Functional characterization of an Orientia tsutsugamushi nucleomodulin

恙虫病东方体核调节素的功能表征

基本信息

批准号：
10117190
负责人：
Jason A Carlyon
金额：
$ 18.63万
依托单位：
VIRGINIA COMMONWEALTH UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-03-02 至 2023-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10117190
关键词：
Africa African Ankyrin Repeat Antibiotics Arabs Area Asia Bacteria Binding Biology CUL1 gene Case Study Cell Nucleus Cells Chiggers Chile Chromosomes Co-Immunoprecipitations Complex Country Data Dendritic Cells Disease Disease Progression Endothelial Cells Etiology Europe Event F Box Domain F-Box Motifs Foundations Funding Gene Expression Genes Genetic Transcription Geography Hela Cells Human Importins Infection Integration Host Factors Intervention Invaded Isoleucine Leukocytes Mediating Microbe Mites Molecular Nuclear Nuclear Import Orientia tsutsugamushi Pathogenesis Pathway interactions Patients Persons Polyubiquitination Populations at Risk Positioning Attribute Proteins Radar Reporting Role Scrub Typhus South America Testing Tissue-Specific Gene Expression Ubiquitination Work Yeasts antimicrobial global health macrophage member microbial mortality mutant neglect novel nuclear transport factor 2 pathogen success trafficking transcription factor transcriptome sequencing ubiquitin-protein ligase yeast two hybrid system

项目摘要

Scrub typhus is a neglected potentially fatal zoonosis that afflicts 1 million persons each year in the Asia Pacific. Recent case reports outside this region signify scrub typhus as a global health threat. The causative agent is Orientia tsutsugamushi (Ot), a mite-transmitted obligate intracellular bacterium that invades leukocytes and endothelial cells. Ot dysregulates host cell gene expression to its advantage, but the responsible bacterial factors are unknown. Ankyrin repeat-containing proteins (Anks) are intracellular microbial effectors that bind specific host proteins to co-opt or subvert eukaryotic functions. Ot carries one of the largest Ank repertories of any microbe. Most Ot Anks carry a second eukaryotic-like motif called the F-box, which interacts with the SCF1 E3 ubiquitin ligase complex that polyubiquitinates proteins. The roles of most Ot Anks and the functional relevance of the F-box to Ot pathobiology are unknown. We discovered that Ot Ank13 robusty traffics into the host cell nucleus. This phenomenon does not involve the canonical importin-driven nuclear import pathway. Rather, it requires isoleucines at the 13th position of its fourth and fifth ankyrin repeats (ARs). This observation suggests that Ank13 uses the RanGDP-dependent RaDAR nuclear import pathway, which, conspicuously, is used by eukaryotic AR-containing transcriptional regulators. Yeast two-hybrid analysis identified the following host proteins as putative Ank13 interacting partners: SCF1 complex members SKP1 and CUL1, and HMG20A, a transcription factor involved in chromosome remodeling that is negatively regulated by polyubiquitination. Thus far, the Ank13-SKP1 and Ank13-CUL1 interactions have been verified by co-immunoprecipitation. Our data support the premise that Ank13 is a nucleomodulin that contributes to the ability of Ot to modulate host cell gene expression. In two specific aims, we will evaluate the hypotheses that (1) Ank13 uses RaDAR to enter the nucleus and (2) Ank13 interacts with and polyubiquitinates HMG20A or other proteins to disrupt host cell gene expression in an F-box-dependent manner to benefit Ot infection. Despite the global biomedical significance of scrub typhus, the host-pathogen interactions that drive disease progression are poorly understood. Nucleomodulin biology is a bourgeoning area of bacterial pathogenesis that is ripe for further exploration. Accomplishing the proposed aims will have a broad and powerful impact on these fields.

恙虫病是一种被忽视的、可能致命的人畜共患病，每年影响亚太地区 100 万人。该地区以外最近的病例报告表明恙虫病是一种全球健康威胁。致病因素是恙虫病东方体 (Ot)，一种由螨虫传播的专性细胞内细菌，可侵入白细胞并内皮细胞。 Ot 调节宿主细胞基因表达对其有利，但负责任的细菌因素未知。含有锚蛋白重复序列的蛋白 (Anks) 是细胞内微生物效应子，可结合特定的宿主蛋白来选择或颠覆真核功能。 Ot 拥有最大的 Ank 曲目之一微生物。大多数 Ot Anks 携带第二个类似真核的基序，称为 F-box，它与 SCF1 E3 相互作用使蛋白质多聚泛素化的泛素连接酶复合物。大多数 Ot Anks 的角色和功能相关性 F-box 与 Ot 病理学的关系尚不清楚。我们发现 Ot Ank13 能够稳健地进入宿主细胞核。这种现象不涉及典型的导入驱动的核导入途径。相反，它在其第四个和第五个锚蛋白重复序列 (AR) 的第 13 位需要异亮氨酸。这一观察表明 Ank13 使用 RanGDP 依赖的 RaDAR 核输入途径，显然，该途径被含有 AR 的真核转录调节因子。酵母二杂交分析确定了以下宿主蛋白质作为假定的 Ank13 相互作用伙伴：SCF1 复合体成员 SKP1 和 CUL1，以及 HMG20A，参与染色体重塑的转录因子，受到多聚泛素化的负调控。因此目前，Ank13-SKP1和Ank13-CUL1的相互作用已通过免疫共沉淀得到验证。我们的数据支持 Ank13 是一种核调节蛋白，有助于 Ot 调节宿主细胞基因的能力表达。在两个具体目标中，我们将评估以下假设：(1) Ank13 使用 RaDAR 进入 (2) Ank13 与 HMG20A 或其他蛋白相互作用并使其多聚泛素化，从而破坏宿主细胞基因以 F-box 依赖性方式表达有利于 Ot 感染。尽管具有全球生物医学意义对于恙虫病，人们对导致疾病进展的宿主与病原体之间的相互作用知之甚少。核调节蛋白生物学是细菌发病机制的一个新兴领域，有待进一步探索。实现拟议的目标将对这些领域产生广泛而强大的影响。