Serine Proteinase Inhibitors (Serpins) in Tick Feeding and Physiology

描述(申请人提供)：在美国，扁虱比任何其他媒介节肢动物传播更多的媒介传播疾病。当前以杀螨剂为基础的蜱类控制策略存在的局限性，威胁到了壁虱传播疾病控制计划的未来可持续性，因此有必要开发替代的壁虱控制策略。我们的长期目标是识别重要的扁虱蛋白，如果这些蛋白被阻止，可能会损害扁虱摄取、获取和传播病原体的能力。一类重要的此类蛋白质是丝氨酸蛋白酶抑制剂，其首字母缩写为“serpin”，这是在大多数生命形式中发现的最大的一类蛋白酶抑制剂。在人类中，蛇参与调节生命所必需的重要的蛋白分解途径，例如，由于自然突变导致许多令人衰弱的疾病而导致的血液凝固。我们最近描述了一个由19只美洲钝角蛇组成的家族，我们将其命名为“Lospins”，这是孤星壁蛇的首字母缩写。序列和表达数据分析表明，19个Lospin cDNA中有10个编码Lospin蛋白，这些蛋白可能在取食扁虱的过程中分泌到扁虱的唾液中。我们的假设是，这10个LSpin在取食扁虱的过程中分泌到宿主体内，以促进扁虱逃避宿主防御，扰乱它们的功能将阻止扁虱取食。在这份提案中，我们将通过两个具体的目标来描述10个LSpin中的6个。第一个是验证六种选定的Lospin蛋白分泌到扁虱唾液中的情况。其基本原理是，在扁虱唾液中检测到的LSpins将代表在吸食扁虱过程中分泌到宿主体内的候选物质，并将被选中进行进一步研究。我们将提出针对候选LSpins的特异性多克隆抗体，并将它们用于收集的扁虱唾液的标准Western blotting分析。第二，我们将抑制在目标1中选择的Lospins的表达，并研究其对蜱类饲养的影响。其基本原理是，抑制一种重要的LSpin将阻止扁虱进食，它将被选为未来的研究对象。美洲按蚊是一种重要的公共卫生有害生物，在美国南部和大西洋各州尤其常见。这项提议的结果的直接效用将是促进我们对这种重要害虫的生理学的了解。这些数据将使我们能够为控制这种疾病的新方法提供理论依据。与公共卫生相关：在美国，扁虱传播的病原体比其他任何媒介节肢动物都要多。当前以杀螨剂为基础的蜱类控制策略存在的局限性，威胁到了未来壁虱传播疾病控制计划的可持续性，因此有必要开发替代的壁虱控制策略。在开发替代控制方法之前，识别重要的壁虱蛋白来调节壁虱的生理和促进壁虱的饲养是很重要的。

DESCRIPTION (provided by applicant): In the United States (US) ticks transmit more vector borne disease agents than any other vector arthropod. Limitations associated with current acaricide based tick control strategies that threaten the future sustainability of tick borne illnesses containment programs, have necessitated the need for development of alternative tick control strategies. Our long-term goals are to identify important tick proteins, which if blocked, can compromise the tick's ability to feed, acquire and transmit disease agents. One important such class of proteins, are serine proteinase inhibitors that are commonly referred to by its acronym "serpin", which is the largest class of proteinase inhibitors that have been found in most forms of life. In humans, serpins are involved in regulation of important of proteolytic pathways that are essential to life such as blood coagulation with serpin malfunctions arising from natural mutations causing numerous debilitating diseases. We have recently described a family of 19 Amblyomma americanum serpins that we have named as "Lospins", an acronym for Lone Star tick serpins. Sequence and expression data analyses have revealed that 10 of the 19 Lospin cDNAs encode for Lospin proteins that are potentially secreted into tick saliva during tick feeding. Our hypothesis is that, the 10 Lospins are secreted into the host during tick feeding to facilitate the tick's escape from host defense and that disrupting their function will prevent the tick from feeding. In this proposal we will characterize six of the 10 Lospins via two specific aims. The first is to validate secretion into tick saliva of six selected Lospin proteins. The rationale is that Lospins detected in tick saliva will represent candidates that are secreted into the host during tick feeding and will be selected for further study. We will raise specific polyclonal antibodies to candidate Lospins and use them in standard western blotting analyses of collected tick saliva. The second, we will be to suppress expression of Lospins selected in aim 1 and study the effects on tick feeding. The rationale is that suppression of an essential Lospin will prevent the tick from feeding and it will be selected for future study. A. americanum is an important public health pest that is commonly encountered tick species particularly in southern and Atlantic states. The immediate utility of results from this proposal will be advancing our knowledge on the physiology of this important pest. This data will position us to rationale new methods to control this tick. PUBLIC HEALTH RELEVANCE: In the United States ticks transmit more vector borne disease agents than any other vector arthropod. Limitations associated with current acaricide based tick control strategies that threaten the future sustainability of containment programs for tick borne illnesses, have necessitated the need for development of alternative tick control strategies. Identification of important tick proteins that regulate tick physiology and facilitate tick feeding is important before alternative tick control methods can be developed.