Functional characterization of an Orientia tsutsugamushi nucleomodulin

恙虫病东方体核调节素的功能表征

• 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
• 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

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潜在致命的人畜共患病，每年在亚太地区影响100万人。 该地区以外的最近病例报告表明，恙虫病是一种全球性的健康威胁。病原体是 恙虫病东方体（Ot），一种螨传播的专性细胞内细菌，侵入白细胞， 内皮细胞OT失调宿主细胞基因表达，以其优势，但负责细菌因素 是未知的。含锚蛋白重复序列的蛋白（Anks）是细胞内微生物效应物， 宿主蛋白质来吸收或破坏真核生物的功能。Ot拥有最大的Ank曲目之一 微生物大多数Ot Anks携带第二个真核生物样基序，称为F盒，与SCF 1 E3相互作用。 多聚泛素化蛋白质的泛素连接酶复合物。大多数Ot Anks的角色和功能相关性 与Ot病理学的关系尚不清楚。我们发现Ot Ank 13能稳健地进入宿主细胞， 原子核这种现象不涉及典型的进口驱动的核进口途径。而是 需要在其第四和第五锚蛋白重复序列（AR）的第13位的异亮氨酸。这一观察结果表明 Ank 13使用RanGDP依赖的RaDAR核输入途径，该途径明显地被 真核生物含有AR的转录调节因子。酵母双杂交分析鉴定了以下宿主 作为假定的Ank 13相互作用伴侣的蛋白质：SCF 1复合物成员SKP 1和CUL 1，以及HMG 20 A，a 参与染色体重塑的转录因子，受多聚泛素化负调控。因此 迄今为止，Ank 13-SKP 1和Ank 13-CUL 1的相互作用已通过免疫共沉淀法得到证实。我们的数据 支持Ank 13是一种核调节蛋白，有助于Ot调节宿主细胞基因的能力的前提 表情在两个具体目标中，我们将评估以下假设：（1）Ank 13使用RaDAR进入 （2）Ank 13与HMG 20 A或其他蛋白相互作用并使其聚泛素化以破坏宿主细胞基因 以F-box依赖性方式表达以有益于Ot感染。尽管具有全球生物医学意义， 恙虫病，宿主-病原体相互作用，驱动疾病的进展知之甚少。 核调节蛋白生物学是细菌致病机制的新兴领域，有待进一步探索。 实现拟议目标将对这些领域产生广泛而强大的影响。