Multifunctional roles of an Orientia tsutsugamushi nucleomodulin

摘要 恙虫病东方体是一种遗传上难以克服的胞内专性细菌，可引起森林斑疹伤寒，一种 全球新出现的高致死率感染。疾病的进展取决于细菌驱动的调节 宿主的抗菌反应使恙虫病原虫具有在白细胞和内皮细胞中存活的能力 细胞。细菌的致病机制在很大程度上是未知的，这突显了我们对宿主- 影响斑疹伤寒转归的病原体相互作用。一系列与真核生物类似的效应器被称为ANKS 关键的恙虫病原虫毒力因子。大多数由N-末端锚定蛋白重复(AR)结构域组成，它介导 与宿主靶标的蛋白质相互作用和C-末端F-box招募宿主SCF E3泛素连接酶 泛素化AR结合蛋白的复合体。ANK相互作用的伙伴和细胞过程 调制方式大多未知。我们发现恙虫病原虫Ank13是一种核调节蛋白。基因 异位表达Ank13的细胞中的表达谱概括了O。 感染了宿主细胞，表明Ank13参与了病原体调节细胞的能力 在转录水平上的过程。感染细胞和表达Ank13的细胞都表现出基因下调 参与免疫反应和Notch信号通路调节的其他过程。一杯酵母二号- 杂交筛选结合免疫共沉淀鉴定宿主MIB1为Ank13结合伙伴。MIB1是一种 规范陷波信号的正向调节器。感染恙虫病原虫的细胞中MIB1水平降低，并且 这在异位表达Ank13的细胞或具有功能失活的F-1的Ank13突变体中被发现 盒。这些数据表明，MIB1的Ank13封存促进了其自身泛素化和蛋白酶体 在感染过程中降解。感染细胞表面的Notch配体呈现改变，并与Notch相关 这些细胞中的基因表达处于静止状态，这表明恙虫病原虫削弱了Notch信号。值得注意的是， 这些相同的基因在异位表达Ank13的细胞中显著下调。一种初步的酵母 毒性抑制因子筛选与宿主转录相关的具有人类同源基因的酵母蛋白 调控通路，包括核糖体和细胞周期调节，以及非典型的Notch信号，如 由Ank13调制。因此，Ank13改变了Notch依赖和非依赖的转录来操纵多个 真核过程。Aim 1将质疑以下假设，即恙虫病原虫Ank13促进MIB1自身- 泛素化/降解以阻止Notch刺激的过程。作为一种补充方法，目标2将 全面定义Ank13在感染过程中调节的宿主靶点和细胞过程的队列。 具体地说，我们将结合无偏酵母抑制物筛选和亲和蛋白质组学分析来鉴定宿主。 Ank13靶向的蛋白质/通路，并将调查它们与恙虫病原虫致病机制的相关性。 总体而言，这一提议将促进我们对核调节蛋白生物学的基本理解，并定义新的 恙虫病原虫靶标的途径，共同对细菌发病机制领域提供了强大的影响。

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.