Multifunctional roles of an Orientia tsutsugamushi nucleomodulin

恙虫病东方体核调节素的多功能作用

基本信息

批准号：
10752156
负责人：
Paige Allen
金额：
$ 6.91万
依托单位：
VIRGINIA COMMONWEALTH UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-12-10 至 2026-12-09
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10752156
关键词：
Affinity Africa African Ankyrin Repeat Antigen Presentation Asia Bacteria Binding Binding Proteins Biological Assay Biology C-terminal Cell Communication Cell Cycle Cell Nucleus Cell physiology Cell surface Cells Chile Co-Immunoprecipitations Coculture Techniques Complement Complex Country Coupled Cytoplasm Data Degradation Pathway Detection Disease Disease Progression Endothelial Cells Europe Exhibits F Box Domain Family Fatality rate Gene Expression Genes Genetic Transcription Geography Homologous Gene Human Immune response Immune signaling Immunity Impairment Infection Invaded Knowledge Leukocytes Ligands Link Mass Spectrum Analysis Mediating N-terminal Notch Signaling Pathway Orientia tsutsugamushi Outcome Pathogenesis Pathway interactions Persons Peru Phenocopy Process Proteins Proteomics Publishing Regulatory Pathway Reporting Ribosomes Role Scrub Typhus Signal Transduction South America Surface Testing Toxic effect Transcript Transcriptional Regulation Transfection United Arab Emirates Vaccines Virulence Factors Western Blotting Yeasts Zoonoses antimicrobial cohort extracellular gene repression immunoregulation interest monocyte mutant notch protein novel pathogen prevent protein protein interaction receptor recruit response screening ubiquitin-protein ligase yeast protein yeast two hybrid system

项目摘要

SUMMARY Orientia tsutsugamushi is a genetically intractable obligate intracellular bacterium that causes scrub typhus, a globally emerging infection with a high fatality rate. Disease progression depends on bacterial-driven modulation of host antimicrobial responses that affords O. tsutsugamushi the ability to survive in leukocytes and endothelial cells. The bacterial mechanisms responsible are largely unknown, highlighting a gap in our knowledge of host- pathogen interactions that influence scrub typhus outcome. A family of eukaryotic-like effectors called Anks are key O. tsutsugamushi virulence factors. Most consist of an N-terminal ankyrin repeat (AR) domain that mediates protein-protein interactions with host targets and a C-terminal F-box that recruits the host SCF E3 ubiquitin ligase complex to ubiquitinate the AR-bound proteins. The interacting partners and cellular processes that the Anks modulate are mostly unknown. We discovered that O. tsutsugamushi Ank13 is a nucleomodulin. Gene expression profiles in cells ectopically expressing Ank13 recapitulate many of those observed for O. tsutsugamushi infected host cells, indicating that Ank13 contributes to the pathogen’s ability to modulate cellular processes at the transcriptional level. Both infected and Ank13-expressing cells exhibit down-regulation of genes involved in immune responses and other processes regulated by the Notch signaling pathway. A yeast two- hybrid screen coupled with co-immunoprecipitation identified host MIB1 as an Ank13 binding partner. MIB1 is a positive regulator of canonical Notch signaling. MIB1 levels are reduced in O. tsutsugamushi infected cells, and this is phenocopied in cells ectopically expressing Ank13 or an Ank13 mutant with a functionally inactivated F- box. These data suggest that Ank13 sequestration of MIB1 promotes its auto-ubiquitination and proteasomal degradation during infection. Notch ligand surface presentation on infected cells is altered and Notch-related gene expression is quiescent in these cells, indicating that O. tsutsugamushi impairs Notch signaling. Notably, these same genes are significantly downregulated in cells ectopically expressing Ank13. A preliminary yeast toxicity suppressor screen implicated yeast proteins that have human homologs involved in host transcription regulatory pathways, including ribosome and cell cycle modulation, and non-canonical Notch signaling, as being modulated by Ank13. Thus, Ank13 alters Notch-dependent and -independent transcription to manipulate multiple eukaryotic processes. Aim 1 will interrogate the hypothesis that O. tsutsugamushi Ank13 promotes MIB1 auto- ubiquitination/degradation to impede Notch-stimulated processes. As a complementary approach, Aim 2 will comprehensively define the cohort of host targets and cellular processes that Ank13 modulates during infection. Specifically, we will couple unbiased yeast suppressor screening and affinity proteomics assays to identify host proteins/pathways targeted by Ank13 and will investigate their relevance to O. tsutsugamushi pathogenesis. Overall, this proposal will advance our fundamental understanding of nucleomodulin biology and define novel pathways targeted by O. tsutsugamushi, together providing a powerful impact to the bacterial pathogenesis field.

概括东方tsutsugamushi是一种遗传上棘手的义务细胞内细菌，会导致磨砂鼠伤寒，a 全球新兴感染，死亡率很高。疾病进展取决于细菌驱动的调节宿主抗菌反应，可为O. tsutsugamushi提供在白细胞和内皮中生存的能力细胞。负责的细菌机制在很大程度上是未知的，这突出了我们对宿主的了解的差距 - 影响磨砂鼠伤疫的病原体相互作用。一个名为ANK的真核生物样效果的家族是关键O. tsutsugamushi病毒因素。大多数由培养的N末端伸缩蛋白重复（AR）域组成蛋白质 - 蛋白质与宿主靶标和C末端F-box募集宿主SCF E3泛素连接酶复合物以泛素化AR结合的蛋白质。 ANKS的相互作用伙伴和蜂窝过程调节大多未知。我们发现O. tsutsugamushi ank13是一种核苷酸。基因通过生态表达ANK13的细胞中的表达谱概括了许多观察到的O。 Tsutsugamushi感染了宿主细胞，表明ANK13有助于病原体调节细胞的能力转录级别的过程。受感染和表达ANK13的细胞均表现出基因的下调参与了Notch信号通路调节的免疫反应和其他过程。两种酵母混合屏幕与共免疫沉淀相结合，将宿主MIB1确定为ANK13结合伙伴。 MIB1是典型凹槽信号的正调节剂。 tsutsugamushi感染细胞中的MIB1水平降低，并且这是在异位表达ANK13或具有功能失活的F-的ANK13突变体的细胞中进行的盒子。这些数据表明，MIB1的ANK13隔离促进其自动泛素化和蛋白酶体感染过程中的降解。受感染细胞上的Notch配体表面表现改变并与Notch相关这些细胞中的基因表达静止，表明O. tsutsugamushi会损害Notch信号。尤其，这些相同的基因在生态表达ANK13的细胞中显着下调。初步酵母毒性抑制剂筛选实施了与宿主转录有关的人类同源物的酵母蛋白调节途径，包括核糖体和细胞周期调制以及非典型的凹口信号传导，为由ANK13调制。那就是ANK13改变了Notch的依赖性和独立的转录，以操纵多个真核过程。 AIM 1将审问O. tsutsugamushi Ank13促进MIB1自动的假设泛素化/降解会阻碍缺口刺激的过程。作为一种完整的方法，AIM 2将全面定义了ANK13在感染过程中调节的宿主靶标和细胞过程的队列。具体而言，我们将融合公正的酵母抑制剂筛选和亲和力蛋白质组学测定，以识别宿主 ANK13靶向的蛋白质/途径将研究其与O. tsutsugamushi发病机理的相关性。总体而言，该提案将提高我们对核动脉蛋白生物学的基本理解并定义新颖 O. tsutsugamushi靶向的途径共同为细菌发病机构提供了强大的影响。