Limiting effects of a tick kunitz protease inhibitor on rickettsial infection

描述(由申请人提供)：成功的立克次体共生变得稳定，因为每个生物体为生存水平而斗争到耐受状态。研究表明，蒙丹立克次体能引起硬蜱--变色立克次体的防御反应。为了在一只扁虱中建立一个生态位，立克次体必须避开这种反应。定义驱动媒介传播疾病传播的生物学对公共卫生有影响。例如，破译壁虱防御和立克次体入侵之间平衡的基础构成了旨在阻断媒介传播疾病传播的研究基础。NIAID在其出版物《21世纪规划》中将节肢动物阻断疾病传播确定为主要研究重点。我的长期目标是了解病媒传播的病原体如何在主动的宿主防御反应存在的情况下定植节肢动物。我们的初步研究支持我们的中心假设，即DV-KPI通过灭活立克次体定植因子来限制硬蜱中肠的立克次体感染。首先，抗体介导的DV-KPI的中和导致立克次体在体外的定植增加。其次，RNAi介导的DV-KPI在硬蜱中的敲除导致蒙丹乳杆菌在中肠的负担增加。第三，细菌亲和力下拉分析表明，DV-KPI与蒙古乳杆菌结合。第四，一项初步的下拉研究表明，DV-KPI沉淀了立克次体配体。为了解决我的长期目标，NIAID的研究重点和中心假设，提出了两个目标。具体目的I-确定DV-KPI是否限制了蒙丹根结线虫的进入和中肠的迁徙。通过抑制DV-KPI功能，我们将进一步测试DV-KPI对立克次体定植和通过硬蜱中肠的迁移的影响。我们还将调查是否进入限制在宿主细胞周界或通过内体逃逸。特异性目的II-确定DV-KPI的立克次体配体(S)。使用下拉和共IP方法，我们将鉴定和确认相互作用的立克次体配体(S)的身份。完成这些目标将有助于确定立克次体相互作用的分子属性，这是导致人类感染的自然传播循环的核心。 相关性(见说明)：观察到的DV-KPI对立克次体生长的影响形成了我们的研究的基础，以调查促成扁虱和立克次体之间耐受状态的因素。利用这一知识开发方法来阻断致病立克次体向人类的传播，解决了NIAID的研究重点，即阻断节肢动物的疾病传播。

DESCRIPTION (provided by applicant): Successful tick-rickettsiae symbioses become stable as each organism's struggle for survival levels to a state of tolerance. Research demonstrates that Rickettsia montanensis elicits a defense response from the hard tick, Dermacentor variabilis. To establish a niche within a tick, rickettsiae must evade that response. Defining the biology that drives the transmission of vector-borne diseases has implications for public health. For instance, deciphering the underpinnings of the balance between tick defense and rickettsial invasion forms the basis of research that aims to interrupt transmission of vector-borne diseases. NIAID identified disruption of disease transmission by arthropods as a major research focus in its publication "Planning for the 21st Century." My long-term goal is to understand how vector-borne pathogens colonize arthropods in the presence of an active host defense response. Our preliminary studies support our central hypothesis that D. variabilis-Kunitz-type protease inhibitor (Dv-KPI) limits rickettsial infection of the tick midgut by inactivating a rickettsial colonization factor. First, antibody-mediated neutralization of Dv-KPI results in increased rickettsial colonization in vitro. Second, RNAi-mediated knockdown of Dv-KPI in ticks results in an increase in R. montanensis burden in the midgut. Third, a bacterial affinity pulldown assay shows that Dv-KPI associates with R. montanensis. Fourth, a preliminary pulldown study suggests that Dv-KPI precipitates a rickettsial ligand. To address my long-term goal, NIAID's research focus and the central hypothesis, two aims are proposed. Specific Aim I - To determine if Dv-KPI limits R. montanensis entry and transmigration of the D. variabilis midgut. By suppressing Dv-KPI function in the tick, we will test further the effect that Dv-KPI has on rickettsial colonization and transmigration though the tick midgut. We will also investigate if entry is limited at the host cell perimeter or through endosomal escape. Specific Aim II - To identify the rickettsial ligand(s) for Dv-KPI. Using pulldown and Co-IP methods, we will identify and confirm the identity of the interacting rickettsial ligand(s). Completion of these aims will help to define the molecular attributes of the tick-rickettsiae interaction, which is central to the natural transmission cycle that leads to human infection. RELEVANCE (See instructions): Dv-KPI's observed affect on rickettsial growth forms the basis of our studies to investigate the factors that contribute to a state of tolerance between the tick and rickettsiae. Exploiting this knowledge to develop methods to interrupt tick transmission of pathogenic rickettsiae to humans addresses NIAID's research focus to disrupt disease transmission by arthropods.