Elucidating the function of a novel antibacterial amidase in Ixodes scapularis

阐明肩胛硬蜱中新型抗菌酰胺酶的功能

• 批准号: 9012761
• 负责人: Joseph David Mougous
• 金额: $ 23.2万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-02-15 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9012761
• 关键词: Amidohydrolases; Anaplasma phagocytophilum; Anti-Bacterial Agents; Arthropods; Bacteria; Biochemical; Biological Assay; Black-legged Tick; Borrelia burgdorferi; Bovine Anaplasmosis; Cell Wall; Chemical Structure; Data; Deer Tick; Disease; Disease Vectors; Endopeptidases; Enzymes; Eukaryota; Evolution; Family; Genes; Glycoside Hydrolases; Gram-Negative Bacteria; Grant; Health; Homologous Gene; Horizontal Gene Transfer; Human; Immune; Immune response; Immune system; In Vitro; Informatics; Ixodidae; Lyme Disease; Maintenance; Measures; Mediating; Membrane; Microbe; Midgut; Midwestern United States; Mites; Muramidase; Mus; Natural Immunity; Order Spirochaetales; Organism; Pattern recognition receptor; Peptides; Peptidoglycan; Physiological; Population; Proteins; Proteobacteria; RNA Interference; Role; Salivary Glands; Structure; Testing; Ticks; Toxin; United States; Work; amidase; antimicrobial; base; crosslink; feeding; human disease; in vitro activity; in vivo; insight; knock-down; member; microbial community; novel; pathogen; permissiveness; research study; response; stem; transmission process; vector

 DESCRIPTION (provided by applicant): Despite the importance of ticks as disease vectors, the immune system of these organisms remains poorly understood. We have discovered a novel antibacterial enzyme found in the hard tick Ixodes scapularis that was horizontally acquired from a bacterium early in the evolution of ticks and mites. This enzyme, which we term Dae2 (domesticated amidase effector 2), is related to a family of peptidoglycan-degrading toxins transferred between bacteria during interbacterial competition. Preliminary in vitro data show that Dae2 is indeed an antibacterial enzyme, and that it retains the DD-endopeptidase peptidoglycan-degrading activity of its characterized bacterial homologs. Additionally, we demonstrate Dae2 is expressed in the midgut and salivary glands of I. scapularis, and that dae2 knockdown reduces the ability of I. scapularis to control replication of the Lyme disease agent Borrelia burgdorferi. In this grant, we propose to define the function and physiological mechanism of action of Dae2 in I. scapularis using in vitro and in vivo approaches. In the first aim of this proposal, we will establish the target range and mechanism of action of Dae2 antibacterial activity in vitro. To accomplish this, we will measure purified Dae2 activity against each major peptidoglycan type. Furthermore, we will test the direct antibacterial capacity of Dae2 against diverse species representing tick-associated microbes in the presence and absence of accessory immune factors present in tick salivary gland and midgut extracts. The second aim of our work is to define the function of Dae2 in vivo. Taking advantage of RNAi-based knockdown strategies, we will determine the contribution of Dae2 to structuring the commensal microbial community of I. scapularis, and define the role of Dae2 in mediating the tick innate immune response to a bacterial pathogen. In total, our studies will provide key insights into the role of Dae2 in the innate immune system of I. scapularis.

 描述（由申请人提供）：尽管蜱作为疾病媒介很重要，但人们对这些生物体的免疫系统仍然知之甚少。我们在硬蜱肩胛硬蜱中发现了一种新型抗菌酶，它是在蜱和螨进化早期从细菌中水平获得的。这种酶，我们称之为 Dae2（驯化酰胺酶效应子 2），与细菌间竞争期间在细菌之间转移的肽聚糖降解毒素家族有关。初步体外数据表明，Dae2 确实是一种抗菌酶，并且它保留了其特征细菌同系物的 DD-内肽酶肽聚糖降解活性。此外，我们证明 Dae2 在肩胛 I. scapularis 的中肠和唾液腺中表达，并且 dae2 敲低降低了肩胛 I. scapularis 控制莱姆病病原体伯氏疏螺旋体复制的能力。在这项资助中，我们建议使用体外和体内方法来定义 Dae2 在 I. scapularis 中的功能和生理作用机制。在本提案的第一个目标中，我们将建立Dae2体外抗菌活性的目标范围和作用机制。为了实现这一目标，我们将测量纯化的 Dae2 活性 每种主要的肽聚糖类型。此外，我们将在蜱唾液腺和中肠提取物中存在或不存在辅助免疫因子的情况下，测试 Dae2 对代表蜱相关微生物的不同物种的直接抗菌能力。我们工作的第二个目标是确定 Dae2 在体内的功能。利用基于 RNAi 的敲低策略，我们将确定 Dae2 对构建肩胛 I. scapularis 共生微生物群落的贡献，并确定 Dae2 在介导蜱对细菌病原体的先天免疫反应中的作用。总的来说，我们的研究将为了解 Dae2 在肩胛冈先天免疫系统中的作用提供重要见解。