Molecular Architecture of the Mosquito Carbon Dioxide Receptor Complex

蚊子二氧化碳受体复合物的分子结构

• 批准号: 9808786
• 负责人: Conor James McMeniman
• 金额: $ 19.54万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-23 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9808786
• 关键词: Aedes; Agonist; Alcohols; Alleles; Anatomy; Animals; Architecture; Behavior; Behavioral; Bicarbonates; Blood; Butanones; C-terminal; CRISPR/Cas technology; Carbon Dioxide; Carbonic Anhydrase Inhibitors; Cells; Chemicals; Complex; Confocal Microscopy; Cues; Culicidae; Cyclohexanones; Dendrites; Detection; Development; Disease Vectors; Dissociation; Electrophysiology (science); Epitopes; Family; Fluorescence; Gases; Genes; Genomics; Genotype; Goals; Grant; Human; Human body; Immunohistochemistry; In Vitro; Individual; Insecta; Ions; Ketones; Label; Ligand Binding; Ligands; Location; Lymph; Maxilla; Mediating; Membrane; Methods; Molecular; Mosquito-borne infectious disease; N-terminal; Names; Nervous system structure; Neurons; Odors; Olfactory Pathways; Pharmacology; Play; Population; Preparation; Property; Protein Family; Proteins; Protons; Resolution; Role; Sensory; Sensory Process; Signal Transduction; Skin; Smell Perception; Specificity; Spermidine; Structure; Supporting Cell; Surface; System; Testing; Transgenic Organisms; Vector-transmitted infectious disease; Yellow Fever; aqueous; capitate bone; carbon dioxide receptor; carbonate dehydratase; cofactor; combat; disorder control; ethyl pyruvate; feeding; genome editing; homologous recombination; improved; insect disease vector; molecular mechanics; mutant; neuronal cell body; novel; olfactory sensory neurons; pyridine; receptor; receptor function; reconstitution; response; screening; trafficking

Blood feeding mosquitoes use sensitively tuned chemosensory systems to locate their vertebrate hosts. At locations far from a target host, anthropophilic mosquitoes predominantly rely on their sense of smell to follow plumes of volatile host emissions emanating from the human body, which include skin odor and carbon dioxide (CO2). In the yellow fever mosquito Aedes aegypti, CO2 is sensed by specialized populations of olfactory sensory neurons (OSNs) housed within capitate peg sensilla on the ventral surface of the maxillary palps. In each CO2-sensitive neuron, three gustatory receptors (Grs) named Gr1, Gr2 and Gr3, are hypothesized to interact to form a CO2 receptor complex mediating detection of this volatile gas, as well as structurally diverse ligands including certain heterocyclics, ketones and alcohols. Whether these three Gr subunits are all functionally required for CO2 receptor complex formation and the broad tuning responses of this neuron to this gas and other odorants, or whether additional signaling cofactors are involved is unknown. Here, we propose to apply integrative methods to functionally define the molecular architecture of the mosquito CO2 receptor complex. We will initially apply single-sensillum electrophysiology to functionally probe responses of Ae. aegypti Gr1, 2 and 3 null mutants to CO2 and a select panel of volatile agonists and antagonists of this receptor complex. Using CRISPR-Cas9 genome editing, we will additionally insert small-epitope tags onto the N– and C–terminal ends of Gr1, 2 and 3 to evaluate the ability of each of these individual Grs to traffic independently from the neuronal cell bodies to the lamellate cpA dendrite using immunohistochemistry. These approaches will reveal Gr subunits that may influence the odor tuning sensitivity or specificity of the complex, or those that may serve as co-receptors for transport of ligand-binding subunits to the sensory membrane. To evaluate a specific role for carbonic anhydrases (CAHs) in CO2 receptor complex function, which we hypothesize may catalyze the conversion of CO2 inside the aqueous sensillar lymph to bicarbonate ions and protons to act as receptor ligands, we will ablate target CAHs expressed in the maxillary palps using genome- editing and pharmacological manipulations. We will further highlight the anatomy of carbonic anhydrase expressing cells in the mosquito olfactory system using transgenic tracing methods to query their potential role in gas detection. CO2 is one of the most universal and important components of odor-baited traps used for surveillance and control of hematophagous insects including mosquito disease vectors. An improved understanding of the molecular architecture of the broadly conserved CO2 receptor complex may facilitate the development of efficient heterologous expression systems that reconstitute the mosquito CO2 receptor complex in vitro. Such an advance may aid in the rapid identification of novel volatile compounds mimicking or antagonizing the effects of CO2 for applied use in vector-borne disease control strategies.

血液进食蚊子使用灵敏的化学水敏系统来定位其脊椎动物宿主。在 肉虫蚊子远离目标主机的位置主要依靠他们的嗅觉来跟随 从人体发出的挥发性宿主排放的羽毛，其中包括皮肤气味和二氧化碳 （CO2）。在黄热病的蚊子埃及埃及埃及，二氧化碳被专业人群的嗅觉感知 感觉神经元（OSN）位于上颌pal的腹侧表面上的大写字母peg Sensora中。 假设每个二氧化碳敏感的神经元，三个称为GR1，GR2和GR3的味觉受体（GRS） 相互作用以形成这种挥发性气体的介导检测的二氧化碳受体复合物，以及结构上的潜水 配体包括某些杂环，酮和醇。这三个GR亚基是否全部 二氧化碳受体复合物形成和该神经元对此的广泛调谐反应所需 气体和其他气味，或是否涉及其他信号辅助因子是未知的。在这里，我们建议 应用集成的方法来定义蚊子二氧化碳受体的分子结构 复杂的。我们最初将应用单感电物质生理学在功能上探测AE的响应。 埃及gr1、2和3零突变体到二氧化碳，以及一组挥发性激动剂和拮抗剂的精选面板 接收器复合体。使用CRISPR-CAS9基因组编辑，我们还将插入小ePitope标签 GR1、2和3的N –和C末端评估这些单个GRS的交通能力的能力 使用免疫组织化学独立于神经元细胞体到层状CPA树突。这些 方法将揭示可能影响复合物的气味调整敏感性或特异性的GR亚基， 或可以用作将配体结合亚基转运到感觉膜的共受体的人。到 评估碳酸酐酶（CAH）在CO2受体复合功能中的特定作用，我们 假设可能会催化二氧化碳在水性感觉淋巴内的转化为碳酸氢盐离子和 质子充当接收器配体，我们将使用基因组在上颌触发中燃烧靶标CAHS 编辑和药物操作。我们将进一步强调碳酸酐酶的解剖结构 使用转基因示踪方法在蚊子嗅觉系统中表达细胞，以查询其潜在作用 在气体检测中。 CO2是用于气味诱饵陷阱的最普遍，最重要的组成部分之一 包括蚊子疾病载体在内的血管绝缘的监测和控制。改进 了解广泛保守的二氧化碳受体复合物的分子结构可能会促进 开发有效的异源表达系统，重建蚊子二氧化碳受体 体外复合物。这样的进步可能有助于快速识别模仿新型挥发性化合物或 拮抗二氧化碳在媒介传播疾病控制策略中应用的影响。