Functional characterization of Orientia tsutsugamushi ankryin repeat proteins

恙虫病东方体锚蛋白重复蛋白的功能表征

基本信息

批准号：
8463116
负责人：
Jason A Carlyon
金额：
$ 7.48万
依托单位：
VIRGINIA COMMONWEALTH UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-05-01 至 2015-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8463116
关键词：
Accounting Acute Affinity Chromatography Afghanistan Anaplasma phagocytophilum Ankyrin Repeat Antibiotic Resistance Antibiotics Applications Grants Asia Bacteria Biological Case Study Cell physiology Cells Chiggers Coiled-Coil Domain Disease Disease Outbreaks Effectiveness Eukaryotic Cell Fever Funding Genes Genome Glutathione S-Transferase Goals Grant Hour In Vitro Infection Japan Knowledge Ligands Locales Location Mammalian Cell Marines Mediating Military Personnel Mites Molecular Native-Born Nature Orientia tsutsugamushi Pakistan Pathogenesis Pathway interactions Pattern Precipitation Prevalence Proteins Recombinants Reporting Rickettsiales Risk Role Scrub Typhus Soldier Staging Stress Testing Therapeutic Training Viral Visit Work base design experience global health in vivo inhibitor/antagonist interest mortality novel overexpression pathogen protein protein interaction small molecule success trafficking typhus vaccine

项目摘要

DESCRIPTION (provided by applicant): Scrub typhus is a potentially fatal disease that is endemic in the Asia-Pacific region, where 1 billion people are at risk for infection and 1 million new cases are reported annually. Scrub typhus is treatable with antibiotics, but reinfections are common and decreased efficacy of antibiotics has been increasingly reported. A recent outbreak among U.S. Marines training at a military base in Japan, and the prevalence of scrub typhus in Afghanistan and Pakistan where U.S. troops are deployed, echoes the risk of U.S. soldiers for the disease. The etiologic agent is the trombiculid mite-transmitted obligate intracellular bacterium, Orientia tsutsugamushi. Despite the global health threat that it poses, O. tsutsugamushi is severely understudied. Indeed, how this pathogen manipulates eukaryotic host cell functions to facilitate its intracellular survival is poorly understood. An emerging theme among many intracellular bacterial pathogens is that they translocate ankryin repeat-containing proteins (Anks) into eukaryotic host cells. The ankryin repeat is one of the most common protein-protein interaction motifs in nature. Anks of other bacterial and viral pathogens traffic t distinct subcellular locations where they interact with host target proteins and mimic or interfere with host cell functions to facilitate pathogen survival. The O. tsutsugamushi genome carries 38 ank genes, and we have determined that all 38 are expressed during infection of mammalian host cells in vitro. Genes encoding Ank4, Ank9, and Ank12_1 are induced at 1 h post-infection, Ank6 at 4 h, and Ank13 at 8 h. We hypothesize that Ank4, Ank6, Ank9, Ank12_1, and Ank13 are important for O. tsutsugamushi to establish infection and have prioritized these proteins for functional studies. Genes encoding ank1, ank5, and ank17 are also expressed during infection and carry coiled-coil domains. The coiled-coil domain is a signature protein-protein interaction motif of numerous bacterial pathogen effectors that mediates interaction with host cell targets. Because Ank1, Ank5, and Ank17 carry both ankyrin repeat and coiled-coil motifs, we will include them in our functional studies. In Aim 1, we will assess if the eight Anks of interest perform distinct effector functions by ectopically expressing the proteins in mammalian host cells and identifying the subcellular locations to which they traffic. To determine if they interact with hos cell factors that are critical for O. tsutsugamushi intracellular survival, we will assess whether ectopically expressed Anks competitively inhibit Orientia survival. In Aim 2, we will capture and identify host cell target proteins that interact with Orientia Anks using in vivo coprecipitation ad affinity chromatography. The proposed work will fill a considerable knowledge gap of how an understudied pathogen of global biomedical importance facilitates its intracellular survival. Since antibiotic resistance among O. tsutsugamushi isolates is increasing and reinfections are common, defining the functional roles of Anks will potentially aid the design of small molecule inhibitors that target specific Anks to treat scrub typhus.

描述（由申请人提供）：恙虫病是一种潜在致命的疾病，在亚太地区流行，该地区有 10 亿人面临感染风险，每年报告 100 万新病例。恙虫病可以用抗生素治疗，但再次感染很常见，而且越来越多的报道称抗生素疗效下降。最近在日本军事基地训练的美国海军陆战队士兵中爆发的疫情，以及美军驻扎地阿富汗和巴基斯坦流行的丛林斑疹伤寒疫情，都与美国士兵感染这种疾病的风险相呼应。病原体是由螨传播的专性细胞内细菌东方恙虫病。尽管它构成了全球健康威胁，O. 恙虫病的研究还很严重。事实上，人们对这种病原体如何操纵真核宿主细胞功能以促进其细胞内生存知之甚少。许多细胞内细菌病原体中的一个新兴主题是它们将含有锚蛋白重复的蛋白质（Anks）易位到真核宿主细胞中。锚蛋白重复序列是自然界中最常见的蛋白质-蛋白质相互作用基序之一。其他细菌和病毒病原体的分支在不同的亚细胞位置运输，在那里它们与宿主靶蛋白相互作用并模仿或干扰具有促进病原体存活的宿主细胞功能。恙虫病基因组携带 38 个 ank 基因，我们已确定所有 38 个均在体外感染哺乳动物宿主细胞期间表达。编码 Ank4、Ank9 和 Ank12_1 的基因在感染后 1 小时诱导，Ank6 在 4 小时诱导，Ank13 在 8 小时诱导。我们假设 Ank4、Ank6、Ank9、Ank12_1 和 Ank13 对于恙虫病引起的感染很重要，并优先考虑这些蛋白质进行功能研究。编码 ank1、ank5 和 ank17 的基因也在感染过程中表达并携带卷曲螺旋结构域。卷曲螺旋结构域是许多细菌病原体效应子的标志性蛋白质-蛋白质相互作用基序，介导与宿主细胞靶标的相互作用。由于 Ank1、Ank5 和 Ank17 均携带锚蛋白重复序列和卷曲螺旋基序，因此我们将把它们纳入我们的功能研究中。在目标 1 中，我们将通过在哺乳动物宿主细胞中异位表达蛋白质并识别它们运输的亚细胞位置来评估八个感兴趣的 Anks 是否执行不同的效应器功能。为了确定它们是否与对恙虫病细胞内存活至关重要的宿主细胞因子相互作用，我们将评估异位表达的 Anks 是否竞争性抑制东方体存活。在目标 2 中，我们将使用体内共沉淀和亲和层析捕获并鉴定与 Orientia Anks 相互作用的宿主细胞靶蛋白。这项拟议的工作将填补一个相当大的知识空白，即一种尚未被研究的具有全球生物医学重要性的病原体如何促进其细胞内存活。自从恙虫病菌株中的抗生素耐药性正在增加，并且再次感染很常见，确定 Anks 的功能作用可能有助于设计针对特定 Anks 的小分子抑制剂来治疗恙虫病。