Keys to prevent cholesterol robbery and infection by intracellular bacteria

描述（由申请人提供）：人类粒细胞无形体病（HGA）是一种新兴的人畜共患病，是北美最普遍的威胁生命的蜱传疾病之一。这种疾病是由细胞内的专性细菌，嗜吞噬细胞无原体感染引起的。鉴于嗜吞噬胞芽胞杆菌引起严重甚至有时致命疾病的倾向，其在世界范围内的流行日益增加，以及现有的治疗选择和预防措施有限，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需的，并且胆固醇是HGA发病机制的关键决定因素，但这种细菌如何获得胆固醇尚不清楚。我们的长期目标是了解无原体如何从宿主细胞获得胆固醇，并应用这一知识来预防和治疗严重的HGA。本文的目的是确定宿主细胞吸收的血清低密度脂蛋白（LDL）中的胆固醇从晚期内体转运到无原体包涵体的途径，这可能揭示新的干预靶点。我们的中心假设是无浆体调节正常的LDL-衍生胆固醇（LDL- chol）在细胞内运输的关键步骤，以适当的胆固醇。为了验证这一假设，具体目的1是确定LDL-CHOL被递送到无原体包涵体的机制。我们的工作假设是，无原体感染上调了含有LDL-CHOL的Niemann-Pick型C- 1 （NPC1）囊泡的一个子集，但不包括溶酶体标记物，这些标记物可运输到无原体包涵体；NPC1功能是LDL-CHOL向细菌传递从而促进感染的必要条件。为了验证工作假设，我们的方法是通过几种独立的方法表征NPC1室和NPC1囊泡交通，以及NPC1减少或功能丧失对嗜吞噬细胞芽胞杆菌胆固醇摄取和感染的影响。具体目的2是确定NPC1囊泡向无原体包涵体转运的机制。我们的工作假设是TGN-SNARE机制参与了含有LDL-CHOL的NPC1囊泡向无原体包裹体的运输，因此是感染所必需的。为了验证这一假说，我们的方法是确定与NPC囊泡运输和系缚蛋白相关的TGN-SNARE复合物的细胞内定位，以及它们对无原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性还没有被用作干预措施发展的基础。关于预期结果，提出的工作将确定LDL-CHOL囊泡交通转移的关键部位，该部位可以被阻断，从而抑制无形体感染而不损害宿主细胞。这些结果预计将产生重要的积极影响，因为所确定的成分和途径极有可能为预防和治疗干预提供新的目标，除了从根本上推进细胞内胆固醇调节领域，这将有助于解决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.