真核生物中的缺失相结合，使这一信号通路成为开发新型抗菌药物的靶标。增加对这些信号转导蛋白的了解可能有助于合理设计药物来对抗病原体。

DESCRIPTION (provided by applicant): The objective of the proposed research is to understand how information concerning environmental phosphate levels is transmitted through the phosphate signal transduction pathway to control gene expression in Escherichia coli. At the heart of this pathway are the response regulator PhoB and the histidine kinase PhoR. A great deal of work has already been accomplished elucidating the mechanisms by which these two proteins function. The focus of this proposal is to understand how two auxiliary proteins, PstSCAB2 and PhoU, are involved in the sensing of phosphate and the control of this signaling pathway. Given the history of research into phosphate regulation, it is somewhat surprising that fundamental questions still exist as to how cells sense phosphate and how that information is transmitted within a cell. PstSCAB2 is a phosphate transporter that also functions as the sensor for the system. PhoU controls the activity of PstSCAB2 and is required for the transmission of the signal from PstSCAB2 to PhoR. Our first hypothesis is that the PstSCAB2 transporter transduces information about environmental phosphate levels through conformational changes that are inherent to the transport process. Our second hypothesis is that PhoU transmits this information by specifically interacting with a particular PstSCAB2 conformation and mediates protein/protein interactions with PhoR. We plan on addressing these hypotheses through both genetic and biochemical approaches. Mutant versions of the transporter will be isolated that "lock" it into various conformations. We will use these versions of the transporter to trap complexes with other proteins using co-elution and co-immunoprecipitation experiments and as well as bacterial two-hybrid analysis. These approaches should prove to be complementary to one another and will provide the greatest opportunity to observe protein/protein interactions within the proposed signaling complex. In the analysis of the role of PhoU, constitutive signaling mutants will be isolated in the absence of the transporter and studied for interactions with PhoR. In addition, highly conserved amino acid residues of PhoU, which must be important to its function, will be mutated and studied for function and cellular localization. PhoU-GFP fusions will also be constructed and used to study localization. The proposed work is important because these signaling proteins are essential for bacteria to survive changing environments - including the human immune system. This feature combined with their absence in higher eukaryotes, makes this signaling pathway a targets for the development of new antimicrobial drugs. An increased understanding of these signal transduction proteins may assist in the rational design of drugs to combat pathogens. PUBLIC HEALTH RELEVANCE: This project, which focuses on determining how bacterial cells sense phosphate to control various survival genes, is relevant to public health because it provides foundational basic research into a fundamental process of bacteria. The phosphate sensory response pathway is essential to many pathogens in their ability to cause disease. Because of this research, strategies and drugs may be developed that will limit a pathogens survival, thereby helping to combat various bacterial infections.