Functional characterization of an Orientia tsutsugamushi nucleomodulin

丛林斑疹伤寒是一种被忽视的潜在致命人畜共患病，每年在亚太地区有100万人患病。 最近该区域以外的病例报告表明，丛林斑疹伤寒是一种全球健康威胁。致病因素是 恙虫病东方体(OT)是一种通过蠕形螨传播的细胞内专性细菌，它入侵白细胞和 内皮细胞。OT对宿主细胞基因表达的失调是有利的，但负责的细菌因素 都是未知的。锚蛋白重复序列包含蛋白(ANK)是细胞内的微生物效应物，可以结合特定的 宿主蛋白来增选或颠覆真核功能。OT拥有最大的Ank曲目之一 微生物。大多数OT ANK带有第二个真核样基序，称为F-box，它与SCF1 E3相互作用 泛素连接酶复合体，可使蛋白质泛素化。大多数OT ANK的作用和功能关联 F-box对OT的致病作用尚不清楚。我们发现OT Ank13强毒进入宿主细胞 原子核。这一现象不涉及典型的进口素驱动的核进口途径。相反，它 在其第四和第五个锚定蛋白重复序列(AR)的第13位需要异亮氨酸。这一观察结果表明 Ank13使用依赖RanGDP的雷达核进口途径，显然，该途径被 真核细胞中含有AR的转录调控因子。酵母双杂交分析鉴定出以下宿主 作为Ank13相互作用伙伴的蛋白质：SCF1复合体成员Skp1和CUL1，以及HMG20A，a 转录因子参与受多泛素化负向调节的染色体重塑。因此， 到目前为止，Ank13-Skp1和Ank13-CUL1的相互作用已经通过免疫共沉淀得到证实。我们的数据 支持Ank13是一种核调节蛋白，有助于OT调节宿主细胞基因的能力 表情。在两个具体目标中，我们将评估以下假设：(1)Ank13使用雷达进入 核和(2)Ank13与HMG20A或其他蛋白质相互作用并使其泛素化以扰乱宿主细胞基因 以F-box依赖的方式表达有利于OT感染。尽管全球生物医学意义重大，但 斑疹伤寒是推动疾病进展的宿主-病原体相互作用，人们对此知之甚少。 核调节蛋白生物学是细菌发病机制的一个新兴领域，有待进一步研究。 实现提出的目标将对这些领域产生广泛和强大的影响。

Scrub typhus is a neglected potentially fatal zoonosis that afflicts 1 million persons each year in the Asia Pacific. Recent case reports outside this region signify scrub typhus as a global health threat. The causative agent is Orientia tsutsugamushi (Ot), a mite-transmitted obligate intracellular bacterium that invades leukocytes and endothelial cells. Ot dysregulates host cell gene expression to its advantage, but the responsible bacterial factors are unknown. Ankyrin repeat-containing proteins (Anks) are intracellular microbial effectors that bind specific host proteins to co-opt or subvert eukaryotic functions. Ot carries one of the largest Ank repertories of any microbe. Most Ot Anks carry a second eukaryotic-like motif called the F-box, which interacts with the SCF1 E3 ubiquitin ligase complex that polyubiquitinates proteins. The roles of most Ot Anks and the functional relevance of the F-box to Ot pathobiology are unknown. We discovered that Ot Ank13 robusty traffics into the host cell nucleus. This phenomenon does not involve the canonical importin-driven nuclear import pathway. Rather, it requires isoleucines at the 13th position of its fourth and fifth ankyrin repeats (ARs). This observation suggests that Ank13 uses the RanGDP-dependent RaDAR nuclear import pathway, which, conspicuously, is used by eukaryotic AR-containing transcriptional regulators. Yeast two-hybrid analysis identified the following host proteins as putative Ank13 interacting partners: SCF1 complex members SKP1 and CUL1, and HMG20A, a transcription factor involved in chromosome remodeling that is negatively regulated by polyubiquitination. Thus far, the Ank13-SKP1 and Ank13-CUL1 interactions have been verified by co-immunoprecipitation. Our data support the premise that Ank13 is a nucleomodulin that contributes to the ability of Ot to modulate host cell gene expression. In two specific aims, we will evaluate the hypotheses that (1) Ank13 uses RaDAR to enter the nucleus and (2) Ank13 interacts with and polyubiquitinates HMG20A or other proteins to disrupt host cell gene expression in an F-box-dependent manner to benefit Ot infection. Despite the global biomedical significance of scrub typhus, the host-pathogen interactions that drive disease progression are poorly understood. Nucleomodulin biology is a bourgeoning area of bacterial pathogenesis that is ripe for further exploration. Accomplishing the proposed aims will have a broad and powerful impact on these fields.