Regulation of Gene Expression in Borrelia burgdorferi

描述(申请人提供)：伯氏疏螺旋体引起莱姆病。伯氏杆菌从其媒介向哺乳动物宿主的传播和哺乳动物的感染需要改变传播和感染所需的一系列基因的表达。基因表达程序的变化是根据与扁虱饲养相关的环境信号而调节的，包括温度升高。基因表达的最大变化是主要外表面脂蛋白的相互调节，包括传播和感染所需的OspC。OspC基因表达的诱导依赖于替代的Sigma因子rpos(C38或CS)。这一应用的中心假设是，rpos是地方病循环的关键调节因子，因此通过几种调节机制作为多个信号的靶标。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向和负向的调控，包括由一个小的调节RNA对温度的反应进行翻译控制，以及由蛋白酶进行翻译后降解。这种调节也被认为是通过顺式作用的DNA超螺旋对温度的反应来调节的。这项建议的长期目标是了解OspC在伯氏杆菌传播和莱姆病发病机制中的功能和调节，这将导致改进诊断、预防和治疗策略，因为OspC的表达是伯氏杆菌引起莱姆病所必需的；这与该机构为减轻人类疾病而追求基础知识的使命相关。为了实现这一目标，提出了以下具体目标：1)确定和表征抑制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；以及3)确定OSPC在传播中的时间需求，并确定RPOS在哺乳动物感染过程中调节OSPC的作用。遗传学、生化、分子、转录学和蛋白质组学方法将被用来检验这些假说。具体地说，编码调控因子的基因将被干扰和/或融合到新的可诱导启动子上，以检测在培养和扁虱-小鼠模型中的功能，将构建报告融合以定量检测基因表达，将通过RNA结合实验来探索小RNA的调控机制，并将通过微阵列和蛋白质鉴定来确定DNA超螺旋对基因表达的整体影响。

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.