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: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=761051
• 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-a and IL-1ß 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 a5ß1 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-1ß without significant cell death. Although the NLRP3 inflammasome and caspase-1/4 activation cause elevated levels of IL-1ß 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-1ß 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-1ß 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.

背景资料：溶组织内阿米巴（Eh）是一种原生动物寄生虫，在99%的感染中无害地定殖在结肠粘液层并以细菌为食。然而，当粘膜屏障被削弱时，Eh侵入结肠并在先天性宿主防御中触发具有高水平的TNF-α和IL-1 β的强烈的宿主促炎反应。活的Eh与免疫细胞的直接接触引起的反应是不同的幅度和质量相比，释放的Eh组分引起的反应。只有与巨噬细胞直接接触的Eh才以Gal-凝集素和Eh半胱氨酸蛋白酶5（EhCP-A5）依赖性方式激活NLRP 3炎性体。半乳糖凝集素在Eh和巨噬细胞之间形成桥，允许Eh表面半胱氨酸蛋白酶EhCP-A5 RGD基序接合，以连接α 5 β 1整联蛋白，从而触发ATP释放，用于激活半胱天冬酶-1依赖性NLRP 3炎性体。Eh还诱导caspase-4活化，其增强caspase-1 CARD结构域的切割，并且两种caspase一起作用以切割gasdermin D（GSDMD），释放N-末端p30孔形成片段以释放生物活性IL-1 β，而没有显著的细胞死亡。尽管NLRP 3炎性小体和caspase-1/4激活导致IL-1 β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启动大规模的促炎反应，以抵御宿主的入侵。我们知识中的这一关键差距是理解Eh如何在肠道中保持无害的殖民者和/或入侵者的关键，在肠道中它由先天宿主反应控制。 研究目的和方法：在接下来的五年里，我将致力于三个互补的目标：（1）确定caspase-1/4的亚基，它们相互作用以增加caspase-1 IL-1 β的生物活性。通过LC-MS/MS测序鉴定被活化的半胱天冬酶-1切割的半胱天冬酶-1 CARD结构域的亚基，并通过在COS 7细胞中用半胱天冬酶-1和IL-1 β检测预测的半胱天冬酶-1切割位点，然后用Eh刺激来完成。(2)探讨Eh刺激的巨噬细胞中caspase-4活化的机制。为此，我们将首先确定caspase-4是否需要双信号模型进行激活，并通过LC-MS/MS鉴定具有酶活性的caspase-4中间体形式。(3)目的：探讨GSDMD在Eh诱导的肠道炎症中的作用。这将通过在结肠中感染Gsdmd+/+和Gsdmd-/-同窝仔并通过半胱天冬酶-3切割和TUNEL测定定量Eh诱导的促炎反应和细胞死亡来实现。 重要性：这项工作将定义如何通过Eh和宿主免疫细胞之间的接触依赖性信号突触激活炎症性caspase-4/1的机制，破译Eh入侵与定植Eh，以区分感染的严重程度并适当地调节免疫反应。