Keys to prevent cholesterol robbery and infection by intracellular bacteria

防止胆固醇抢劫和细胞内细菌感染的关键

• 批准号: 8270716
• 负责人: YASUKO RIKIHISA
• 金额: $ 38.13万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-01 至 2017-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8270716
• 关键词: Anaplasma; Anaplasma phagocytophilum; Animal Diseases; Bacteria; Binding; Blood; Bovine Anaplasmosis; Cells; Cholesterol; Cholesterol Homeostasis; Clinical; Complex; Data; Dependency; Destinations; Disease; Doxycycline; Endoplasmic Reticulum; Endosomes; Goals; Gram-Negative Bacteria; Human; Infection; Intervention; Knowledge; LDL Cholesterol Lipoproteins; Learning; Life; Low Density Lipoprotein Receptor; Low-Density Lipoproteins; Lysosomes; Mammalian Cell; Measures; Membrane; Membrane Proteins; Membrane Structure and Function; Methods; Mission; Mus; North America; Nuclear Pore Complex; Outcome; Pathogenesis; Pathway interactions; Peptidoglycan; Prevalence; Prevention; Prevention strategy; Preventive; Proteins; Public Health; Regulation; Research; Rickettsia; Risk; SNAP receptor; Serum; Site; Testing; Therapeutic Intervention; Tick-Borne Diseases; Ticks; Time; Transport Vesicles; Vesicle; Work; Zoonoses; base; cholesterol trafficking; disability; granulocyte; human disease; innovation; late endosome; loss of function; novel; pathogen; prevent; prophylactic; receptor; receptor mediated endocytosis; robbery; syntaxin 16; therapy development; trafficking; trans-Golgi Network; treatment strategy; uptake; vesicle-associated membrane protein

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. PUBLIC HEALTH RELEVANCE: The application is important to public health because Anaplasma phagocytophilum is now recognized one of the most prevalent life-threatening tick-borne disease in North America. At this time, treatment and prevention strategies are limited. Our work is to characterize how this bacterium, and related bacteria, hijacks intracellular cholesterol trafficking to promote bacterial survival. The results from this study will reveal nove targets for treatment and prevention. Furthermore, what is learned will contribute to a broader understanding of how essential cholesterol is regulated inside mammalian cells. Thus, the proposed research is relevant to the part of NIH's mission that pertains to developing fundamental knowledge that will reduce the burden of human disability.

描述（由申请方提供）：人粒细胞无形体病（HGA）是一种新兴的人畜共患病，是北美最流行的危及生命的蜱虫病之一。这种疾病是由专性细胞内细菌，嗜吞噬细胞无形体感染引起的。由于A.嗜吞噬细胞菌引起严重的和有时致命的疾病，其在世界各地的日益增加的流行，以及有限的治疗选择和可用的预防措施，迫切需要了解这种病原体及其发病机制。虽然已知胆固醇对该细菌是必需的，并且胆固醇是HGA发病机制的关键决定因素，但该细菌如何获得胆固醇尚不清楚。我们的长期目标是了解无形体如何从宿主细胞中获得胆固醇，并将这些知识应用于预防和治疗严重的HGA。本文的目的是确定宿主细胞摄取的血清低密度脂蛋白（LDL）中的胆固醇从晚期内体运输到无形体包涵体的途径，这可能揭示一种新的干预靶点。我们的中心假设是，无形体在关键步骤调节正常LDL衍生的胆固醇（LDL-CHOL）细胞内运输，以适当的胆固醇。为了检验这一假设，具体目标1是确定LDL-CHOL递送至无形体包涵体的机制。我们的工作假设是，无形体感染上调了含有LDL-CHOL但不含溶酶体标记物的尼曼-匹克C- 1型（NPC 1）囊泡的一个子集，后者转运至无形体包涵体; NPC 1功能是将LDL-CHOL递送至细菌所必需的，从而促进感染。为了验证工作假设，我们的方法是通过几种独立的方法来表征NPC 1隔室和NPC 1囊泡交通，以及NPC 1减少或功能丧失对A.嗜吞噬细胞菌胆固醇摄取和感染。具体目标2是确定NPC 1囊泡交通无形体包涵体的机制。我们的工作假设是，TGN-SNARE机制参与了含有LDL-CHOL的NPC 1囊泡向无形体包涵体的转运，因此是感染所必需的。为了验证工作假设，我们的方法是确定与NPC囊泡运输和拴系蛋白相关的TGN-陷阱复合物的细胞内定位，以及它们对无形体胆固醇摄取和感染的要求。我们的方法是创新的，因为细菌的胆固醇依赖性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以阻断的LDL-CHOL囊泡运输转移的关键部位，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，因为所鉴定的组分和途径极有可能为预防和治疗干预提供新的靶点，此外还从根本上推进了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稳态异常问题。 公共卫生关系：该应用对公共卫生很重要，因为嗜吞噬细胞无形体现在被认为是北美最流行的威胁生命的蜱传疾病之一。目前，治疗和预防策略有限。我们的工作是表征这种细菌和相关细菌如何劫持细胞内胆固醇运输以促进细菌存活。这项研究的结果将揭示新的治疗和预防目标。此外，所学到的将有助于更广泛地了解必需胆固醇在哺乳动物细胞内是如何调节的。因此，拟议的研究是相关的NIH的使命的一部分，涉及到发展的基础知识，将减少人类残疾的负担。