Essential role of Hk1/Rrp1 TCS for survival of Borrelia burgdorferi in ticks

Hk1/Rrp1 TCS 对蜱中伯氏疏螺旋体存活的重要作用

• 批准号: 9303275
• 负责人: MELISSA J CAIMANO
• 金额: $ 19.94万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-24 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9303275
• 关键词: ATP-Binding Cassette Transporters; Anabolism; Arthropod Vectors; Arthropods; Attention; Bacteria; Bacteria sigma factor KatF protein; Biogenesis; Blood; Borrelia; Borrelia burgdorferi; Borrelia burgdorferi OspE protein; Carbon; Categories; Consensus; Crystallization; Data; Defect; Development; Genes; Genetic Transcription; Glycerol; Glycolysis; Growth; Human; Hybrids; In Vitro; Individual; Larva; Lead; Length; Ligand Binding; Ligands; Lipoproteins; Lyme Disease; Maintenance; Mammals; Mediating; Melissa; Membrane; Metabolic; Metabolism; Methodology; Midgut; Modeling; Molecular Conformation; Nature; Nymph; Operon; Order Spirochaetales; Organism; Output; Peptidoglycan; Phase; Phenotype; Phospholipids; Phosphotransferases; Physiological Adaptation; Population; Predisposition; Principal Investigator; Process; Proteomics; Public Health; Publishing; Quantitative Reverse Transcriptase PCR; RNA; Regulon; Role; Sensory; Sigma Factor; Signal Transduction; Site-Directed Mutagenesis; Source; Stress; Structure; Surface; System; Testing; Ticks; Tissue-Specific Gene Expression; Venus; Virulence; Work; bactericide; base; cell envelope; chitobiose; diguanylate cyclase; disorder risk; enzootic; feeding; fly; gene product; insight; killings; mutant; novel strategies; periplasm; programs; protein-histidine kinase; response; risk minimization; sensor; sensor histidine kinase; stem; stressor; success; transcriptome; transcriptome sequencing; transmission process; uptake; vector

Project Summary/Abstract The Hk1/Rrp1 Two component system (TCS) in Borrelia burgdorferi (Bb) is comprised of a membrane-bound hybrid sensory histidine kinase (Hk1) and a cytoplasmic response regulator with diguanylate cyclase activity (Rrp1). Bb lacking either Hk1 or Rrp1 are destroyed within ticks during the blood meal. In the current application, we will investigate how the sensory and effector components of the Hk1/Rrp1 TCS work together to promote survival of Bb in feeding ticks. In Aim One, we focus on the Hk1's three tandem periplasmic sensor SBP domains (D1-3). Using a site- directed mutagenesis approach, we will determine the contribution(s) of individual SBP domains to activation of Rrp1 in vitro and during the acquisition and transmission blood meals. To better understand how the Hk1 sensor domains function, we will build upon our success crystalizing D1 by solving the structures for D2, D3 and the full-length sensor. Results from structural analyses will help to refine the predicted ligand binding pockets for D2 and D3 and provide insight into how the three domains interface within the native HK. While there is consensus on the dramatic nature of the hk1/rrp1 phenotype, we have only a limited understanding of how the output of this TCS, c-di-GMP, promotes survival of Bb in ticks. RNA-Seq and proteomic analyses of Rrp1- deficient organisms lead us to hypothesize that c-di-GMP signaling promotes metabolic and physiological adaptation to the blood meal as a means of evading killing by exogenous stressors generated during the feeding process. In our second Aim, we will test this hypothesis by manipulating in vitro conditions to more closely mirror those encountered by Bb in the fed midgut. In addition, we also will mutagenize select c-di-GMP-regulated cell envelope associated genes and phenotypically characterize the resulting mutants during acquisition and transmission blood meals. Our proposal will methodologically advance our understanding of how Bb perceives and responds to environmental signals encountered at the tick-mammal interface. Although beyond the scope of this proposal, our work has clear implications for the development of novel approaches to curtail Lyme disease, a rapidly expanding threat to public health.

项目摘要/摘要 Borrelia Burgdorferi（BB）中的HK1/RRP1两个组件系统（TCS）已完成 膜结合的杂种感觉组氨酸激酶（HK1）和细胞质反应调节剂 与二甘氨酸酸环化酶活性（RRP1）。缺乏HK1或RRP1的BB在壁虱中被销毁 在血餐期间。在当前的应用程序中，我们将研究感觉和效应器 HK1/RRP1 TC的组件共同促进BB在喂食滴答中的生存。目标 第一，我们专注于HK1的三个串联传感器SBP域（D1-3）。使用网站 - 定向诱变方法，我们将确定单个SBP域的贡献 在体外以及在获取和传播血液中激活RRP1。更好 了解HK1传感器域的运作方式，我们将建立在成功的基础上 通过求解D2，D3和全长传感器的结构。结构分析的结果 将有助于完善D2和D3的预测配体结合袋，并洞悉 本机HK中的三个域接口如何。虽然在戏剧性上达成共识 HK1/RRP1表型的性质，我们对如何输出的方式有限 该TCS（C-DI-GMP）促进了tick中BB的生存。 RRP1-的RNA-seq和蛋白质组学分析 不足的生物会导致我们假设C-DI-GMP信号传导促进了代谢和 生理适应血液粉，作为逃避外源压力杀死的一种手段 在进食过程中生成。在我们的第二个目标中，我们将通过 操纵体外条件，以更紧密地反映BB在美联储中遇到的那些 Midgut。此外，我们还将诱变选择C-DI-GMP调节的细胞包膜相关 基因和表达在获取和传播过程中所产生的突变体的表征 血餐。我们的建议将在方法论上促进我们对BB感知的理解 并响应tick妈妈接口遇到的环境信号。虽然 除了该提议的范围之外，我们的工作对新颖的发展具有明显的影响 减少莱姆病的方法，这是对公共卫生的迅速扩大的威胁。