Limiting effects of a tick kunitz protease inhibitor on rickettsial infection

描述（由申请人提供）：成功的蜱-立克次体共生体变得稳定，因为每个生物体的生存水平的斗争，以一个国家的宽容。研究表明，蒙大拿立克次体是硬蜱Dermacentor variabilis的防御反应。为了在蜱虫体内建立一个生态位，立克次体必须避开这种反应。确定推动病媒传播疾病传播的生物学因素对公共卫生具有重要意义。例如，破译蜱虫防御和立克次体入侵之间平衡的基础构成了旨在中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》中确定了节肢动物破坏疾病传播的主要研究重点。“我的长期目标是了解媒介传播的病原体如何在存在主动宿主防御反应的情况下定殖节肢动物。我们的初步研究支持我们的中心假设，D。变异库尼茨型蛋白酶抑制剂（Dv-KPI）通过灭活立克次体定殖因子来限制蜱中肠的立克次体感染。首先，抗体介导的Dv-KPI中和导致体外立克次体定殖增加。其次，RNAi介导的蜱中Dv-KPI的敲低导致R.中肠中的Montanensis负担。第三，细菌亲和下拉试验表明Dv-KPI与R.蒙大拿人。第四，初步的下拉研究表明，Dv-KPI沉淀立克次体配体。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。具体目标I -确定Dv-KPI是否限制R。montanensis的进入和迁移。变异中肠通过抑制蜱中的Dv-KPI功能，我们将进一步测试Dv-KPI对立克次体通过蜱中肠的定殖和迁移的影响。我们还将研究进入是否局限于宿主细胞周边或通过内体逃逸。具体目标II -鉴定Dv-KPI的立克次体配体。使用下拉和Co-IP方法，我们将识别和确认相互作用的立克次体配体的身份。这些目标的完成将有助于确定蜱-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周期的核心。 相关性（参见说明）：Dv-KPI对立克次体生长的观察影响构成了我们研究的基础，以调查促成蜱和立克次体之间耐受状态的因素。利用这些知识来开发中断蜱传播致病性立克次体给人类的方法，解决了NIAID的研究重点，以破坏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.