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（由申请人提供）：人类粒细胞无形体病（HGA）是一种新兴的人畜共患病，也是北美最流行的威胁生命的蜱传疾病之一。这种疾病是由细胞内必需细菌嗜吞噬细胞无形体感染引起的。鉴于嗜吞噬细胞放线菌容易引起严重甚至有时致命的疾病，其在世界各地的患病率不断增加，而且可用的治疗选择和预防措施有限，因此迫切需要了解这种病原体及其发病机制。尽管已知胆固醇对于这种细菌至关重要，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形体如何从宿主细胞获取胆固醇，并应用这些知识来预防和治疗严重的 HGA。这里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涵体运输到无形体包涵体的路径，这可能揭示一个新的干预目标。我们的中心假设是，无形体在关键步骤中调节正常 LDL 衍生胆固醇 (LDL-CHOL) 细胞内运输，以调节胆固醇。为了检验这一假设，具体目标 1 是确定 LDL-CHOL 传递至无形体内含物的机制。我们的工作假设是，无形体感染上调了含有 LDL-CHOL 的 Niemann-Pick C-1 型 (NPC1) 囊泡子集，但不上调溶酶体标记物，后者会转运至无形体内含物； NPC1 功能是将 LDL-CHOL 传递给细菌所必需的，从而促进感染。为了检验工作假设，我们的方法是通过几种独立的方法来表征 NPC1 区室和 NPC1 囊泡运输，以及 NPC1 减少或功能丧失对嗜吞噬细胞胆固醇摄取和感染的影响。具体目标 2 是确定 NPC1 囊泡运输至无形体内含物的机制。我们的工作假设是，TGN-SNARE 机制参与将含有 LDL-CHOL 的 NPC1 囊泡转运至无形体包涵体，因此是感染所必需的。为了检验工作假设，我们的方法是确定与 NPC 囊泡转运和束缚蛋白相关的 TGN-SNARE 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需求。我们的方法是创新的，因为细菌的胆固醇依赖性尚未被用作开发干预措施的基础。就预期结果而言，拟议的工作将确定可被阻断的 LDL-CHOL 囊泡运输转移的关键位点，从而在不损害宿主细胞的情况下抑制无形体感染。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很可能为预防和治疗干预提供新的目标，此外还从根本上推进细胞内胆固醇调节领域，这将有助于解决日益严重的胆固醇稳态异常问题。我们。 公共健康相关性：该应用对公共健康非常重要，因为嗜吞噬细胞无形体现已被认为是北美最常见的威胁生命的蜱传疾病之一。此时，治疗和预防策略是有限的。我们的工作是描述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治疗和预防的新目标。此外，所学到的知识将有助于更广泛地了解哺乳动物细胞内必需胆固醇的调节机制。因此，拟议的研究与 NIH 使命的一部分相关，即发展基础知识以减轻人类残疾的负担。