Development of Paratransgenic Ticks for Disease Control

在温带气候地区，由硬虱传播的人类疾病病例正在上升，并且数量超过了蚊子传播的疾病。 在北美，肩部硬蜱作为疏螺旋体的媒介，被列为最受公共卫生关注的扁虱 伯氏杆菌(Bb，莱姆病病原体)，无浆体吞噬细胞性贫血(AP，人类无浆体病原体)， 和波瓦桑或鹿虱病毒(波瓦桑脑炎的病原体)等。在这次重新提交中，我们将 利用我们在立克次体基因组学方面的进展，创造出能够在扁虱中表达转基因的立克次体。 转基因方法目前还不适用于扁虱，部分原因是它们长达数月到一年的世代。 泰晤士报。我们产生非共生扁虱的能力与我们的膜饲养器相结合，用来重建扁虱 改造布氏乳杆菌并用AP和BB实验挑战它们史无前例 有可能剖析扁虱、它们的共生体和它们传播的病原体之间的相互作用。 共生体在形成节肢动物的免疫系统方面起着至关重要的作用。这突显了有必要 在研究副转基因策略的可能性时，了解扁虱免疫系统是如何工作的 用于壁虱传播疾病的控制。扁虱的免疫系统还没有被很好地理解，但是共同研究人员佩德拉博士 在更新项目中，发现了与昆虫免疫系统的根本区别。 我们建议的主要假设是：1)肩袖立克次体的共生体 Buchneri)在调节这种扁虱与病原体的关系方面发挥了核心作用，这种关系决定了它的能力 2)扁虱免疫系统是这三合会的重要中介。为了解剖 关于相互作用的组成部分，我们将解决以下目标： 目的1)确定布氏立克次体在硬体适合性、排除其他立克次体、对AP和BB易感性方面的作用。 目的2)制备能够利用Himar1定植硬蜱的布氏丝虫转座子突变体 转座子设计用于随后使用重组酶介导盒交换(RMCE)进行替换。 目的3)用编码抗AP纳米抗体的表达盒替换Himar1转座子 具有定殖能力的布氏根结线虫突变体；测定重组肩胛藻对AP的敏感度。 目的4.1)确定我肩周肌中布氏杆菌的存在是否影响IMD途径的活性。 目的4.2)确定在AP和BB定植肩周炎期间改变免疫信号的宿主因素 在布赫内里在场或不在场的情况下。 在拟议的项目结束时，我们将获得关于布氏酵母作为一种 扁虱的必需营养素的提供者，作为扁虱免疫系统成熟所必需的一个因素， 并作为病原体获取和传播的控制器。我们将进一步定义满足以下条件 必须满足将转基因的布氏罗非氏菌导入肩周炎的条件，以便有效地 可以设计出异基因移植的方法。

Tick-borne human disease cases are rising and outnumber mosquito-borne diseases in temperate climates. In North America, Ixodes scapularis ranks as the tick of highest public health concern as vector of Borrelia burgdorferi (Bb, an agent of Lyme disease), Anaplasma phagocytophilum (Ap, agent of human anaplasmosis), and Powassan or deer tick virus (agent of Powassan encephalitis), among others. In this resubmission, we will take advantage of our advances in rickettsial genomics to create rickettsiae able to express transgenes in ticks. Transgenic approaches are not yet available for ticks, in part hindered by their months- to year-long generation times. Our ability to generate aposymbiotic ticks coupled with our membrane feeder to reconstitute ticks with transformed R. buchneri and challenge them experimentally with Ap and Bb has opened unprecedented possibilities to dissect the interactions between ticks, their symbionts, and the pathogens they transmit. Symbionts play a fundamental role in shaping an arthropod's immune system. This highlights the need to understand how the tick immune system works when investigating the possibility of a paratransgenic strategy for tick-borne disease control. The tick immune system is not well understood, but Dr. Pedra, a co-investigator in the renewal project, has discovered fundamental differences from the insect immune system. The overarching hypotheses of our proposal are 1) that the rickettsial symbiont of I. scapularis (Rickettsia buchneri) plays a core role in regulating this tick's relationship with pathogens that determines its capacity to be a vector, and 2) that the tick immune system is an important intermediary in this triad. In order to dissect the interacting components, we will address the following aims: Aim 1) Determine R. buchneri role in tick fitness, exclusion of other rickettsiae, susceptibility to Ap and Bb. Aim 2) Prepare R. buchneri transposon mutants that are competent to colonize ticks using a Himar1 transposon designed for subsequent replacement using Recombinase Mediated Cassette Exchange (RMCE). Aim 3) Replace the Himar1 transposon with an expression cassette encoding an anti-Ap Nanobody in colonization competent R. buchneri mutants; determine susceptibility of reconstituted I. scapularis to Ap. Aim 4.1) Determine whether R. buchneri presence in I. scapularis affects the activity of the IMD pathway. Aim 4.2) Identify host factors that alter immune signaling during colonization of I. scapularis by Ap and Bb in the presence or absence of R. buchneri. At the conclusion of the proposed project we will have gained detailed knowledge about R. buchneri as a provider of essential nutrients to ticks, as a factor required for maturation of a functional tick immune system, and as a controller of pathogen acquisition and transmission. We will further have defined the conditions that must be met to introduce a genetically modified R. buchneri into I. scapularis so that an effective paratransgenesis approach could be devised.