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: 8415504
• 负责人: YASUKO RIKIHISA
• 金额: $ 35.84万
• 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/8415504
• 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.

描述（由申请人提供）：人类粒细胞肿瘤病（HGA）是新兴的人畜共患病，也是北美最普遍的威胁性壁虱传播性。该疾病是由强制性细胞内细菌（纳普氏吞噬细胞吞噬的感染）引起的。鉴于吞噬大麻的倾向是引起严重疾病，有时是致命的疾病，其在世界范围内的患病率增加，并且可用的治疗选择和预防措施有限，因此需要了解这种病原体及其发病机理。尽管众所周知，胆固醇对于该细菌至关重要，而胆固醇是HGA发病机理的关键决定因素，但该细菌如何获得胆固醇是未知的。我们的长期目标是了解Anaplasma如何从宿主细胞中获取胆固醇，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从晚期内体到Anaplasma夹杂物吸收的血清低密度脂蛋白（LDL）中胆固醇的路径，这可能揭示了新的干预靶标。我们的中心假设是，Anaplasma调节了正常的LDL衍生胆固醇（LDL-Chol）细胞内交通，以便为了适当的胆固醇。为了检验这一假设，具体目的1是确定LDL-Chol递送到厌氧菌夹杂物的机制。我们的工作假设是，Anaplasma感染上调了niemann-pick型C-1（NPC1）囊泡的子集，其中含有LDL-Chol，但不含溶酶体标记物，这些标记物运输于anaplasma夹杂物。 NPC1功能是LDL-Chol递送到细菌所必需的，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方法来表征NPC1室和NPC1囊泡流量，以及NPC1降低或功能丧失对吞噬细胞脂肪杆菌胆固醇摄取和感染的影响。具体目的2是确定NPC1囊泡流向adlasma夹杂物的机制。我们的工作假设是TGN-SNARE机械参与了含有LDL-Chol到Anaplasma夹杂物的NPC1囊泡的运输，因此需要感染。为了检验工作假设，我们的方法是确定与NPC囊泡转运和绑扎蛋白相关的TGN-SNARE复合物的细胞内定位，以及它们对anaplasma胆固醇摄取和感染的需求。我们的方法具有创新性，因为细菌的胆固醇依赖性尚未用作开发干预措施的基础。关于预期的结果，提出的工作将确定可以阻止的LDL-Chol囊泡流量转移的关键部位，从而导致抑制Anaplasma感染而不会损害宿主细胞。预计此类结果将产生重要的积极影响，因为确定的成分和途径极有可能为预防性和治疗性干预提供新的靶标，除了从根本上促进细胞内胆固醇调节领域的领域，这将有助于美国在美国的胆固醇异常稳态的问题，