Multifunctional roles of an Orientia tsutsugamushi nucleomodulin

总结 恙虫病东方体是一种遗传学上难治的专性细胞内细菌，可引起恙虫病， 全球新出现的高致死率感染。疾病进展取决于细菌驱动的调节 宿主的抗菌反应提供了O.恙虫病在白细胞和内皮细胞中存活的能力 细胞细菌的机制在很大程度上是未知的，突出了我们对宿主知识的空白。 病原体相互作用影响恙虫病的结果。一个名为Anks的真核生物样效应子家族， 关键O。恙虫病毒力因子大多数由N-末端锚蛋白重复（AR）结构域组成， 与宿主靶标的蛋白质-蛋白质相互作用和募集宿主SCF E3泛素连接酶的C末端F盒 复合物以泛素化AR结合蛋白。相互作用的伙伴和细胞过程， 调制方式大多未知。我们发现O.恙虫病Ank 13是一种核调节蛋白。基因 异位表达Ank 13的细胞中的表达谱重现了O. 恙虫病感染的宿主细胞，表明Ank 13有助于病原体调节细胞增殖的能力， 在转录水平上的过程。感染和表达Ank 13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。一个酵母二- 结合免疫共沉淀杂交筛选鉴定宿主MIB 1为Ank 13结合配偶体。MIB 1是一个 典型Notch信号传导的正调节因子。在O.恙虫病感染细胞，和 这在异位表达Ank 13或具有功能性失活的F-的Ank 13突变体的细胞中表现出来 盒子这些数据表明，MIB 1的Ank 13螯合促进其自身泛素化和蛋白酶体降解。 在感染过程中降解。Notch配体在感染细胞上的表面呈递被改变并且与Notch相关 基因表达在这些细胞中处于静止状态，表明O.恙虫病损害Notch信号传导。值得注意的是， 这些相同的基因在异位表达Ank 13的细胞中显著下调。一种初步的酵母 毒性抑制筛选涉及具有参与宿主转录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节，以及非经典Notch信号传导， 由Ank 13调制。因此，Ank 13改变Notch依赖性和非依赖性转录，以操纵多个 真核生物的过程目的1将质疑的假设，O。Tsutsugamushi Ank 13推出MIB 1自动 泛素化/降解以阻碍Notch刺激的过程。作为补充办法，目标2将 全面定义宿主靶标和Ank 13在感染期间调节的细胞过程的队列。 具体而言，我们将偶联无偏酵母抑制子筛选和亲和蛋白质组学分析来鉴定宿主 Ank 13靶向的蛋白/通路，并将研究它们与O.恙虫病发病机制 总的来说，这一建议将推进我们对核调节蛋白生物学的基本理解，并定义新的核调节蛋白。 O.恙虫病，一起提供了一个强大的影响，细菌致病领域。

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.