Regulation of gene expression in Borrelia burgdorferi

描述（由申请人提供）：莱姆病是由伯氏疏螺旋体感染引起的。伯氏疏螺旋体通过由蜱虫媒介和脊椎动物宿主组成的地方性动物循环在自然界中维持。蜱虫向哺乳动物的传播和哺乳动物感染的建立需要感知外部信号并通过调节基因表达程序作出反应。这种基因表达的变化主要是由替代sigma因子RpoS介导的。本应用的中心假设是RpoS是控制向哺乳动物宿主传播和感染的主要转录调节因子，因此是几种调节机制的目标。我们建议剖析控制下游靶标上rpoS表达和rpoS活性的机制。具体来说，我们假设一种新的rpoS转录本在传播中具有独特的作用，并由最近发现的核糖核酸内切酶处理；信号分子四磷酸鸟苷和相关的调节蛋白在整个地方性动物周期中全面影响rpos介导的转录和翻译；一个小的调控RNA影响sigma因子的选择性和活性。本提案的长期目标是了解RpoS在蜱-哺乳动物传播和疾病发病机制中的作用和调控，这将导致改进诊断、预防和治疗策略，因为RpoS是伯氏疏螺旋体引起莱姆病所必需的；这与该机构为减轻人类疾病而“寻求基础知识”的使命有关。为实现这一目标，提出了以下具体目标：1)确定新型rpoS转录物的作用和处理机制；2)阐明四磷酸鸟苷在动物地方病循环中的作用靶点和功能；3)表征调节sigma因子选择性的小RNA。遗传学、生物化学、转录组学和蛋白质组学方法将被用来测试这些假设。具体来说，编码调节因子（或调节因子的产生）的基因将被破坏和/或融合到一个诱导启动子中，以在蜱鼠模型中测定体内功能。

DESCRIPTION (provided by applicant): Lyme disease is caused by infection with the spirochete Borrelia burgdorferi. B. burgdorferi is maintained in nature through an enzootic cycle comprising a tick vector and vertebrate host. Transmission from the tick to the mammal and establishment of the mammalian infection require sensing external signals and responding by regulating programs of gene expression. This change in gene expression is predominantly mediated by the alternative sigma factor RpoS. The central hypothesis of this application is that RpoS is the principal transcriptional regulator controlling transmission to and infection of the mammalian host, and thus serves as the target of several regulatory mechanisms. We propose to dissect the mechanisms controlling rpoS expression and RpoS activity on downstream targets. Specifically, we hypothesize that a novel rpoS transcript has a distinct role in transmission and is processed by a recently identified endoribonuclease; the signaling molecule guanosine tetraphosphate, and associated regulatory proteins, globally affect RpoS-mediated transcription and translation throughout the enzootic cycle; and a small regulatory RNA affects sigma factor selectivity and activity. The long-term objective of this proposal is to understand th role and regulation of RpoS in tick-to-mammal transmission and disease pathogenesis, which will lead to improved diagnostic, prevention, and treatment strategies because RpoS is required for B. burgdorferi to cause Lyme disease; this is relevant to the mission of the agency to "seek fundamental knowledge" for the sake of alleviating human disease. The following specific aims are proposed toward achieving this objective: 1) determine the role of, and processing mechanism for, the novel rpoS transcript; 2) elucidate the targets and function of guanosine tetraphosphate during the enzootic cycle; and 3) characterize a small RNA that regulates sigma factor selectivity. Genetic, biochemical, transcriptomic, and proteomic approaches will be utilized to test these hypotheses. Specifically, genes encoding regulatory factors (or the production of regulatory factors) will be disrupted and/or fused to an inducible promoter to assay in vivo function in a tick-mouse model.