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

基本信息

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
项目类别：
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

项目摘要

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（由适用提供）：尽管tick虫作为疾病媒介很重要，但这些生物的免疫系统仍然知之甚少。我们已经发现了一种在硬滴答ixodes肩cap骨中发现的一种新型抗菌酶，该酶在tick和螨的演变早期从细菌中水平获取。我们称这种酶为DAE2（驯化的amidase效应子2），与在细菌间竞争期间在细菌之间转移在细菌之间的薄荷糖毒素家族有关。初步的体外数据表明，DAE2确实是一种抗菌酶，并且保留了其特征性细菌同源物的DD-耐肽酶降解活性。此外，我们证明了dae2在肩cap骨的中肠和唾液腺中表达，并且DAE2敲低降低了肩cap骨的能力控制莱姆病剂的复制能力。在这笔赠款中，我们建议使用体外和体内方法来定义DAE2在I. capularis中的作用和物理机理。在该提案的第一个目的中，我们将在体外建立DAE2抗菌活性的目标范围和作用机理。为此，我们将测量针对纯化的DAE2活动每种主要的PepperyDoglycan类型。此外，我们将测试DAE2的直接抗菌能力对代表tick唾液腺和中肠提取物中存在和不存在附属免疫因子的潜水物种的潜水物种。我们工作的第二个目的是定义DAE2在体内的功能。利用基于RNAi的敲低策略，我们将确定DAE2对构造肩cap骨的共生微生物群落的贡献，并确定DAE2在介导tick tick tick tick tick tick tick tick tick tick tick tick sishate sive sianate simpons sminate simenate simpose sminate sive siante sival sival sival sive sianate siment sival sival simensal syrougn commuciate struction strument策略。总的来说，我们的研究将为DAE2在肩cap骨的先天免疫系统中的作用提供关键见解。