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

埃里希体TRP120效应子在抑制宿主细胞凋亡中的Notch配体模拟功能特征

• 批准号: 10294941
• 负责人: LaNisha Patterson
• 金额: $ 3.48万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10294941
• 关键词: Academia; Amino Acids; Apoptosis; Apoptosis Inhibitor; Apoptotic; Atomic Force Microscopy; Autophagocytosis; B-Lymphocytes; Binding; Blood Coagulation Disorders; Case Study; Caspase; Cells; Cellular biology; Cleaved cell; Communicable Diseases; Databases; Development; Disease Progression; Ehrlichia; Ehrlichia chaffeensis; Ehrlichiosis; Ensure; Epidermal Growth Factor Receptor; Etiology; FBXW7 gene; Future; Genome; Goals; Gram-Negative Bacteria; Health; Homeostasis; Hospitalization; Host Defense; Human; Immune; Immune Evasion; In Vitro; Infection; Inhibition of Apoptosis; Kidney Failure; Knock-out; Knowledge; Laboratories; Life; Ligands; Link; Liver Failure; MHC Class II Genes; Meningitis; Metalloproteases; Mitochondria; Modeling; Molecular; Molecular Mimicry; Notch Signaling Pathway; Outcome; Pathogenicity; Pathology; Pathway interactions; Patients; Play; Proteins; Recombinant Proteins; Reporting; Research; Research Personnel; Research Project Grants; Resources; Respiratory Failure; Role; Sequence Analysis; Sequence Homology; Signal Pathway; Signal Transduction; Surface Plasmon Resonance; T-Cell Development; Tandem Repeat Sequences; Testing; Therapeutic; Time; Training; improved; inhibitor/antagonist; innate immune mechanisms; macrophage; mimicry; notch protein; novel; pathogen; pathogenic bacteria; protein protein interaction; receptor; synthetic peptide; targeted treatment; tick-borne

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.

项目总结/摘要 人嗜单核细胞埃立克体病（Human monocytotropic ehrllichiosis，HME）是一种由细胞内革兰氏阴性杆菌引起的蜱传人畜共患传染病 细菌，查菲埃里希体。自2000年以来，仅报告了200例埃立克体病，但在2017年， 报告的病例数已增至1 642例。值得注意的是，报告的病例数被低估， 100-由于报告不足和诊断不足而折叠。HME导致43-62%的患者住院 疾病的进展可能导致危及生命的结果，包括呼吸衰竭、脑膜炎， 肾衰竭、肝衰竭和凝血病。这项研究旨在揭示所利用的分子机制 由E. Chaffeensis通过病原体模仿宿主蛋白质来重新利用宿主免疫防御以进行感染 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。特别是几 病原体已经进化出特异性的相互作用基序，其模仿宿主基序以促进宿主-病原体相互作用 来剥削主机我们实验室最近的研究表明，宿主-病原体相互作用， 通过串联重复蛋白（TRP）效应子TRP 120发生，TRP 120能够直接激活 陷波信号。Notch激活已被证明在各种其他功能中发挥重要作用，包括 先天免疫机制，如自噬和凋亡。最近的研究表明， 宿主细胞凋亡是E. chaffeensis生存。Notch的激活有助于 通过稳定抗凋亡蛋白X连锁细胞凋亡抑制剂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，TRP 120直接与ADAM 17相互作用，ADAM 17是一个Notch。 金属蛋白酶和FBW 7，Notch拮抗剂。此外，TRP 120与ADAM 17和TRP 120的共定位可以被认为是一种特异性的蛋白质。 Notch-1受体已被证实。使用序列同源性数据库，我们已经证明了 TRP 120的串联重复结构域中的基序（TRP 120-TR）和几种Notch配体的序列同源性。 此外，我们已经证明了TRP 120-TR结构域是Notch在E.恰菲 目前已鉴定出35个氨基酸的TRP 120 Notch激活基序。我们研究了表达水平 以确定Notch激活是否可能导致XIAP的稳定性。结果 证实在感染的较晚时间点XIAP表达增加。在本研究中， 为了实现我们的主要研究目标，下一步我们将使用 表面等离子体共振（SPR）和原子力显微镜（AFM）与TRP 120重组蛋白， 合成肽以定义精确的TRP 120 Notch配体模拟基序并表征TRP 120-Notch 受体相互作用我们将进一步使用体外研究来确定Notch激活在细胞凋亡过程中的功能作用。 E.查菲线虫感染在完成这个项目的过程中，我将接受广泛的培训， 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 以及未来作为学术界独立研究人员所需的资源。