Defining the pathobiological roles of Orientia tsutsugamushi Ank proteins

恙虫病东方体是一种专性细胞内细菌，是恙虫病的病原体， 死亡率很高的疾病，每年导致大约100万新病例。丛林斑疹伤寒长期以来 被认为是亚太地区的地方病，但与旅行无关的病例和病原体的检测 在多个大陆的流行表明这种疾病是一种新出现的全球健康威胁。锚蛋白重复序列是 自然界中最常见的蛋白质相互作用基序。O.恙虫病颠覆先天和适应 免疫力使用其含有锚蛋白重复序列的效应子（Anks）的干部，其中许多还携带PRANC/F- 框基序，其与SCF泛素连接酶共选择。Ank 1和Ank 6抑制NF-κB在细胞核中的积聚， 以锚蛋白重复序列和PANC/F-box依赖性方式的NF-κ B依赖性基因表达。两个效应器 结合宿主细胞NF-κB抑制剂p105，并阻止其TNFα诱导的降解。显然，Ank 1和 Ank 6泛素化Crybg 3，影响p105稳定性的宿主激酶。进一步筛查显示，13名Anks 拮抗NF-κB，其中一些结合p105，而另一些不结合。因此，多个O.恙虫病 通过不同的重叠机制抑制NF-κB。Ank 5促进泛素化和蛋白酶体降解 NLRC 5是MHC-I基因表达的反式激活因子，可有效降低MHC-I水平。我们的研究结果表明 Ank免疫调节使O.恙虫病赢得了，至少在最初，它与宿主细胞的拔河比赛， 建立感染。然而，Anks调节NF-κB、NLRC 5和其他靶点的详细机制尚不清楚。 仍然知之甚少。事实上，虽然我们的数据建立了O。恙虫病利用Anks来改变宿主 细胞泛素化，这种策略的程度，修饰靶点的身份，以及特定的功能结果， 定义不好。由于细菌的遗传难治性，Ank介导的体内毒力尚未被证实。 追求。最后，几个额外的Anks靶向未知的宿主途径，也可能影响O。 恙虫病病理学为了填补这些知识空白，我们建议（1）破译机制， O.恙虫病Anks抑制NF-κB，并将采用一种新的体内筛选作为该方法的一部分;（2）解剖 Ank 5如何促进NLRC 5降解以阻断MHC-I表达;以及（3）鉴定新的宿主细胞途径， Anks调节的泛素变化。我们的研究成果将定义新的和以前的 细胞内病原体调节宿主免疫力的未知机制。总的来说，这项工作将 广泛而强大的影响。

Orientia tsutsugamushi is an obligate intracellular bacterium and causative agent of scrub typhus, a severe illness with a high fatality rate that results in approximately 1 million new cases each year. Scrub typhus has long been considered endemic to the Asia-Pacific region, but non-travel related cases and detection of the pathogen on multiple continents signify the disease as an emerging global health threat. The ankyrin repeat is one of the most-common protein-protein interaction motifs in nature. O. tsutsugamushi subverts innate and adaptive immunity using its cadre of ankyrin repeat-containing effectors (Anks), many of which also carry a PRANC/F- box motif that co-opts SCF ubiquitin ligases. Ank1 and Ank6 inhibit NF-κB accumulation in the nucleus to impair NF-κB-dependent gene expression in an ankyrin repeat- and PRANC/F-box-dependent manner. Both effectors bind the host cell NF-κB inhibitor, p105, and prevent its TNFα-induced degradation. Conspicuously, Ank1 and Ank6 ubiquitinate Crybg3, a host kinase that influences p105 stability. Further screening revealed that 13 Anks antagonize NF-κB, some of which bind p105 and others of which do not. Thus, multiple O. tsutsugamushi Anks inhibit NF-κB by distinct, overlapping mechanisms. Ank5 promotes ubiquitination and proteasomal degradation of NLRC5, a transactivator of MHC-I gene expression, to potently reduce MHC-I levels. Our findings indicate that Ank immunomodulation enables O. tsutsugamushi to win, at least initially, its tug-of-war with host cells to establish infection. Yet, the detailed mechanisms by which Anks modulate NF-κB, NLRC5, and other targets remain poorly understood. Indeed, while our data establish that O. tsutsugamushi uses Anks to alter the host cell ubiquitome, the extent of this strategy, identity of modified targets, and specific functional outcomes are poorly defined. Due to the bacterium’s genetic intractability, Ank-mediated virulence in vivo has not been pursued. Finally, several additional Anks target unknown host pathways that also likely influence O. tsutsugamushi pathobiology. To fill these knowledge gaps, we propose to (1) decipher the mechanisms by which O. tsutsugamushi Anks inhibit NF-κB and will employ a novel in vivo screen as part of this approach; (2) dissect how Ank5 promotes NLRC5 degradation to block MHC-I expression; and (3) identify new host cell pathways and ubiquitome changes that Anks modulate. The culmination of our studies will define novel and previously unsurmised mechanisms by which intracellular pathogens modulate host immunity. Overall, this work will have a broad and powerful impact.