弓形虫是一种细胞内寄生虫,可以激活NLRP1炎性体,导致Lewis大鼠巨噬细胞焦亡,但是潜在的机制还没有很好的理解。在这项研究中,我们进行了全基因组CRISPR筛选,并鉴定了致密颗粒蛋白GRA35,GRA42和GRA43作为介导Lewis大鼠巨噬细胞死亡的弓形虫效应子.GRA35位于寄生虫液泡膜上,它与宿主E3泛素连接酶ITCH相互作用。抑制蛋白酶体活性或ITCH敲除可防止弓形虫感染的Lewis大鼠巨噬细胞的焦亡,与“NLRP1功能退化模型”一致。\"然而,没有证据表明ITCH直接泛素化或与大鼠NLRP1相互作用。我们还发现GRA35-ITCH相互作用影响IFNγ激活的人成纤维细胞中的弓形虫适应性,可能是由于ITCH在招募泛素和寄生虫限制因子RNF213到寄生虫液泡膜中的作用。这些发现确定了宿主E3泛素连接酶ITCH在介导效应子触发免疫中的新作用,一个关键概念,涉及识别细胞内病原体和启动宿主先天免疫反应。IMPORTANCEEffector触发的免疫代表了一种先天免疫防御机制,在感知和控制细胞内病原体感染中起着至关重要的作用。Lewis大鼠的NLRP1炎性体可以检测弓形虫感染,这引发了感染的巨噬细胞的突增,并消除了寄生虫的复制生态位。本文报道的工作表明,宿主E3泛素连接酶ITCH能够识别并与位于寄生虫-宿主界面上的弓形虫效应蛋白GRA35相互作用,导致Lewis大鼠巨噬细胞NLRP1炎性体活化。此外,ITCH-GRA35相互作用有助于在IFNγ刺激的人成纤维细胞中限制弓形虫。因此,这项研究为理解宿主E3泛素连接酶介导的病原体识别和限制提供了有价值的见解。
Toxoplasma gondii is an intracellular parasite that can activate the NLRP1 inflammasome leading to macrophage pyroptosis in Lewis rats, but the underlying mechanism is not well understood. In this study, we performed a genome-wide CRISPR screen and identified the dense granule proteins GRA35, GRA42, and GRA43 as the Toxoplasma effectors mediating cell death in Lewis rat macrophages. GRA35 localizes on the parasitophorous vacuole membrane, where it interacts with the host E3 ubiquitin ligase ITCH. Inhibition of proteasome activity or ITCH knockout prevented pyroptosis in Toxoplasma-infected Lewis rat macrophages, consistent with the \"NLRP1 functional degradation model.\" However, there was no evidence that ITCH directly ubiquitinates or interacts with rat NLRP1. We also found that GRA35-ITCH interaction affected Toxoplasma fitness in IFNγ-activated human fibroblasts, likely due to ITCH\'s role in recruiting ubiquitin and the parasite-restriction factor RNF213 to the parasitophorous vacuole membrane. These findings identify a new role of host E3 ubiquitin ligase ITCH in mediating effector-triggered immunity, a critical concept that involves recognizing intracellular pathogens and initiating host innate immune responses.IMPORTANCEEffector-triggered immunity represents an innate immune defense mechanism that plays a crucial role in sensing and controlling intracellular pathogen infection. The NLRP1 inflammasome in the Lewis rats can detect Toxoplasma infection, which triggers proptosis in infected macrophages and eliminates the parasite\'s replication niche. The work reported here revealed that host E3 ubiquitin ligase ITCH is able to recognize and interact with Toxoplasma effector protein GRA35 localized on the parasite-host interface, leading to NLRP1 inflammasome activation in Lewis rat macrophages. Furthermore, ITCH-GRA35 interaction contributes to the restriction of Toxoplasma in human fibroblasts stimulated by IFNγ. Thus, this research provides valuable insights into understanding pathogen recognition and restriction mediated by host E3 ubiquitin ligase.