Molecular basis for spotted fever group Rickettsia vector competence in ticks

描述（由申请人提供）：立克次体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虫传播途径（水平和垂直）的复合因素影响着人类立克次体的生态学和流行病学。我们的长期目标是阐明由蜱虫传播立克次体的核心分子机制。尽管蜱在自然界中与多种立克次体物种重叠分布，但单个蜱种并不感染多种立克次体物种。因此，正在验证的广泛假设是，斑点热组立克次体（SFG）的水平和垂直传播需要独特的蜱源因素。初步证据表明，立克次体传播取决于蜱虫/立克次体的适当关系和立克次体的致病性。众所周知，蜱对感染立克次体有积极反应；然而，蜱衍生分子的调节在多大程度上是蜱体内立克次体存活所必需的尚不清楚。因此，本研究的实验重点是通过对不同蜱和立克次体物种的比较分析，描述蜱/立克次体关系的特异性、立克次体致病性与传播的关系，并确定与立克次体感染相关的蜱源分子。以前未定义的蜱虫/立克次体相互作用参数将在以下具体目标中确定。1)描述致病性和非致病性SFG立克次体在蜱宿主体内的水平和垂直传播特征。六种SFG立克次体以前未知的传播动力学将在弱视蜱和革螨蜱中进行评估。立克次体从感染立克次体的蜱向脊椎动物宿主的水平传播将使用蜱感染的实验室模型来确定。采用荧光原位杂交和实时荧光定量PCR （qPCR）技术评估立克次体垂直传播效率，评估SFG立克次体携带对蜱体适合度的影响。2)在典型和非典型蜱/立克次体关系中，研究蜱源分子与SFG立克次体感染过程中的相互作用。蛋白-蛋白相互作用测定将用于鉴定整个蜱虫组织中存在的立克次体结合蛋白。研究将利用配对蜱虫组织和SFG立克次体进行比较拉下和蛋白质结合分析。我们将通过包括粘附/入侵阻断试验、免疫/抗菌活性评估和rna介导干扰（RNAi）在内的生物测定来鉴定关键蜱虫蛋白并描述它们在立克次体建立中的作用。还将评估个别立克次体物种对特定分子的利用。尽管蜱与多种立克次体物种重叠分布，但在自然界中，单个蜱类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，并描述蜱媒介能力对斑点热立克次体的分子机制，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.