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（由申请方提供）：尽管蜱虫作为疾病载体很重要，但对这些生物体的免疫系统仍知之甚少。我们在硬蜱肩胛硬蜱中发现了一种新的抗菌酶，这种酶是在蜱和螨的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），与细菌间竞争期间细菌之间转移的肽聚糖降解毒素家族有关。初步的体外数据表明，Dae2确实是一种抗菌酶，它保留了DD-内肽酶肽聚糖降解活性，其特征在于细菌同系物。此外，我们证明Dae2在I的中肠和唾液腺表达。dae2基因敲除降低了I.肩胛肌以控制莱姆病病原体伯氏疏螺旋体的复制。在本研究中，我们拟阐明Dae2在I.使用体外和体内方法研究肩胛肌。在本提案的第一个目标中，我们将建立Dae2体外抗菌活性的靶点范围和作用机制。为了实现这一点，我们将测量纯化的Dae2活性， 每种主要的肽聚糖类型。此外，我们将测试的直接抗菌能力Dae2对不同物种的代表蜱相关微生物的存在和不存在的辅助免疫因子存在于蜱唾液腺和中肠提取物。我们工作的第二个目的是确定Dae2在体内的功能。利用基于RNAi的敲除策略，我们将确定Dae2对构建I.肩胛肌，并定义Dae2在介导蜱对细菌病原体的先天免疫应答中的作用。总之，我们的研究将为Dae2在I.肩胛肌