Orientia tsutsugamushi modulation of host cell ubiquitination machinery

描述（由申请人提供）：恙虫病是一种被忽视的疾病，威胁着亚太地区的10亿居民，每年造成100万新感染。其 死亡率可高达50%。这种疾病是第二次世界大战和越南冲突中发病的重要原因，目前威胁着在中东服役的美国士兵。病原体是恙螨传播的专性细胞内细菌恙虫病东方体，它定植于微血管内皮细胞、巨噬细胞和中性粒细胞。拟议的工作将促进对Orientia如何促进其在不同宿主细胞类型中生存的理解，这一主题长期以来一直是一个隐喻性的“黑匣子”。锚蛋白重复序列是自然界中最常见的蛋白质-蛋白质相互作用基序。许多细胞内细菌和病毒病原体将含锚蛋白重复序列的蛋白质（Anks）易位到真核宿主细胞中。Anks与靶蛋白相互作用以调节宿主细胞功能。O.恙虫病病毒编码38个Anks，其中几个在C-末端还携带另一个真核生物样基序，F-盒。我们将这些称为Ank/F-box蛋白。真核F盒结合SKP 1，这是SCF 1泛素连接酶复合物的一部分，其催化泛素转移到蛋白质上以靶向它们在蛋白酶体中降解。Ank/F-box蛋白的二分结构可以想象地使它们能够通过其Ank结构域结合蛋白质底物，并且使用其F-box基序结合SKP 1以指导SCF 1介导的底物的泛素化和随后的蛋白酶体降解。我们发现东方体在组织培养细胞中建立感染时表达的8个ank基因中有5个编码Ank/F-box蛋白。在目的1中，我们将评估我们的假设，即Ank/F-box蛋白与SKP 1相互作用，以促进SCF 1介导的宿主细胞蛋白的泛素化。我们还将鉴定由于与Ank/F-box蛋白相互作用而变得泛素化的蛋白质。鉴于其广泛的Ank库，我们假设Orientia表达特定的Anks建立感染内皮细胞，巨噬细胞和中性粒细胞。在目标2中，我们将通过鉴定病原体在3种生物学相关宿主细胞中建立感染时表达的Anks来填补一个重要的知识空白。

DESCRIPTION (provided by applicant): Scrub typhus is a neglected disease that threatens the 1 billion inhabitants of the Asia-Pacific rim and causes 1 million new infections annually. Its mortality rate can be as high as 50%. The disease was a significant cause of morbidity in World War II and the Vietnam conflict and presently threatens U.S. soldiers serving in the Middle East. The causative agent is the chigger-transmitted obligate intracellular bacterium, Orientia tsutsugamushi, which colonizes microvascular endothelial cells, macrophages, and neutrophils. The proposed work will advance understanding of how Orientia facilitates its survival in its diverse host cell types, a subject that has long-remained a metaphoric "black box". The ankyrin repeat is the most common protein-protein interaction motif in nature. Many intracellular bacterial and viral pathogens translocate ankyrin repeat-containing proteins (Anks) into eukaryotic host cells. The Anks interact with target proteins to modulate host cell functions. O. tsutsugamushi encodes 38 Anks, several of which also carry another eukaryotic-like motif, the F-box, in their C-termini. We refer to these as Ank/F-box proteins. Eukaryotic F-boxes bind SKP1, a part of the SCF1 ubiquitin ligase complex, which catalyzes the transfer of ubiquitin onto proteins to target them for degradation in the proteasome. The bipartite structure of Ank/F-box proteins conceivably enables them to bind protein substrates via their Ank domains and, using their F-box motifs, bind SKP1 to direct SCF1-mediated ubiquitination and subsequent proteasomal degradation of the substrates. We discovered that 5 of the 8 ank genes that Orientia expresses as it establishes infection in tissue culture cells encode Ank/F-box proteins. In Aim 1, we will evaluate our hypothesis that the Ank/F-box proteins interact with SKP1 to promote SCF1- mediated ubiquitination of host cell proteins. We will also identify the proteins that become ubiquitinated as a result of interacting with Ank/F-box proteins. Given its extensive Ank repertoire, we hypothesize that Orientia expresses specific Anks to establish infection in endothelial cells, macrophages, and neutrophils. In Aim 2, we will close an important knowledge gap by identifying the Anks that the pathogen expresses as it establishes infection in each of the 3 biologically relevant host cells.