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: 10571846
• 负责人: Jason A Carlyon
• 金额: $ 57.32万
• 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/10571846
• 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; Genes; Genetic; Hospitalization; 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; 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.

丛林斑疹伤寒是一种新出现的潜在致命的全球健康威胁。大约有100万新病例 每年报告。病原体是恙虫病东方体，一种专性细胞内细菌， 白细胞和内皮细胞导致血管塌陷、器官衰竭和死亡。治疗选择 有限，没有预防性疫苗。O.恙虫病作为一种病原体， 调节宿主免疫和其他途径。负责机制不明，突出表明需要 以更好地了解恙虫病宿主-病原体相互作用。锚蛋白重复序列（AR）是一种蛋白质， 蛋白质相互作用基序，普遍存在于整个自然界。O.恙虫病拥有世界上最大的 含AR的效应子（Anks）在细菌中表达，并在感染期间表达所有这些效应子，强调了它们的 对细胞内存活和毒力的重要性。大多数Orientia Anks携带一个C末端F盒基序， 宿主泛素连接酶。我们发现O.恙虫病Ank 1和Ank 6在一种免疫抑制剂中阻碍NF-κB通路， AR和F框依赖方式。两者均结合并阻止宿主NF-κB抑制剂p105的降解。Ank1 Ank 6 AR模拟EPRAP的AR，EPRAP是一种稳定p105的宿主蛋白，并且泛素化Crybg 3是一种宿主激酶 影响p105的稳定性。进一步筛选显示，共有13种Anks拮抗NF-κB，其中一些 其结合p105而其它不结合。因此，多种Ank通过不同的重叠机制抑制NF-κB。我们 发现O.恙虫病通过协调蛋白酶体降解NLRC 5降低MHC-I水平， MHC-I基因表达的反式激活因子，并将这种现象与Ank 5联系起来。Ank 1、Ank 5和Ank 6 抑制先天性和获得性免疫的特征很差。我们确定东方蚁改变了宿主细胞 泛素，但这种策略的程度，修饰的目标，和感染结果的身份是未经探索的。 最后，其他Anks靶向未知的真核途径，也可能影响O。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，并使用两个 创新的屏幕，绕过O。恙虫病遗传难治性作为我们方法的一部分（目标1）; 剖析Ank 5如何促进NLRC 5降解以阻断MHC-I表达（Aim 2）;并鉴定新的宿主细胞 Anks调节的途径和泛素化改变（Aim 3）。每一个新发现的宿主 Ank相互作用为O.探讨恙虫病的发病机制。总的来说，我们将推进基本面 了解O。恙虫病-宿主相互作用，定义了细胞内病原体 调节免疫力，确定新的恙虫病治疗靶点， 设计AR蛋白作为生物医学，具有广泛而强大的影响。