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：Entamoeba Histolictica（EH）是一种原生动物寄生虫，无害地将结肠粘液层和以细菌为食，在99％的感染中。但是，当粘膜屏障被削弱时，EH入侵了结肠并触发强大的宿主促炎反应，而天生的宿主防御中的TNF-A和IL-1ß高水平。与免疫细胞的直接接触与免疫细胞的直接接触引起的响应在大小和质量上与释放的EH成分引起的响应相比均不同。仅在与巨噬细胞直接接触时，EH在gal骨蛋白和EH半胱氨酸蛋白酶5（EHCP-A5）依赖性方式中激活NLRP3炎症体。 gal骨蛋白在EH和巨噬细胞之间形成桥梁，从而使EH表面半胱氨酸蛋白酶EHCP-A5 RGD基序接合以将A5ß1整合素结合以触发ATP释放，以激活Caspase-1依赖性NLRP3炎症体。 EH还诱导caspase-4激活，从而增强了caspase-1卡域的清除，并且两种caspase均起作用以清除Gasdermin d（GSDMD），从而释放了N端P30孔形成片段，以释放生物活性IL-1ß而没有明显的细胞死亡。尽管NLRP3炎性体和caspase-1/4激活会导致IL-1ß释放水平升高，但我们仍然不了解在与巨噬细胞接触巨噬细胞时，在天生的宿主防御侵害中与巨噬细胞接触时会发起大规模的促炎反应。在我们的知识上，这种关键差距是理解EH如何在肠道中保持无害的殖民者和/或入侵者的关键，在该肠道中，它受到先天宿主反应的控制。研究目的和方法：在接下来的五年中，我将解决三个完整的目标：（1）确定caspase-1/4的亚基相互作用，以增加caspase-1 IL-1ß生物活性。这将通过识别通过LC-MS/MS测序通过激活的caspase-1裂解的caspase-1卡域的亚基，并通过在COS7细胞中用caspase-1和IL-1ß转染预测的caspase-1裂解位点，然后用EH刺激EH。 （2）研究caspase-4激活在EH刺激的巨噬细胞中的机理。为此，我们将首先确定caspase-4是否需要两种信号模型进行激活，并通过酶促活性来识别LC-MS/MS的caspase-4中间形式。 （3）量化GSDMD在EH诱导的肠炎中的作用。这将通过感染结肠中的GSDMD+/+和GSDMD - / - 同窝室，并通过Caspase-3裂解和TUNEL分析来量化EH诱导的促炎反应和细胞死亡。意义：这项工作将定义炎症caspase-4/1如何通过EH和宿主免疫电池之间的接触依赖性信号传导突触解释EH侵袭与殖民EH的EH的机制，以区分感染的严重程度和调整免疫免疫。