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.

项目总结/摘要 伯氏疏螺旋体（Borrelia burgdorferi，Bb）中的Hk 1/Rrp 1双组分系统（TCS）由以下组成： 膜结合杂合感觉组氨酸激酶（Hk 1）和细胞质反应调节剂 具有二鸟苷酸环化酶活性（Rrp 1）。缺乏Hk 1或Rrp 1的Bb在蜱内被破坏 在血餐中。在当前的应用中，我们将研究感觉器和效应器如何 Hk 1/Rrp 1 TCS的组分一起工作以促进Bb在进食蜱中的存活。在Aim中 第一，我们重点研究了Hk 1的三个串联的周质传感器SBP结构域（D1-3）。使用网站- 定向诱变方法，我们将确定单个SBP结构域的贡献 Rrp 1在体外以及在采集和传输血液过程中的激活。更好地 了解Hk 1传感器域的功能，我们将在成功实现D1的基础上再接再厉 通过解决D2、D3和全长传感器的结构。结构分析结果 将有助于完善D2和D3的预测配体结合口袋， 这三个领域如何在本地香港内部相互作用。虽然人们对戏剧性的 由于p53 hk 1/p53 rrp 1表型的性质，我们对p53 hk 1/p53 rp 1表型的输出如何产生的了解有限。 该TCS，c-di-GMP，促进Bb在蜱中的存活。Rrp 1 - 3的RNA-Seq和蛋白质组学分析 缺陷的生物体使我们假设c-di-GMP信号传导促进代谢， 对血餐的生理适应，作为逃避外源性应激源杀死的手段 是在喂养过程中产生的。在我们的第二个目标中，我们将通过以下方式来检验这一假设： 操纵体外条件，以更接近地反映Bb在美联储中遇到的情况 中肠此外，我们还将诱变选择c-di-GMP调节的细胞包膜相关的 基因和表型特征的收购和传播过程中产生的突变体 血餐我们的建议将在方法论上推进我们对Bb如何感知的理解 并响应在蜱-哺乳动物界面处遇到的环境信号。虽然 在这个建议的范围之外，我们的工作对小说的发展有明显的影响。 遏制莱姆病的方法，莱姆病是对公共卫生的迅速扩大的威胁。