Regulation of Gene Expression in Borrelia burgdorferi

伯氏疏螺旋体基因表达的调控

• 批准号: 7744019
• 负责人: D. SCOTT SAMUELS
• 金额: $ 35.02万
• 依托单位: UNIVERSITY OF MONTANA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-05 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7744019
• 关键词: Bacteria; Bacteria sigma factor KatF protein; Biochemical; Biological Assay; Borrelia burgdorferi; Diagnostic; Gene Expression; Gene Expression Regulation; Genes; Genetic; Infection; Knowledge; Lead; Lyme Disease; Mammals; Mission; Molecular; Names; OspC protein; Pathogenesis; Peptide Hydrolases; Prevention; Principal Investigator; Protein Microchips; Proteomics; RNA; RNA Binding; Regulation; Reporter; Research; Role; Sigma Factor; Signal Transduction; Small RNA; Superhelical DNA; Temperature; Testing; Ticks; enzootic; feeding; human disease; improved; mouse model; outer surface lipoprotein; programs; promoter; research study; response; transcriptomics; transmission process; treatment strategy; vector

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.

描述（由申请人提供）：伯氏疏螺旋体细菌引起莱姆病。伯氏疏螺旋体从其蜱载体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是根据与蜱进食相关的环境信号（包括温度升高）进行调节的。基因表达中最显着的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 sigma 因子 RpoS（C38 或 CS）。该应用的中心假设是，RpoS 是地方性动物循环的关键调节因子，因此通过多种调节机制充当多个信号的目标。具体假设是，RpoS 和 OspC 合成通过多种反式作用机制受到正向和负向调节，包括响应温度的小调节 RNA 的翻译控制以及蛋白酶的翻译后降解。还假设这种调节是通过响应温度的顺式作用 DNA 超螺旋来调节的。该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节，这将改善诊断、预防和治疗策略，因为伯氏疏螺旋体需要 OspC 的表达。伯氏杆菌引起莱姆病；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）识别和表征抑制 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； 3) 确定 OspC 在传播中的时间要求，并确定 RpoS 在哺乳动物感染过程中调节 OspC 的作用。将利用遗传、生化、分子、转录组和蛋白质组方法来检验这些假设。具体来说，编码调节因子的基因将被破坏和/或融合到新的诱导型启动子上，以测定培养物和蜱小鼠模型中的功能，构建报告融合体以定量测定基因表达、小RNA的调节机制将通过RNA结合实验来探测，DNA超螺旋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。