Keys to prevent cholesterol robbery and infection by intracellular bacteria

描述(申请人提供)：人类粒细胞无形体病(HGA)是一种新出现的人畜共患病，是北美最常见的威胁生命的壁虱疾病之一。这种疾病是由感染细胞内必备细菌--吞噬细胞无形体引起的。鉴于吞噬细胞性假丝酵母菌容易引起严重的、有时甚至是致命的疾病，它在世界各地的流行率越来越高，可供选择的治疗方法和预防措施有限，因此迫切需要了解这种病原体及其发病机制。虽然已知胆固醇对这种细菌是必不可少的，而且胆固醇是HGA发病的关键决定因素，但这种细菌是如何获得胆固醇的尚不清楚。我们的长期目标是了解无浆体是如何从宿主细胞获得胆固醇的，并将这一知识应用于预防和治疗严重的HGA。本研究的目的是确定宿主细胞摄取的血清低密度脂蛋白(LDL)中的胆固醇如何从晚期内含体运输到无浆包涵体，这可能揭示一个新的干预靶点。我们的中心假设是，无浆体在一个关键步骤调节正常的低密度脂蛋白衍生胆固醇(LDL-Chol)的细胞内运输，以便调节胆固醇。为了验证这一假说，特定的目标1是确定低密度脂蛋白-胆固醇被运送到无浆包涵体的机制。我们的工作假设是，无浆体感染上调了含有低密度脂蛋白-胆固醇的Niemann-Pick C-1(NPC1)囊泡的子集，但不上调溶酶体标记物，后者运输到无浆体包涵体；NPC1功能是低密度脂蛋白-胆固醇传递到细菌所必需的，从而促进感染。为了验证这一工作假说，我们的方法是用几种独立的方法来表征NPC1隔室和NPC1囊泡运输，以及NPC1功能减少或丧失对吞噬弧菌胆固醇摄取和感染的影响。具体目的2是确定NPC1的囊泡运输到无浆包涵体的机制。我们的工作假设是TGN-SNARE机制参与了含有低密度脂蛋白-胆固醇的NPC1囊泡到无浆包涵体的运输，因此是感染所必需的。为了验证这一工作假设，我们的方法是确定与鼻咽癌囊泡运输和拴系蛋白相关的TGN-SNARE复合体在细胞内的定位，以及它们对无浆体胆固醇摄取和感染的需求。我们的方法是创新的，因为细菌对胆固醇的依赖还没有被用作开发干预措施的基础。关于预期结果，拟议的工作将确定可被阻断的低密度脂蛋白-胆固醇囊泡交通改道的关键部位，从而在不损害宿主细胞的情况下抑制无浆体感染。这些结果预计将产生重要的积极影响，因为已确定的成分和途径极有可能为预防和治疗干预提供新的靶点，此外还将从根本上推进细胞内胆固醇调节领域，这将有助于美国日益严重的胆固醇异常稳态问题。

DESCRIPTION (provided by applicant): Human granulocytic anaplasmosis (HGA) is an emerging zoonosis, and one of the most prevalent life- threatening tick-borneillnesses in North America. This disease is caused by infection with the obligatory intracellular bacterium, Anaplasma phagocytophilum. Given the propensity of A. phagocytophilum to cause severe and sometimes deadly diseases, its increasing prevalence throughout the world, and limited treatment choices and preventive measures available, there is a critical need to understand this pathogen and its pathogenesis. Although it is known that cholesterol is essential for this bacterium, and cholesterol is a critical determinant of HGA pathogenesis, how this bacterium acquires cholesterol is unknown. Our long-term goal is to understand how Anaplasma acquires cholesterol from host cells and apply this knowledge to prevent and treat severe HGA. The objective here is to determine the path by which cholesterol in serum low-density lipoprotein (LDL) taken up by host cells traffics from late endosomes to Anaplasma inclusions, which may reveal a novel target for intervention. Our central hypothesis is that Anaplasma modulates the normal LDL- derived cholesterol (LDL-CHOL) intracellular traffic at a critical step in order to appropriate cholesterol. To test this hypothesis, Specific aim 1 is to determine the mechanism by which LDL-CHOL is delivered to Anaplasma inclusions. Our working hypothesis is that Anaplasma infection up-regulates a subset of Niemann-Pick type C- 1 (NPC1) vesicles containing LDL-CHOL, but not lysosomal markers, which traffics to the Anaplasma inclusions; NPC1 function is required for LDL-CHOL delivery to bacteria, thus promoting infection. To test the working hypothesis, our approach is to characterize the NPC1 compartment and NPC1 vesicle traffic by several independent methods, and the effects of NPC1 reduction or loss-of function on A. phagocytophilum cholesterol uptake and infection. Specific aim 2 is to determine the mechanism by which NPC1 vesicles traffic to Anaplasma inclusions. Our working hypothesis is that TGN-SNARE machinery is involved in transport of NPC1 vesicles containing LDL-CHOL to Anaplasma inclusions, and therefore is required for infection. To test the working hypothesis, our approach is to determine the intracellular localization of TGN-SNARE complexes associated with NPC vesicle transport and tethering proteins, and their requirement for Anaplasma cholesterol uptake and infection. Our approach is innovative, because cholesterol dependency of bacteria has not been used as a basis for the development of interventions. With respect to expected outcomes, the work proposed will identify the critical site of diversion of LDL-CHOL vesicular traffic that can be blocked, resulting in inhibition of Anaplasma infection without harming host cells. Such results are expected to have an important positive impact because the identified components and pathways are highly likely to provide new targets for prophylactic and therapeutic intervention in addition to fundamentally advancing the field of intracellular cholesterol regulation that will help growing problems of abnormal cholesterol homeostasis in the U.S.