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.

Scrub Typhus是一种新兴而可能致命的全球健康威胁。大约一百万个新案件是 每年报告。病因学剂是东方tsutsugamushi，是一种强性的细胞内细菌，感染了 白细胞和内皮细胞导致血管塌陷，器官衰竭和死亡。治疗选择是 有限，没有预防疫苗。 O. tsutsugamushi作为病原体的成功在于它的能力 调节宿主免疫和其他途径。负责任的机制未知，强调了需求 为了更好地了解磨砂鼠伤寒宿主 - 病原体的相互作用。 Ankyrin重复（AR）是蛋白质 蛋白质相互作用基序在整个自然界中都是普遍的。 O. tsutsugamushi是最大的武器库之一 细菌中含AR的效应（ANK），并在感染过程中表达所有效果 对于细胞内生存和病毒的重要性。大多数Orientia Anks携带C末端F-box主题 宿主泛素连接酶。我们发现O. tsutsugamushi ank1和ank6阻碍了NF-κB途径 Ar-和F-box依赖性方式。结合并防止宿主NF-κB抑制剂的降解，p105。 ank1 和ANK6 ARS模仿EPRAP的ARS，一种稳定P105的宿主蛋白和泛素酸Crybg3（宿主激酶） 这影响了P105稳定性。进一步的筛选显示，总共有13个ANK拮抗NF-κB，其中一些 绑定p105和其他绑定。这是多个ANK通过不同的重叠机制抑制NF-κB的。我们 发现O. tsutsugamushi通过策划NLRC5的蛋白酶体降解来降低MHC-I水平 MHC-I基因表达的反式激活剂，并将这种现象与ANK5联系起来。 ANK1，ANK5和ANK6如何 抑制先天和适应性免疫学的特征很差。我们确定东方ank会改变宿主单元 泛素体，但是这种策略的程度，修改目标的身份和感染结果是出乎意料的。 最后，其他ANK的目标是未知的真核途径，这也可能影响O. tsutsugamushi病理生物学。 为了填补这些知识差距，我们将破译ANK抑制NF-κB的机制，并使用两个 作为我们方法的一部分，绕过O. tsutsugamushi遗传性的创新屏幕（AIM 1）； 剖析ANK5如何促进NLRC5降解以阻断MHC-I表达（AIM 2）；并识别新的主机单元 途径和泛素会改变ANK的调节（AIM 3）。每个新发现的主机的贡献 - 将询问与O. tsutsugamushi发病机理的ANK相互作用。总体而言，我们将提高基本 了解O. tsutsugamushi-host相互作用，定义了细胞内病原体的新机制 调节免疫力，识别新的磨砂鼠伤寒性治疗靶标，并受益于资产阶级的概念 将AR蛋白设计为生物医学，以产生广泛而强大的影响。