Identification of bacterial genes required for transmission of tick-borne pathoge

描述（由申请方提供）：蜱传疾病，包括无形体病、埃立克体病和几种立克次体病，在发病率不断上升的情况下仍然控制不力。阻断蜱传播是预防疾病的潜在关键控制点。然而，这些病原体能够成功地定殖蜱虫的机制代表了我们知识中的一个重大空白，阻碍了在疾病预防中利用这一控制点的能力。拟议研究的目标是确定病原体分子所需的定植和成功传播的硬蜱。病原体传播依赖于从脊椎动物宿主到蜱的成功转变，其包括进入根本不同的宿主细胞类型（包括蜱中肠和唾液腺的细胞）内并在其中复制。通过转录组学和蛋白质组学，已经鉴定了无形体属和埃里希体属在蜱定殖期间特异性上调的病原体蛋白。然而，测试单个蛋白质和功能途径的功能要求所需的遗传工具要么不可用，要么正在开发中。与蜱传立克次体病原体的这一局限性相反，通过突变体文库筛选利用基因组序列数据已经允许快速鉴定广泛细菌病原体中特定功能所需的基因。利用土拉热弗朗西丝菌土拉热亚种（Francisella tularensis subsp. novicida，确定F.杀螨素能定殖于安氏革蜱，并在蜱蜕皮至成年期间在蜱中肠内存活。一旦成年蜱以第二个宿主为食，F。杀螨剂传播到蜱的唾液腺，并传播到哺乳动物宿主，从而完成类似于无形体属、埃立克体属和立克次体属的传播循环。重要的是，突变体对蜱的定殖是非随机的;因此，除非存在特异性影响定殖蜱的能力的缺陷，否则定殖蜱的突变体群体将反映血餐中的突变体群体。这允许特异性检测不能定殖的突变体，从而检测传播所需的基因。我们假设，所需的基因将分组到广泛适用于细胞内，蜱传播的病原体的功能途径。通过靶向基因敲除和跨物种互补实验，可以确定蜱传播的广泛保守要求。这项研究与R21机制的探索性/发展性是一致的，因为它利用了一个新的模型系统来寻找传播所需的基因和基因通路。拟议的研究可能会导致新的和潜在的广泛适用的战略，以防止蜱传疾病的传播。 公共卫生相关性：根据CDC提供的最新数据，蜱传疾病的发病率，包括无形体病，埃立克体病和落基山斑疹热，一直在稳步上升（CDC蜱病网站）。本研究的目的是确定细菌病原体如何在蜱虫中定植，长期目标是开发预防蜱虫定植的新方法，从而预防蜱传疾病。

DESCRIPTION (provided by applicant): Tick-borne diseases, including anaplasmosis, ehrlichiosis, and several rickettsial diseases, remain poorly controlled amid increasing incidence. The interruption of tick transmission is a potential critical control point for disease prevention. However, the mechanisms by which these pathogens are able to successfully colonize the tick represent a significant gap in our knowledge, impeding the ability to exploit this control point in disease prevention. The goal of the proposed research is to identify pathogen molecules required for colonization of and successful transmission by ixodid ticks. Pathogen transmission relies on the successful transition from the vertebrate host into the tick, which includes the entry into and replication within radically different host cell types including cells of the tick midgut and salivary glands. Through transcriptomics and proteomics, pathogen proteins specifically up-regulated during tick colonization have been identified for both Anaplasma and Ehrlichia. However, the genetic tools needed to test the functional requirement of individual proteins and functional pathways are either unavailable or in development for these organisms. In contrast to this limitation for tick-borne rickettsial pathogens, exploitation of genomic sequence data through mutant library screens has allowed for rapid identification of genes required for specific functions in a broad array of bacterial pathogens. Using a transposon mutant library of Francisella tularensis subsp. novicida, we established that F. novicida can colonize Dermacentor andersoni nymphs, and survive within the tick midgut during the molt to adulthood. Once the adult ticks are fed on a second host, the F. novicida disseminate to the tick salivary glands, and are transmitted to the mammalian host, thus completing a transmission cycle similar to Anaplasma, Ehrlichia, and Rickettsia. Importantly, colonization of the tick by mutants is non-random; thus, unless a defect is present which specifically affects the ability to colonize the tick, the population of mutants colonizing the tick will reflect the population of mutants in the blood meal. This allows for the specific detection of mutants unable to colonize and thus genes required for transmission. We hypothesize that the required genes will group into functional pathways broadly applicable to intracellular, tick-transmitted pathogens. Through both targeted gene knockout and cross-species complementation experiments, broadly conserved requirements for tick transmission can be identified. This research is consistent with the exploratory/developmental nature of the R21 mechanism because it utilizes a new model system to search for genes and gene pathways required for transmission. The proposed studies may lead to novel and potentially broadly applicable strategies to prevent the transmission of tick-borne diseases. PUBLIC HEALTH RELEVANCE: According to the most recent data available from the CDC, the incidence of tick-borne disease, including anaplasmosis, ehrlichiosis and Rocky Mountain spotted fever, has been steadily increasing (CDC tick diseases website). The objective of this research is to identify how bacterial pathogens colonize the tick, with the long-term goal of developing new means of preventing tick colonization, and thus preventing tick-borne disease.