Endoplasmic Reticulum Stress-Sensing Mechanism Is Activated in Entamoeba histolytica upon Treatment with Nitric Oxide

The Endoplasmic Reticulum stores calcium and is a site of protein synthesis and modification. Changes in ER homeostasis lead to stress responses with an activation of the unfolded protein response (UPR). The Entamoeba histolytica endomembrane system is simple compared to those of higher eukaryotes, as a canonical ER is not observed. During amoebiasis, an infection of the human intestine and liver by E. histolytica, nitric oxide (NO) triggers an apoptotic-like event preceded by an impairment of energy production and a loss of important parasite pathogenic features. We address the question of how this ancient eukaryote responds to stress induced by immune components (i.e. NO) and whether stress leads to ER changes and subsequently to an UPR. Gene expression analysis suggested that NO triggers stress responses marked by (i) dramatic up-regulation of hsp genes although a bona fide UPR is absent; (ii) induction of DNA repair and redox gene expression and iii) up-regulation of glycolysis-related gene expression. Enzymology approaches demonstrate that NO directly inhibits glycolysis and enhance cysteine synthase activity. Using live imaging and confocal microscopy we found that NO dramatically provokes extensive ER fragmentation. ER fission in E. histolytica appears as a protective response against stress, as it has been recently proposed for neuron self-defense during neurologic disorders. Chronic ER stress is also involved in metabolic diseases including diabetes, where NO production reduces ER calcium levels and activates cell death. Our data highlighted unique cellular responses of interest to understand the mechanisms of parasite death during amoebiasis.内质网（Endoplasmic Reticulum, ER）可储存钙离子，同时是蛋白质合成与修饰的核心场所。内质网稳态失衡会触发应激反应，激活未折叠蛋白反应（unfolded protein response, UPR）。溶组织内阿米巴（Entamoeba histolytica）的内膜系统相较于高等真核生物更为简化，尚未观察到典型的内质网结构。溶组织内阿米巴可感染人体肠道与肝脏引发阿米巴病，该感染过程中，一氧化氮（nitric oxide, NO）会诱导类凋亡事件，此事件发生前会伴随能量产生受损以及寄生虫重要致病特征的丧失。本研究旨在探究这一古老真核生物如何响应免疫组分（如NO）诱导的应激，以及应激是否会引发内质网结构改变并进而激活未折叠蛋白反应。基因表达分析结果显示，NO可触发具有以下特征的应激反应：其一，尽管未检测到典型的未折叠蛋白反应，热休克蛋白基因（hsp genes）仍出现显著上调；其二，DNA修复与氧化还原相关基因的表达被诱导激活；其三，糖酵解相关基因的表达水平上升。酶学实验证实，NO可直接抑制糖酵解过程，并增强半胱氨酸合酶的活性。通过活细胞成像与共聚焦显微镜观察，我们发现NO可显著诱导广泛的内质网碎片化。正如近期针对神经系统疾病中神经元自我防御机制的研究提出的观点，溶组织内阿米巴的内质网分裂似乎是一种应激保护性响应。慢性内质网应激同样与包括糖尿病在内的代谢性疾病密切相关，而NO的产生可降低内质网内钙离子水平并激活细胞死亡程序。本研究的数据揭示了溶组织内阿米巴独特的细胞应激响应机制，有助于深入解析阿米巴病过程中寄生虫死亡的分子机制。