Integration of Small RNAs in Control of Salmonella Pathogenicity Island 1

项目摘要 感知和应对环境条件中各种动态变化的能力对于 细菌适合度许多信号必须通过细胞调节系统整合，以促进细胞的生长。 对这些变化条件的最佳反应。沙门氏菌是一种重要的模式生物， 了解基因调控和细菌致病机理。沙门氏菌致病的一个必要条件是 将效应蛋白通过编码于 沙门氏菌致病岛1（SPI 1）。SPI 1已经成为了解细菌如何利用 不同的全球监管体系，以应对众多的环境信号。我们的长期目标是 在系统水平上了解控制SPI 1的信号如何在转录，后 转录和翻译后水平，以允许适当的时机和幅度的SPI 1基因 表情我们已经制定了一个新的模型的SPI 1调节电路，其中三个AraC样 调节因子HilD、HilC和RtsA在复杂的前馈调节环中起作用以控制hilA的表达， 编码SPI 1结构基因的直接调节子。大部分监管投入都整合在 HilD水平，包括在hilD mRNA翻译或稳定性，而其他调控系统绕过HilD， 直接控制hilA。我们假设多种信号控制着SPI 1毒力的翻译 调节子hilD和hilA，并且这种调节大部分由小RNA（sRNA）介导。的确， 我们广泛的初步数据已经揭示了调节hilD和hilA翻译或mRNA的sRNAs， 稳定性和计算预测表明，许多其他的sRNA作用于这些目标。这 包括通过hilD mRNA的独特的300个核苷酸的3'非翻译区的调节。具体目标 该建议的主要内容是：1.识别和分类与编码主要SPI 1的mRNA碱基配对的sRNA 毒力调节因子hilA和hilD。我们已经开发了一系列的报告融合，使我们能够测试 通过sRNA调节并精确确定其作用位点。2.确定sRNA如何调节SPI 1 在分子水平上。生化和遗传实验将定义sRNA-mRNA碱基配对相互作用， 以及阐明特定的调节机制。3.阐明sRNAs对沙门氏菌的影响 生理学和毒力调节。表达分析和上位性测试将确定 sRNA进入调节SPI 1表达的整体生理框架。单细胞分析将测试 sRNA对SPI 1诱导或关闭动力学的影响。我们将把这些信息整合到我们的 现有的数学模型来扩展我们对信号积分的整体理解。的调节 SP1 T3 SS作为整合宿主环境信号以控制复合体的范例 毒力表型我们的工作揭示了控制这个系统的分子机制， 详细的模型为其他系统，并最终将是至关重要的，我们了解这一重要的病原体。

PROJECT SUMMARY The ability to sense and respond to diverse and dynamic changes in environmental conditions is crucial for bacterial fitness. Numerous signals must be integrated by cellular regulatory systems in order to promote optimal responses to those changing conditions. Salmonella is an important model organism for understanding genetic regulation and bacterial pathogenesis. A requisite for Salmonella to cause disease is the direct injection of effector proteins into host cells via a Type Three Secretion System (T3SS) encoded on Salmonella Pathogenicity Island 1 (SPI1). SPI1 has become a paradigm for understanding how bacteria use diverse global regulatory systems to respond to numerous environmental signals. Our long term goal is to understand at a systems level how the signals that control SPI1 are integrated at transcriptional, post- transcriptional and post-translational levels to allow the appropriate timing and magnitude of SPI1 gene expression. We have formulated a new model for the SPI1 regulatory circuit in which the three AraC-like regulators HilD, HilC, and RtsA act in a complex feed-forward regulatory loop to control expression of hilA, encoding the direct regulator of the SPI1 structural genes. Much of the regulatory input is integrated at the level of HilD, including at hilD mRNA translation or stability, while additional regulatory systems bypass HilD to directly control hilA. We hypothesize that multiple signals control translation of central SPI1 virulence regulators, hilD and hilA, and that much of this regulation is mediated by small RNAs (sRNAs). Indeed, our extensive preliminary data have revealed sRNAs that regulate both hilD and hilA translation or mRNA stability, and computational predictions suggest numerous additional sRNAs that act on these targets. This includes regulation via the unique 300 nucleotide 3' untranslated region of the hilD mRNA. The specific aims of this proposal are to: 1. Identify and classify sRNAs that base pair with mRNAs encoding major SPI1 virulence regulators, hilA and hilD. We have developed a series of reporter fusions that allow us to test regulation by sRNAs and precisely determine their site(s) of action. 2. Determine how sRNAs regulate SPI1 at a molecular level. Biochemical and genetic experiments will define sRNA-mRNA base pairing interactions, as well as elucidate specific regulatory mechanisms. 3. Elucidate the impacts of sRNAs on Salmonella physiology and virulence regulation. Expression analysis and tests of epistasis will place the identified sRNAs into the overall physiological framework regulating SPI1 expression. Single cell analysis will test the effects of sRNAs on the dynamics of SPI1 induction or shut-down. We will integrate this information into our existing mathematical model to expand our overall understanding of signal integration. The regulation of the SP1 T3SS serves as a paradigm for the integration of host environmental signals to control a complex virulence phenotype. Our work to uncover the molecular mechanisms controlling this system serves as a detailed model for other systems and will ultimately be critical to our understanding of this important pathogen.