Characterizing Notch Ligand Mimic Function of the Ehrlichia TRP120 Effector in Suppression of Host Cell Apoptosis

项目摘要/摘要 人单核细胞增多性埃立克体病(HME)是一种由细胞内革兰氏阴性菌引起的人畜共患病。 细菌，查菲埃利希氏菌。自2000年以来，只报告了200例埃立克体病，但在2017年 报告的病例数量增加到1,642例。值得注意的是，报告的病例数量被低估了 由于报告不足和诊断不足，增加了100倍。43-62%的病例中HME导致患者住院 疾病的发展可能导致危及生命的后果，包括呼吸衰竭、脑膜炎、 肾功能衰竭、肝功能衰竭和凝血功能障碍。这项研究旨在揭开分子机制的面纱 查菲埃希氏菌通过病原体模拟宿主蛋白感染改变宿主免疫防御的目的 和生存。分子拟态是病原体利用的一种有据可查的生存策略。尤其是，有几个 病原体已经进化出特定的相互作用基序，模仿宿主基序来促进宿主与病原体的相互作用 用于开发宿主机器。我们实验室最近的研究表明，宿主与病原体之间的相互作用 通过串联重复蛋白(Trp)效应器TRP120发生，TRP120能够直接激活 凹槽信号。缺口激活已被证明在各种其他功能中发挥着重要作用，包括 先天免疫机制，如自噬和细胞凋亡。最近的研究表明， 宿主细胞凋亡是查菲埃菲斯利用的一种生存机制。激活Notch被证明是有帮助的 通过稳定X连锁细胞凋亡抑制蛋白的表达来抑制细胞凋亡 (XIAP)。有趣的是，我们的实验室已经证明，TRP120可以直接与Notch的ADAM17相互作用 金属蛋白酶和Notch拮抗剂FBW7。此外，TRP120与ADAM17和 Noch-1受体已被证实。使用序列同源性数据库，我们已经证明 TRP120(TRP120-TR)串联重复结构域中的一个基序与几个Notch配体的序列同源性。 此外，我们还证明了TRP120-TRR结构域是查菲埃菲斯体内Notch激活所必需的 目前已经确定了一个由35个氨基酸组成的TRP120 Notch激活基序。我们研究了基因的表达水平 以确定Notch激活是否潜在地导致XIAP的稳定性。结果 在感染后的时间点，XIAP的表达增加。在目前的研究中，这是 为了实现我们的主要研究目标，下一步，我们将使用 TRP120重组蛋白的表面等离子体共振(SPR)和原子力显微镜(AFM) 用于确定TRP120-Notch配体模拟基序和鉴定TRP120-Notch的合成肽 受体相互作用。我们将进一步使用体外研究来确定Notch激活在 查菲乳杆菌感染。在完成这个项目时，我将接受广泛的培训，这将使我的 在细胞生物学、病理学和传染病领域的知识，并将提供必要的培训 以及未来在学术界作为独立研究员取得进步所需的资源。

PROJECT SUMMARY/ABSTRACT Human monocytotropic ehrlichiosis (HME) is a tick-borne zoonosis caused by the intracellular gram-negative bacterium, Ehrlichia chaffeensis. Since 2000, only 200 cases of ehrlichiosis were reported, however in 2017 the number of reported cases has increased to 1,642. Notably, the number of reported cases is underestimated by 100-fold due to underreporting and underdiagnosing. HME results in patient hospitalization in 43–62% of cases and progression of the disease can result in life threatening outcomes, including respiratory failure, meningitis, renal failure, hepatic failure, and coagulopathies. This study aims to unmask the molecular mechanisms utilized by E. chaffeensis to repurpose host immune defenses through pathogen mimicry of host proteins for infection and survival. Molecular mimicry is a well-documented survival stategy utilized by pathogens. In particular, several pathogens have evolved specific interaction motifs that mimic host motifs to facilitate host-pathogen interactions for exploitation of host machinery. Recent studies by our laboratory have shown host-pathogen interactions to occur through the tandem repeat protein (TRP) effector, TRP120, which is able to directly activate Notch signaling. Notch activation has been shown to play significant roles in various other functions, including innate immune mechanisms such as autophagy and apoptosis. Recent studies have demonstrated inhibition of host cell apoptosis as a mechanism utilized by E. chaffeensis for survival. Activation of Notch has shown to assist in inhibition of apoptosis by stabilizing expression of an anti-apoptotic protein, X-Linked Inhibitor of Apoptosis (XIAP). Interestingly, TRP120 has been shown, by our laboratory, to directly interact with ADAM17, a Notch metalloprotease, and FBW7, a Notch antagonist. Further, colocalization of TRP120 with both ADAM17 and the Notch-1 receptor has been demonstrated. Using sequence homology databases, we have demonstrated sequence homology of a motif in the tandem repeat domain of TRP120 (TRP120-TR) and several Notch ligands. Moreover, we have demonstrated the TRP120-TR domain is necessary for Notch activation during E. chaffeensis and have currently identified a 35-amino acid TRP120 Notch activation motif. We investigated expression levels of XIAP during infection to determine if Notch activation is potentially leading to stability of XIAP. Results demonstrated an increase in XIAP expression at later time points of infection. In the present study, which is the next step toward achieving our main research goal, we will perform protein-protein interaction studies using surface plasmon resonance (SPR) and atomic force microscopy (AFM) with TRP120 recombinant proteins and synthetic peptides to define the precise TRP120 Notch ligand mimic motif and characterize TRP120-Notch receptor interaction. We will further use in vitro studies to determine the functional role of Notch activation during E. chaffeensis infection. The extensive training I will receive while completing this project will advance my knowledge in the fields of cell biology, pathology and infectious disease, and will provide the necessary training and resources needed to advance as an independent researcher in academia in the future.