Modeling Entamoeba histolytica host-parasite interactions

背景：溶组织内阿米巴(Eh)是一种原生动物寄生虫，在99%的感染中以细菌为食，无害地定植于结肠粘液层。然而，当粘膜屏障减弱时，Eh侵入结肠并触发强大的宿主促炎反应，在固有宿主防御中产生高水平的TNF-和IL-1。与释放的Eh组分引起的反应相比，活的Eh与免疫细胞的直接接触引起的反应在幅度和质量上都不同。只有与巨噬细胞直接接触的Eh才以半乳糖凝集素和Eh半胱氨酸蛋白酶5(EHCP-A5)依赖的方式激活NLRP3炎症体。半乳糖凝集素在Eh和巨噬细胞之间形成一座桥梁，使Eh表面半胱氨酸蛋白酶EHCP-A5RGD基序与51整合素结合，从而触发ATP释放，激活依赖caspase-1的NLRP3炎症体。Eh还可诱导caspase-4激活，促进caspase-1卡区的裂解，两个caspase共同作用裂解Gasdermin D(GSDMD)，释放N端的P30成孔片段，用于释放具有生物活性的IL-1，而不会造成明显的细胞死亡。尽管NLRP3炎症体和caspase-1/4的激活导致IL-1释放水平升高，但我们仍不清楚Eh与巨噬细胞接触时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，Eh由天生的宿主反应控制。 研究目标和方法：在接下来的五年里，我将解决三个互补的目标：(1)确定caspase-1/4的亚基相互作用，以提高caspase-1，IL-1的生物活性。这将通过LC-MS/MS测序鉴定被激活的caspase-1切割的caspase-1卡域的亚基，并通过将预测的caspase-1切割位点与caspase-1和IL-1一起导入COS7细胞，然后用Eh刺激。(2)探讨Eh刺激巨噬细胞激活caspase-4的机制。为此，我们将首先确定caspase-4是否需要双信号模型来激活，并用具有酶活性的LC-MS/MS鉴定caspase-4中间体。(3)定量研究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-/-来实现，并通过caspase-3裂解和TUNEL试验来定量Eh诱导的促炎反应和细胞死亡。 意义：这项工作将定义炎性caspase-4/1激活的机制，通过Eh和宿主免疫细胞之间的接触依赖信号突触来破译Eh的侵袭和定植的Eh，以区分感染的严重程度并适当地调节免疫反应。

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.