Functional characterization of Orientia tsutsugamushi ankryin repeat proteins

描述（由申请人提供）：恙虫病是一种潜在的致命疾病，在亚太地区流行，有10亿人面临感染风险，每年报告100万例新病例。恙虫病可以用抗生素治疗，但再感染很常见，越来越多的报道称抗生素的疗效下降。最近在日本一个军事基地训练的美国海军陆战队爆发疫情，以及在部署美军的阿富汗和巴基斯坦流行的丛林斑疹伤寒，都反映了美国士兵感染这种疾病的风险。病原体是恙螨传播的专性细胞内细菌，恙虫病东方体。尽管它对全球健康构成威胁，O。 对恙虫病的研究严重不足。事实上，这种病原体如何操纵真核宿主细胞的功能，以促进其细胞内的生存知之甚少。许多细胞内细菌病原体中的一个新兴主题是它们将含锚蛋白重复序列的蛋白（Anks）转运到真核宿主细胞中。锚蛋白重复序列是自然界中最常见的蛋白质相互作用基序之一。其他细菌和病毒病原体的锚在不同的亚细胞位置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。手术恙虫病基因组携带38个ANK基因，我们已经确定所有38个ANK基因在体外感染哺乳动物宿主细胞时表达。在感染后1小时诱导编码Ank 4、Ank 9和Ank12_1的基因，在4小时诱导编码Ank 6的基因，并且在8小时诱导编码Ank 13的基因。我们假设Ank 4、Ank 6、Ank 9、Ank12_1和Ank 13对O.恙虫病建立感染，并优先考虑这些蛋白质的功能研究。编码ank 1、ank 5和ank 17的基因也在感染过程中表达，并携带卷曲螺旋结构域。卷曲螺旋结构域是介导与宿主细胞靶标相互作用的许多细菌病原体效应物的标志性蛋白质-蛋白质相互作用基序。由于Ank 1、Ank 5和Ank 17同时携带锚蛋白重复序列和卷曲螺旋基序，因此我们将其纳入功能研究。在目标1中，我们将评估八个感兴趣的Anks是否通过在哺乳动物宿主细胞中异位表达蛋白质并鉴定它们运输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对O.恙虫病细胞内生存，我们将评估是否异位表达的Anks竞争性抑制东方体生存。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Anks相互作用的宿主细胞靶蛋白。拟议的工作将填补一个相当大的知识空白，如何研究全球生物医学重要性的病原体促进其细胞内生存。以来 O.随着恙虫病分离株的增加和再感染的普遍，确定Anks的功能作用将可能有助于设计靶向特定Anks的小分子抑制剂来治疗恙虫病。

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.