Arthropod host-dependent influence on rickettsial pathogenicity

描述（由申请人提供）：猫立克次体最初于1991年在美国被确定为人类病原体；而且，随着全世界越来越多地认识到，现在人们认识到，在撒哈拉以南非洲的非疟疾疾病中，由猫科动物引起的人类感染占发烧原因的6%。我们的长期目标是阐明对跳蚤传播立克次体至关重要的分子机制，以便更好地了解跳蚤传播的立克次体疾病的流行病学并确定干预点。除了是一种水平传播的蚤传病原体外，猫毛霉也是一种垂直维持的非吸血昆虫的内共生体。对于节肢动物传播的立克次体，预计其毒力水平取决于垂直传播和水平传播对维持立克次体的相对重要性。因此，正在验证的中心假设是，存在对立克次体毒力和传播至关重要的宿主依赖机制。在节肢动物种群中，狐猴区分寄主环境并通过水平或垂直途径成功传播和维持的机制尚不清楚。本研究的实验重点是通过对不同节肢动物宿主和节肢动物-宿主界面上猫鼠立克次体和节肢动物依赖性因素的比较和功能分析，来描述立克次体和节肢动物依赖性因素对成功水平传播的影响。猫R. felis在跳蚤宿主中传播的生物学和分子事件尚不清楚，这对于清楚了解跳蚤传播的立克次体病的流行病学和确定干预点至关重要。以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。猫蚤与非食血节肢动物立克次体转录物表达谱的比较。利用RNA-seq技术，将在感染猫R.的跳蚤、跳蚤吸血过程中以及在共食跳蚤之间水平传播过程中沉积到血液中的立克次体转录物中鉴定表达的立克次体转录物。将与本构感染的书柜进行比较。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，并评估其对水平传播的要求。利用体内系统，我们将能够验证假设，即在跳蚤血液获取过程中，猫鼠具有水平传播所必需的基本转录谱。目标2。立克次体水平传播猫蚤源性唾液腺分泌因子的测定。唾液腺转录谱已在猫蚤中得到初步表征，唾液腺转录谱被鉴定为分泌分子，将在饲养过程中对感染猫蚤的猫蚤进行评估

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.