Integration of Small RNAs in Control of Salmonella Pathogenicity Island 1

项目总结 感知和响应环境条件的各种动态变化的能力对 细菌适合性。细胞调控系统必须整合大量信号，才能促进 对这些不断变化的条件做出最佳反应。沙门氏菌是一种重要的模式生物 了解遗传调控和细菌致病机制。沙门氏菌致病的必要条件是 通过三型分泌系统(T3SS)将效应蛋白直接注射到宿主细胞 沙门氏菌致病岛1(SPI1)。SPI1已经成为了解细菌如何利用 多样化的全球监管体系，以应对众多环境信号。我们的长期目标是 在系统水平上理解控制SPI1的信号是如何在转录、后转录和转录后整合的 转录和翻译后水平允许SPI1基因的适当时机和大小 表情。我们已经制定了一个新的SPI1调节电路模型，在这个模型中，三个AraC样蛋白 调节子Hild、HilC和RTSA在复杂的前馈调控环中作用，以控制Hila的表达， 编码SPI1结构基因的直接调节因子。大部分监管意见都集成在 HILD的水平，包括在HILD mRNA的翻译或稳定，而额外的调控系统绕过HILD以 直接控制希拉。我们假设多个信号控制中心SPI1毒力的翻译 调节者，希尔德和希拉，这一调节的大部分是由小RNA(SRNA)介导的。的确， 我们广泛的初步数据揭示了调节HILD和HILA翻译或mRNAs的sRNA 稳定性和计算预测表明，许多额外的sRNA作用于这些靶点。这 包括通过Hild mRNA独特的300核苷酸3‘非翻译区进行调控。具体目标 这一建议的目的是：1.识别碱基对与编码主要SPI1的mRNAs 毒力监管者，希拉和希尔德。我们已经开发了一系列的记者融合，使我们能够测试 由sRNA进行调控，并准确地确定它们的作用部位(S)。2.确定sRNA如何调控SPI1 在分子水平上。生物化学和遗传学实验将定义sRNA-mRNA碱基配对的相互作用， 以及阐明具体的调控机制。3.阐明sRNA对沙门氏菌的影响 生理和毒力调节。上位性的表达分析和测试将把已识别的 SRNA进入调控SPI1表达的整体生理框架。单细胞分析将测试 SRNAs对SPI1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 现有的数学模型拓展了我们对信号集成的整体理解。对法律的监管 SP1 T3SS用作集成宿主环境信号以控制综合体的范例 毒力表型。我们发现控制这一系统的分子机制的工作是一个 其他系统的详细模型，最终将对我们理解这一重要病原体至关重要。

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.