Modeling Entamoeba histolytica host-parasite interactions

溶组织内阿米巴宿主-寄生虫相互作用建模

• 批准号: RGPIN-2019-04136
• 负责人: Chadee, Khrisendath
• 金额: $ 4.01万
• 依托单位: University of Calgary
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=714646
• 关键词: Modeling; Entamoeba; histolytica; host; parasite

Background: Entamoeba histolytica (Eh) is a protozoan parasite that harmlessly colonizes the colonic mucus layer and feed on bacteria in 99% of infection. However when mucosal barriers are weakened, Eh invades the colon and triggers a robust host pro-inflammatory response with high levels of TNF- and IL-1 in innate host defense. Direct contact of live Eh with immune cells elicits a response that is different in both magnitude and quality compared to responses elicited by released Eh components. Only Eh in direct contact with macrophages activate the NLRP3 inflammasome in a Gal-lectin and Eh cysteine protease 5 (EhCP-A5) dependent manner. Gal-lectin forms a bridge between Eh and macrophages allowing engagement of Eh surface cysteine proteinase EhCP-A5 RGD motif to ligate 51 integrin to trigger ATP release for the activation of caspase-1 dependent NLRP3 inflammasome. Eh also induce caspase-4 activation that enhanced the cleavage of caspase-1 CARD domains and both caspase acted together to cleave gasdermin D (GSDMD), liberating the N-terminal p30 pore-forming fragment for the release of bioactive IL-1 without significant cell death. Although the NLRP3 inflammasome and caspase-1/4 activation cause elevated levels of IL-1 release we still do not understand how large-scale pro-inflammatory responses are initiated upon Eh contact with macrophage in innate host defense against invasion. This crucial gap in our knowledge is key to understanding how Eh remains a harmless colonizer and/or invader in the gut where it is controlled by innate host responses. Research Aims and Approach: Over the next five years I will address three complementary aims: (1) To determine the subunits of caspase-1/4 that interact with one another to increase caspase-1 IL-1 bioactivity. This will be done by identifying the subunits of caspase-1 CARD domains that are cleaved by activated caspase-1 by LC-MS/MS sequencing and by transfecting predicted caspase-1 cleavage sites with caspase-1 and IL-1 in COS7 cells followed by stimulation with Eh. (2) To investigate the mechanism of caspase-4 activation in Eh-stimulated macrophages. To do this we will first determine if caspase-4 requires a two-signal model for activation and to identify the caspase-4 intermediate forms by LC-MS/MS with enzymatic activity. (3) To quantify what role GSDMD plays in Eh-induced intestinal inflammation. This will be achieved by infecting Gsdmd+/+ and Gsdmd-/- littermates in the colon and quantifying Eh-induced pro-inflammatory responses and cell death by caspase-3 cleavage and TUNEL assay. Significance: This work will define the mechanisms of how inflammatory caspase-4/1 activation through a contact-dependent signaling synapse between Eh and host immune cells deciphers Eh invasion versus colonized Eh to distinguish the severity of infection and tune immune responses appropriately.

背景：溶组织内阿米巴（Eh）是一种寄生于结肠黏液层的原生动物，99%的感染以细菌为食。然而，当粘膜屏障被削弱时，Eh侵入结肠并在先天宿主防御中触发高水平的TNF-和IL-1的强大的宿主促炎反应。活的Eh与免疫细胞直接接触所引起的反应，与释放的Eh成分所引起的反应相比，在强度和质量上都不同。只有与巨噬细胞直接接触的Eh才能以gal凝集素和Eh半胱氨酸蛋白酶5 （EhCP-A5）依赖的方式激活NLRP3炎性体。凝集素在Eh和巨噬细胞之间形成桥梁，允许Eh表面半胱氨酸蛋白酶EhCP-A5 RGD基序连接51整合素，从而触发ATP释放，激活caspase-1依赖性NLRP3炎性体。Eh还诱导caspase-4激活，增强caspase-1 CARD结构域的裂解，两种caspase共同作用裂解gasdermin D (GSDMD)，释放n端p30成孔片段，释放生物活性IL-1，而不会导致显著的细胞死亡。尽管NLRP3炎性体和caspase-1/4激活导致IL-1释放水平升高，但我们仍然不清楚Eh与巨噬细胞接触后如何在先天宿主防御入侵中启动大规模的促炎反应。这一重要的知识缺口是理解Eh如何在肠道中保持无害的殖民者和/或入侵者的关键，在肠道中它受先天宿主反应的控制。