Defining the pathobiological roles of Orientia tsutsugamushi Ank proteins

确定恙虫病东方体 Ank 蛋白的病理生物学作用

• 批准号: 10455792
• 负责人: Jason A Carlyon
• 金额: $ 46.57万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-12 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10455792
• 关键词: ANK1 gene; Africa; Ankyrin Repeat; Antigen-Presenting Cells; Asia; Bacteria; Binding; Biological Assay; Cell Nucleus; Cell physiology; Cells; Cellular biology; Communities; Complex; Coupled; Cowpox; Cytoplasm; Data; Defense Mechanisms; Detection; Disease; Etiology; Event; F Box Domain; F-Box Motifs; Fatality rate; Funding; Gene Expression; Genetic; Immune; Immunity; Immunology; Immunoprecipitation; Impairment; Infection; Knowledge; Lysine; Mass Spectrum Analysis; Mediating; Microbe; Middle East; Natural Immunity; Nature; Orientia tsutsugamushi; Pathogen detection; Pathogenesis; Pathway interactions; Phosphotransferases; Proteins; Proteomics; Research; Role; SKP Cullin F-Box Protein Ligases; Scrub Typhus; South America; TNF gene; Time; Toxic effect; Trans-Activators; Ubiquitin; Ubiquitination; Virulence; Virulence Factors; War; Work; Yeasts; adaptive immunity; antimicrobial; cell type; cohort; functional outcomes; gain of function; global health; immunoregulation; in vivo; inhibitor/antagonist; innovation; microbial; mortality; multicatalytic endopeptidase complex; neglect; novel; pathogen; pathogenic bacteria; prevent; protein protein interaction; response; screening

Orientia tsutsugamushi is an obligate intracellular bacterium and causative agent of scrub typhus, a severe illness with a high fatality rate that results in approximately 1 million new cases each year. Scrub typhus has long been considered endemic to the Asia-Pacific region, but non-travel related cases and detection of the pathogen on multiple continents signify the disease as an emerging global health threat. The ankyrin repeat is one of the most-common protein-protein interaction motifs in nature. O. tsutsugamushi subverts innate and adaptive immunity using its cadre of ankyrin repeat-containing effectors (Anks), many of which also carry a PRANC/F- box motif that co-opts SCF ubiquitin ligases. Ank1 and Ank6 inhibit NF-κB accumulation in the nucleus to impair NF-κB-dependent gene expression in an ankyrin repeat- and PRANC/F-box-dependent manner. Both effectors bind the host cell NF-κB inhibitor, p105, and prevent its TNFα-induced degradation. Conspicuously, Ank1 and Ank6 ubiquitinate Crybg3, a host kinase that influences p105 stability. Further screening revealed that 13 Anks antagonize NF-κB, some of which bind p105 and others of which do not. Thus, multiple O. tsutsugamushi Anks inhibit NF-κB by distinct, overlapping mechanisms. Ank5 promotes ubiquitination and proteasomal degradation of NLRC5, a transactivator of MHC-I gene expression, to potently reduce MHC-I levels. Our findings indicate that Ank immunomodulation enables O. tsutsugamushi to win, at least initially, its tug-of-war with host cells to establish infection. Yet, the detailed mechanisms by which Anks modulate NF-κB, NLRC5, and other targets remain poorly understood. Indeed, while our data establish that O. tsutsugamushi uses Anks to alter the host cell ubiquitome, the extent of this strategy, identity of modified targets, and specific functional outcomes are poorly defined. Due to the bacterium’s genetic intractability, Ank-mediated virulence in vivo has not been pursued. Finally, several additional Anks target unknown host pathways that also likely influence O. tsutsugamushi pathobiology. To fill these knowledge gaps, we propose to (1) decipher the mechanisms by which O. tsutsugamushi Anks inhibit NF-κB and will employ a novel in vivo screen as part of this approach; (2) dissect how Ank5 promotes NLRC5 degradation to block MHC-I expression; and (3) identify new host cell pathways and ubiquitome changes that Anks modulate. The culmination of our studies will define novel and previously unsurmised mechanisms by which intracellular pathogens modulate host immunity. Overall, this work will have a broad and powerful impact.

恙虫病东方体是一种专性胞内细菌，也是严重的丛林斑疹伤寒的病原体。 这种疾病的死亡率很高，每年导致大约100万新病例。丛林斑疹伤寒已久 被认为是亚太地区的地方病，但与旅行无关的病例和病原体的检测 在多个大洲发生的这种疾病意味着这种疾病是一种新兴的全球健康威胁。Ankyrin Repeat是 自然界中最常见的蛋白质相互作用基序。恙虫病原虫颠覆先天和适应性 利用其含有锚蛋白重复序列的效应器(ANK)进行免疫，其中许多效应器还携带PRANC/F- 框基序，辅以SCF泛素连接酶。ANK1和ANK6抑制NF-κB在细胞核内的积聚 依赖于核因子-κB的基因以锚定蛋白重复和PrANC/F-box依赖的方式表达。两个效应器 结合宿主细胞的NF-κB抑制物p105，并阻止其由肿瘤坏死因子α诱导的降解。值得注意的是，ANK1和 Ank6泛素化Crbg3，一种影响p105稳定性的宿主激酶。进一步的筛查显示，13个ANK 拮抗核因子-κB，其中一些结合p105，另一些不结合。因此，多株恙虫病原虫 通过不同的、重叠的机制抑制核因子-κB。Ank5促进泛素化和蛋白酶体降解 NLRC5，一种MHC-I基因表达的反式激活因子，有效地降低MHC-I水平。我们的发现表明 Ank的免疫调节使恙虫病原虫赢得了与宿主细胞的拉锯战，至少在一开始是这样 确定感染。然而，ANK调节NF-κB、NLRC5和其他靶标的详细机制 人们对此仍然知之甚少。事实上，尽管我们的数据表明？使用ANK来改变宿主 细胞泛素组、这一策略的范围、修饰靶标的识别和特定的功能结果 定义不明确。由于细菌的遗传难治性，Ank介导的体内毒力尚未被 被追捕。最后，几个额外的ANK针对未知的宿主途径，这些途径也可能影响O。 恙虫病病原生物学。为了填补这些知识空白，我们建议(1)破译 ？ Ank5如何促进NLRC5的降解以阻断MHC-I的表达；以及(3)识别新的宿主细胞途径和 ANK调节的无所不在的变化。我们研究的结果将定义小说和以前的 细胞内病原体调节宿主免疫的未知机制。总体而言，这项工作将具有 一种广泛而强大的影响。