Serine Proteinase Inhibitors (Serpins) in Tick Feeding and Physiology

描述（由申请人提供）：在美国，蜱虫比任何其他媒介节肢动物传播更多的媒介传播疾病媒介。目前基于杀螨剂的蜱虫控制策略的局限性威胁到蜱虫传播疾病控制计划的未来可持续性，因此有必要开发替代蜱虫控制策略。我们的长期目标是确定重要的蜱虫蛋白质，如果这些蛋白质被阻断，就会损害蜱虫进食、获取和传播病原体的能力。其中一类重要的蛋白质是丝氨酸蛋白酶抑制剂，通常以其首字母缩略词“serpin”命名，这是在大多数生命形式中发现的最大一类蛋白酶抑制剂。在人类中，蛇形蛋白参与了对生命至关重要的蛋白质水解途径的调节，如血液凝固，蛇形蛋白的功能障碍是由自然突变引起的，导致许多衰弱性疾病。我们最近描述了一个由19只Amblyomma americanum蛇组成的家族，我们将其命名为“Lospins”，这是Lone Star蜱蛇的首字母缩略词。序列和表达数据分析显示，19个Lospin cdna中有10个编码Lospin蛋白，这些蛋白可能在蜱虫进食时分泌到蜱虫唾液中。我们的假设是，在蜱虫取食过程中，10个lospin被分泌到宿主体内，以帮助蜱虫逃离宿主的防御，而破坏它们的功能将阻止蜱虫取食。在本建议中，我们将通过两个具体目标来描述10个lospin中的6个。首先是验证六种选定的Lospin蛋白在蜱虫唾液中的分泌。其基本原理是，在蜱虫唾液中检测到的lospin将代表在蜱虫进食过程中分泌到宿主体内的候选物质，并将被选择用于进一步研究。我们将培养针对候选lospin的特异性多克隆抗体，并将其用于收集的蜱虫唾液的标准western blotting分析。第二，我们将抑制目标1中选择的lospin的表达，并研究其对蜱食的影响。其基本原理是，抑制一种基本的Lospin将阻止蜱虫进食，它将被选择用于未来的研究。美洲蜱是一种重要的公共卫生害虫，是南部和大西洋各州常见的蜱类。这一建议的结果的直接效用将促进我们对这一重要害虫生理学的认识。这些数据将使我们找到控制这种蜱虫的新方法。公共卫生相关性：在美国，蜱虫比任何其他媒介节肢动物传播更多的媒介传播疾病媒介。目前基于杀螨剂的蜱虫控制策略的局限性威胁到蜱虫传播疾病控制计划的未来可持续性，因此有必要开发替代蜱虫控制策略。在开发替代蜱虫控制方法之前，鉴定调节蜱虫生理和促进蜱虫摄食的重要蜱虫蛋白非常重要。

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.