Elucidating the function of a novel antibacterial amidase in Ixodes scapularis

 描述(申请人提供)：尽管扁虱作为疾病媒介的重要性，但这些生物的免疫系统仍然知之甚少。我们在硬蜱中发现了一种新的抗菌酶，这种酶是从扁虱和螨类进化早期的一种细菌水平获得的。这种酶，我们称之为DAE2(驯化的酰胺酶效应器2)，与一类在细菌间竞争过程中在细菌之间转移的肽聚糖降解毒素家族有关。初步的体外实验数据表明，Dae2确实是一种抗菌酶，并且它保留了其特征细菌同源物的DD-内肽酶降解肽聚糖的活性。此外，我们证明Dae2在我肩部的中肠和唾液腺中表达，并且dae2的敲除降低了I.肩部控制莱姆病病原体伯氏疏螺旋体的复制的能力。在这项授权中，我们建议使用体外和体内方法来确定Dae2在肩周肌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 每一种主要的肽聚糖类型。此外，我们将测试DAE2对不同物种的直接抗菌能力，这些微生物代表着硬蜱唾液腺和中肠提取液中存在和不存在的辅助免疫因子。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的基因敲除策略，我们将确定Dae2在构建肩胛单螺旋体共生微生物群落中的贡献，并确定Dae2在介导扁虱对细菌病原体的先天免疫反应中的作用。总而言之，我们的研究将为Dae2在肩周炎先天免疫系统中的作用提供关键的见解。

 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.