Functional characterization of Orientia tsutsugamushi ankryin repeat proteins

描述(申请人提供)：斑疹伤寒是一种潜在的致命疾病，在亚太地区流行，那里有10亿人面临感染的风险，每年报告100万新病例。斑疹伤寒可以用抗生素治疗，但再感染很常见，抗生素疗效下降的报道越来越多。最近在日本一个军事基地训练的美国海军陆战队爆发，以及美军部署的阿富汗和巴基斯坦丛林斑疹伤寒的流行，呼应了美国士兵患这种疾病的风险。其病原菌为恙螨传播的细胞内专性细菌--恙虫病东方体。尽管它对全球健康构成了威胁，但O. 恙虫病的研究严重不足。事实上，这种病原体是如何操纵真核宿主细胞的功能以促进其细胞内生存的，人们对此知之甚少。在许多细胞内的细菌病原体中，一个新的主题是它们将含有ankryin重复序列的蛋白(ANK)转运到真核宿主细胞中。Ankryin重复序列是自然界中最常见的蛋白质相互作用基序之一。其他细菌和病毒病原体在不同的亚细胞位置运输，在那里它们与宿主目标蛋白相互作用并模仿或干扰 具有促进病原体生存的宿主细胞功能。恙虫病原虫基因组携带38个ANK基因，我们已经确定这38个基因在体外感染哺乳动物宿主细胞的过程中都有表达。编码Ank4、Ank9和Ank12_1的基因在感染后1h被诱导，Ank6在4h被诱导，Ank13在8h被诱导，我们推测Ank4、Ank6、Ank9、Ank12_1和Ank13对恙虫病原虫的感染是重要的，并优先对这些蛋白进行功能研究。编码ANK1、ank5和ank17的基因也在感染过程中表达，并携带卷曲结构域。螺旋卷曲结构域是许多细菌病原体效应器的标志性蛋白质-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同时携带Ankyrin重复序列和螺旋线圈基序，我们将把它们包括在我们的功能研究中。在目标1中，我们将评估八个感兴趣的ANK是否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到的亚细胞位置来执行不同的效应器功能。为了确定它们是否与宿主细胞因子相互作用，这些宿主细胞因子对钉螺的细胞内生存至关重要，我们将评估异位表达的ANK是否竞争性地抑制东方体的生存。在目标2中，我们将使用体内共沉淀和亲和层析来捕获和鉴定与东方体相互作用的宿主细胞靶蛋白。这项拟议的工作将填补关于全球生物医学重要性的未被充分研究的病原体如何促进其细胞内生存的相当大的知识空白。自.以来 钩端螺旋体分离株对抗生素的耐药性正在增加，再感染也很常见，明确ANK的功能作用可能有助于设计针对特定ANK的小分子抑制剂来治疗森林斑疹伤寒。

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.