Orientia tsutsugamushi Ank-host interactions in scrub typhus pathogenesis

恙虫病东方体在恙虫病发病机制中的Ank-宿主相互作用

• 批准号: 10413474
• 负责人: Jason A Carlyon
• 金额: $ 59.1万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-15 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10413474
• 关键词: ANK1 gene; Ankyrin Repeat; Annual Reports; Antigen-Presenting Cells; Bacteria; Binding; Biological Assay; Blood Vessels; C-terminal; Case Study; Cell Nucleus; Cell physiology; Cells; Cellular biology; Cessation of life; Complex; Coupled; Cytoplasm; Data; Defense Mechanisms; Development; Disease; Dominant-Negative Mutation; Endothelial Cells; Etiology; Evolution; F Box Domain; F-Box Motifs; Gene Expression; Genetic; Hospitalization; Immune; Immune response; Immunity; Immunoprecipitation; Impairment; Infection; Invaded; Knowledge; Leukocytes; Life; Link; Lysine; Mammalian Cell; Mass Spectrum Analysis; Mediating; Microbe; Mus; Natural Immunity; Nature; Organ failure; Orientia tsutsugamushi; Outcome; Pathogenesis; Pathway interactions; Persons; Phosphotransferases; Preventive vaccine; Proteins; Role; SKP Cullin F-Box Protein Ligases; Scrub Typhus; Sum; System; Testing; Time; Toxic effect; Trans-Activators; Trees; Ubiquitin; Ubiquitination; Virulence; Virulence Factors; War; Yeasts; adaptive immunity; cell type; cohort; design; functional mimics; functional outcomes; gain of function; global health; immunoregulation; in vivo; inhibitor; innovation; insight; microbial; mimicry; multicatalytic endopeptidase complex; novel; pathogen; pathogenic bacteria; prevent; protein protein interaction; screening; success; therapeutic target; ubiquitin ligase

Scrub typhus is an emerging and potentially fatal global health threat. Approximately one million new cases are reported annually. The etiologic agent is Orientia tsutsugamushi, an obligate intracellular bacterium that infects leukocytes and endothelial cells resulting in vascular collapse, organ failure, and death. Treatment options are limited and no preventative vaccine exists. The success of O. tsutsugamushi as a pathogen lies in its ability to modulate host immunity and other pathways. The responsible mechanisms are unknown, highlighting the need for a better understanding of scrub typhus host-pathogen interactions. The ankyrin repeat (AR) is a protein- protein interaction motif that is prevalent throughout nature. O. tsutsugamushi has one of the largest arsenals of AR-containing effectors (Anks) among bacteria and expresses all of them during infection, underscoring their importance for intracellular survival and virulence. Most Orientia Anks carry a C-terminal F-box motif that co-opts host ubiquitin ligases. We discovered that O. tsutsugamushi Ank1 and Ank6 impede the NF-κB pathway in an AR- and F-box-dependent manner. Both bind and prevent the degradation of host NF-κB inhibitor, p105. Ank1 and Ank6 ARs mimic those of EPRAP, a host protein that stabilizes p105, and ubiquitinate Crybg3, a host kinase that influences p105 stability. Further screening revealed that a total of 13 Anks antagonize NF-κB, some of which bind p105 and others do not. Thus, multiple Anks inhibit NF-κB by distinct, overlapping mechanisms. We found that O. tsutsugamushi lowers MHC-I levels by orchestrating proteasomal degradation of NLRC5, a transactivator of MHC-I gene expression, and linked this phenomenon to Ank5. How Ank1, Ank5, and Ank6 inhibit innate and adaptive immunity is poorly characterized. We established that Orientia Anks alter the host cell ubiquitome, but the extent of this strategy, identity of modified targets, and infection outcomes are unexplored. Finally, other Anks target unknown eukaryotic pathways that also likely influence O. tsutsugamushi pathobiology. To fill these knowledge gaps, we will decipher the mechanisms by which Anks inhibit NF-κB and use two innovative screens that circumvent O. tsutsugamushi genetic intractability as part of our approach (Aim 1); dissect how Ank5 promotes NLRC5 degradation to block MHC-I expression (Aim 2); and identify new host cell pathways and ubiquitome changes that Anks modulate (Aim 3). The contribution of each newly discovered host- Ank interaction to O. tsutsugamushi pathogenesis will be interrogated. Overall, we will advance fundamental understanding of O. tsutsugamushi-host interactions, define novel mechanisms by which intracellular pathogens modulate immunity, identify new scrub typhus therapeutic targets, and benefit the bourgeoning concept of designed AR proteins as biomedicals to have a broad and powerful impact.

恙虫病是一种新出现的、可能致命的全球健康威胁。新增病例约一百万 每年报告一次。病原体是恙虫病东方体（Orientia tsutsugamushi），一种专性细胞内细菌，可感染 白细胞和内皮细胞导致血管塌陷、器官衰竭和死亡。治疗方案有 有限且不存在预防性疫苗。恙虫病作为病原体的成功在于它能够 调节宿主免疫和其他途径。负责机制尚不清楚，突出需要 为了更好地了解恙虫病宿主与病原体的相互作用。锚蛋白重复序列​​（AR）是一种蛋白质 自然界中普遍存在的蛋白质相互作用基序。恙虫病拥有最大的武器库之一 细菌中含有 AR 的效应子 (Anks)，并在感染过程中表达所有这些效应子，强调了它们的作用 对细胞内存活和毒力的重要性。大多数 Orientia Anks 带有 C 端 F 盒基序，可以选择 宿主泛素连接酶。我们发现恙虫病 Ank1 和 Ank6 会阻碍 NF-κB 通路 AR 和 F 盒相关的方式。两者都能结合宿主 NF-κB 抑制剂 p105 并防止其降解。安克1 Ank6 ARs 模仿 EPRAP（一种稳定 p105 的宿主蛋白）和泛素化 Crybg3（一种宿主激酶） 影响 p105 的稳定性。进一步筛选发现，共有 13 个 Anks 拮抗 NF-κB，其中一些 其中结合 p105 而其他则不结合。因此，多个 Anks 通过不同的、重叠的机制抑制 NF-κB。我们 发现恙虫病通过协调 NLRC5 的蛋白酶体降解来降低 MHC-I 水平，NLRC5 是一种 MHC-I 基因表达的反式激活因子，并将这种现象与 Ank5 联系起来。 Ank1、Ank5 和 Ank6 如何 抑制先天性和适应性免疫的特征尚不清楚。我们确定 Orientia Anks 改变宿主细胞 泛素组，但这种策略的范围、修改靶标的身份和感染结果尚未被探索。 最后，其他 Anks 靶向未知的真核途径，这些途径也可能影响恙虫病病理学。 为了填补这些知识空白，我们将破译 Anks 抑制 NF-κB 的机制，并使用两种方法 作为我们方法的一部分，规避恙虫病遗传难易性的创新筛选（目标 1）； 剖析 Ank5 如何促进 NLRC5 降解以阻断 MHC-I 表达（目标 2）；并识别新的宿主细胞 Anks 调节的通路和泛素组变化（目标 3）。每个新发现的宿主的贡献- Ank 与恙虫病发病机制的相互作用将受到质疑。总体而言，我们将推进基础 了解恙虫病与宿主相互作用，定义细胞内病原体的新机制 调节免疫力，确定新的恙虫病治疗靶点，并有利于新兴的概念 将 AR 蛋白设计为生物医学，以产生广泛而强大的影响。