Arthropod host-dependent influence on rickettsial pathogenicity

描述（由申请人提供）：猫立克次体最初于1991年在美国被鉴定为人类病原体;随着全球范围内认识的增加，现在认识到由猫立克次体引起的人类感染。在撒哈拉以南非洲地区，猫占非疟疾发热病因的6%。我们的长期目标是阐明跳蚤传播立克次体的关键分子机制，以便更好地了解跳蚤传播立克次体疾病的流行病学，并确定干预点。R.猫也是一种垂直维持的非吸血昆虫内共生体。对于节肢动物传播的立克次体属，毒力水平预期取决于垂直传播和水平传播对立克次体维持的相对重要性。因此，正在测试的中心假设是，有宿主依赖性机制是必不可少的立克次体的毒力和传播。研究了R.猫区分宿主环境，并执行水平或垂直路线，以成功地在节肢动物种群中传播和维持，这一点尚不清楚。本实验的重点是通过对立克次体的比较和功能分析，阐明立克次体和节肢动物依赖的因素对成功的水平传播的贡献。猫在不同的节肢动物宿主，并在节肢动物宿主界面。研究了该菌传播的生物学和分子生物学事件。跳蚤宿主中的猫的定义很差，对于清楚地了解跳蚤传播的立克次体疾病的流行病学和确定干预点至关重要。先前未定义的跳蚤立克次体感染参数将在以下具体目标中确定目标1。与非吸血节肢动物相比，猫蚤中立克次体转录本表达谱的特征。使用RNA-seq，表达的立克次体转录本将在R.猫感染的跳蚤，在跳蚤吸血期间，以及在立克次体中，在共食跳蚤之间的水平传播期间沉积到血液中。将与体质性感染的书虱进行比较。随后，独特的立克次体转录本将通过靶向基因破坏进行功能表征，并评估其水平传播的要求。利用体内系统，我们将能够测试的假设，R。猫具有在跳蚤血粉采集期间水平传播所必需的基本转录物谱。目标二。立克次体水平传播所必需的猫蚤唾液腺分泌因子的测定唾液腺转录谱已初步确定在猫蚤和唾液腺转录鉴定为分泌分子将评估在R。在喂食期间感染猫蚤的猫 期差异表达的转录本与R。猫感染跳蚤的功能特征是RNA介导的干扰，并评估这些跳蚤衍生因子水平传播的必要性。利用水平传播的生物测定方法，对蚤源因子是R.猫将被测试。

DESCRIPTION (provided by applicant): Rickettsia felis was originally identified in the United States as a human pathogen in 1991; and, with increased recognition worldwide, it is now realized that human infections caused by R. felis account for up to 6% of the causes of fever among non-malarial conditions in sub-Saharan Africa. Our long-term goal is to elucidate the molecular mechanisms that are critical to rickettsial transmission by fleas in order to better understand the epidemiology of flea-borne rickettsial diseases and identify points of intervention. In addition to being a horizontally transmitted flea-borne pathogen, R. felis is alsoa vertically maintained endosymbiont of non-hematophagous insects. For arthropod-borne Rickettsia spp., the level of virulence is expected to be governed by the relative importance of vertical and horizontal transmission for maintenance of the rickettsiae. Therefore, the central hypothesis being tested is that there are host-dependent mechanisms that are essential to rickettsial virulence and transmission. The mechanism by which R. felis differentiates the host environment and executes either horizontal or vertical routes for successful transmission and maintenance in arthropod populations are not known. The experimental focus of this proposal is to delineate the Rickettsia- and arthropod-dependent factors contributing to successful horizontal transmission through comparative and functional analyses of R. felis in different arthropod hosts, and at the arthropod-host interface. The biological and molecular events underlying the transmission of R. felis in flea hosts are poorly defined and are critical to clearl understanding the epidemiology of flea-borne rickettsial diseases and identify points of intervention. The previously undefined parameters of rickettsial infection in fleas will be determined in the following specific aims Aim 1. Characterization of rickettsial transcript expression profile in cat fleas compared to non- hematophagous arthropods. Using RNA-seq, expressed rickettsial transcripts will be identified in R. felis- infected fleas, during flea hematophagy, and in rickettsiae deposited into blood during horizontal transmission between cofeeding fleas. Comparisons will be made to constitutively infected booklice. Subsequently, unique rickettsial transcripts will be functionally characterized via targeted gene disruption and their requirement for horizontal transmission assessed. Utilizing in vivo systems, we will be able to test the hypothesis that R. felis has an essential transcript profile necessary for horizontal transmission during flea bloodmeal acquisition. Aim 2. Determination of cat flea-derived salivary gland secreted factors essential for horizontal transmission of Rickettsia. The salivary gland transcription profile has been initially characterized in cat fleas and salivary gland transcripts identified as secreted molecules will be assessed in R. felis-infected cat fleas during the feeding period. Differentially expressed transcripts associated with R. felis infection of fleas will be functionally characterized by RNA-mediated interference and the necessity of these flea- derived factors for horizontal transmission assessed. Utilizing a horizontal transmission bioassay, the hypothesis that flea-derived factors are essential to successful horizontal transmission of R. felis will be tested.