Molecular basis for spotted fever group Rickettsia vector competence in ticks

描述（由申请人提供）：立克次体是美国最严重的蜱传疾病之一的病原体。立克次体致病性和蜱传播途径（水平和垂直）的复合因素影响着人类立克次体病的生态学和流行病学。我们的长期目标是阐明蜱传播立克次体的分子机制。尽管自然界中蜱与多种立克次体物种的分布重叠，但单个蜱物种并不感染多种立克次体物种。因此，正在测试的广泛假设是，独特的蜱源性因子是成功的水平和垂直传播的斑点热群（SFG）立克次体所必需的。初步证据表明，立克次体的传播取决于适当的蜱/立克次体关系和立克次体的致病性。同样已知的是，蜱对感染立克次体有积极反应;然而，蜱内立克次体存活所需的蜱源性分子的调节程度尚不清楚。因此，本研究的重点是通过对不同种类的蜱和立克次体的比较分析，阐明蜱/立克次体关系的特异性、立克次体致病性与传播的关系，以及确定与立克次体感染相关的蜱源性分子。之前未定义的蜱/立克次体相互作用参数将在以下特定目标中确定。1)表征蜱宿主体内致病性和非致病性SFG立克次体的水平和垂直传播。将在钝眼蜱和革蜱中评估6种SFG立克次体的先前未知传播动力学。将使用实验室蜱虫感染模型确定立克次体从立克次体感染蜱虫到脊椎动物宿主的水平传播。将通过荧光原位杂交和定量实时PCR（qPCR）评估立克次体垂直传播的效率，并评估SFG立克次体携带对蜱适合度的影响。2)在典型和非典型蜱/立克次体关系中，检查蜱衍生分子和SFG立克次体在感染期间的相互作用。蛋白质-蛋白质相互作用试验将用于鉴定整个蜱组织中存在的立克次体结合蛋白。研究将利用配对蜱组织和SFG立克次体进行比较下拉和蛋白结合试验。我们将确定关键的蜱蛋白，并通过生物测定，包括粘附/侵袭阻断试验，免疫/抗菌活性评估和RNA介导的干扰（RNAi），描绘他们在建立立克次体中的作用。还将评估立克次体各物种对特定分子的利用。公共卫生相关性尽管蜱与多种立克次体物种的分布重叠，但在自然界中，单个蜱物种不会感染多种立克次体物种。我们将研究蜱/立克次体关系的特异性，并描述蜱媒对斑点热立克次体能力的分子机制，以更好地了解蜱传立克次体疾病的生态学和流行病学。

DESCRIPTION (provided by applicant): Rickettsia rickettsii is the causative agent of one of the most severe tick-borne diseases in the United States. The compounding factors of rickettsial pathogenicity and route of tick transmission (horizontal and vertical) influence the ecology and epidemiology of human rickettsioses. Our long term goal is to elucidate the molecular mechanisms that are central to rickettsial transmission by ticks. Despite the overlapping distribution of ticks with multiple Rickettsia species in nature, individual tick species are not infected with a diverse range of rickettsial species. Therefore, the broad hypothesis being tested is that unique tick-derived factors are required for successful horizontal and vertical transmission of spotted fever group (SFG) Rickettsia. Preliminary evidence indicates rickettsial transmission is dependent on the appropriate tick/Rickettsia relationship and the pathogenic nature of the Rickettsia. It is also known that ticks actively respond to the infecting rickettsiae; however, the extent to which modulation of tick-derived molecules is required for rickettsial survival within the tick is not known. Thus, the experimental focus of this proposal is to delineate the specificity of tick/Rickettsia relationships, the relationship between rickettsial pathogenicity and transmission, and to define the tick-derived molecules associated with rickettsial infection via comparative analysis of different tick and Rickettsia species. The previously undefined parameters of tick/Rickettsia interactions will be determined in the following specific aims. 1) Characterize the horizontal and vertical transmission of pathogenic and nonpathogenic SFG Rickettsia in tick hosts in vivo. The previously unknown kinetics of transmission for six species of SFG Rickettsia will be assessed in Amblyomma and Dermacentor ticks. Horizontal transmission of rickettsiae from Rickettsia-infected ticks to vertebrate hosts will be determined using a laboratory model of tick infestation. The efficiency of vertical transmission of Rickettsia will be assessed by fluorescence in situ hybridization and quantitative real-time PCR (qPCR) and the influence of SFG Rickettsia carriage on tick fitness will be assessed. 2) Examine the interaction between tick-derived molecules and SFG Rickettsia during infection in typical and atypical tick/Rickettsia relationships. Protein-protein interaction assays will be used to identify rickettsial binding proteins present in whole tick tissues. Studies will utilize paired tick tissues and SFG rickettsiae in comparative pull-down and protein binding assays. We will identify key tick proteins and delineate their role in the establishment of Rickettsia via bioassays including adhesion/invasion blocking assays, immune/antimicrobial activity assessment, and RNA-mediated interference (RNAi). The utilization of specific molecules by individual species of Rickettsia will also be assessed. PUBLIC HEALTH RELEVANCE Despite the overlapping distribution of ticks with multiple Rickettsia species, in nature, individual tick species are not infected with a diverse range of rickettsial species. We will examine the specificity of tick/Rickettsia relationships and delineate the molecular mechanisms of tick vector competence for spotted fever Rickettsia to better understand the ecology and epidemiology of tick-borne rickettsial diseases.