Regulation of Gene Expression in Borrelia burgdorferi

描述（由申请人提供）：伯氏疏螺旋体引起莱姆病。伯氏疏螺旋体从其蜱虫媒介传播到哺乳动物宿主并感染哺乳动物需要改变传播和感染所需的一组基因的表达。基因表达程序的变化受到与蜱虫进食相关的环境信号的调节，包括温度升高。基因表达中最显著的变化是主要外表面脂蛋白的相互调节，包括OspC，这是传播和感染所必需的。ospC基因表达的诱导依赖于备选sigma因子RpoS （C38或CS）。本应用的中心假设是RpoS是地方性动物周期的关键调节剂，因此通过几种调节机制作为多种信号的靶标。具体的假设是，RpoS和OspC的合成通过几种反式作用机制受到正调控和负调控，包括响应温度的小调节RNA的翻译控制和蛋白酶的翻译后降解。这种调节也被假设是通过顺式作用的DNA超卷绕来调节的，以响应温度。本提案的长期目标是了解OspC在伯氏疏螺旋体传播和莱姆病发病机制中的功能和调控，从而改进诊断、预防和治疗策略，因为伯氏疏螺旋体引起莱姆病需要OspC的表达；这与该机构为减轻人类疾病而追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)确定并描述抑制RpoS和/或OspC合成的因素；2)识别和表征诱导RpoS和/或OspC合成的因素；3)明确OspC传播的时间要求，确定RpoS在哺乳动物感染过程中调控OspC的作用。遗传学、生物化学、分子学、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，编码调控因子的基因将被破坏和/或融合到新的可诱导启动子中，以在培养和小鼠模型中检测功能；报告基因融合将被构建以定量检测基因表达；小RNA的调控机制将通过RNA结合实验来探索；DNA超缠绕对基因表达的整体影响将通过微阵列和蛋白质鉴定来确定。

DESCRIPTION (provided by applicant): The bacterium Borrelia burgdorferi causes Lyme disease. Transmission of B. burgdorferi from its tick vector to a mammalian host and infection of the mammal require changes in the expression of a suite of genes required for transmission and infection. The change in the program of gene expression is regulated in response to environmental signals associated with tick feeding, including increased temperature. The most dramatic change in gene expression is the reciprocal regulation of the major outer surface lipoproteins, including OspC, which is required for transmission and infection. The induction of ospC gene expression is dependent on the alternative sigma factor RpoS (C38 or CS). The central hypothesis of this application is that RpoS is the key regulator of the enzootic cycle and thus serves as the target of multiple signals through several regulatory mechanisms. The specific hypotheses are that RpoS and OspC syntheses are positively and negatively regulated through several trans-acting mechanisms, including translational control by a small regulatory RNA in response to temperature and post-translational degradation by a protease. The regulation is also hypothesized to be regulated through cis-acting DNA supercoiling in response to temperature. The long-term objective of this proposal is to understand the function and regulation of OspC in transmission of B. burgdorferi and pathogenesis of Lyme disease, which will lead to improved diagnostic, prevention, and treatment strategies because expression of OspC is required for B. burgdorferi to cause Lyme disease; this is relevant to the mission of the agency to pursue fundamental knowledge for the sake of alleviating human disease. The following specific aims are proposed toward achieving this objective: 1) identify and characterize factors that repress RpoS and/or OspC syntheses; 2) identify and characterize factors that induce RpoS and/or OspC syntheses; and 3) define the temporal requirement for OspC in transmission and determine the role of RpoS in regulating OspC during mammalian infection. Genetic, biochemical, molecular, transcriptomic, and proteomic approaches will be utilized to test these hypotheses. Specifically, genes encoding regulatory factors will be disrupted and/or fused to the new inducible promoter to assay function both in culture and in a tick-mouse model, reporter fusions will be constructed to quantitatively assay gene expression, the regulatory mechanism of the small RNA will be probed by RNA binding experiments, and the global effect of DNA supercoiling on gene expression will be determined by microarray and protein identification.