Signalling in Apicomplexan Parasites Laboratory, The Francis Crick Institute, London, United Kingdom.
High-Throughput Screening Science Technology Platform, The Francis Crick Institute, London, United Kingdom.
PLoS Biol. 2023 Jul 17;21(7):e3002202. doi: 10.1371/journal.pbio.3002202. eCollection 2023 Jul.
Toxoplasma gondii secretes protein effectors to subvert the human immune system sufficiently to establish a chronic infection. Relative to murine infections, little is known about which parasite effectors disarm human immune responses. Here, we used targeted CRISPR screening to identify secreted protein effectors required for parasite survival in IFNγ-activated human cells. Independent screens were carried out using 2 Toxoplasma strains that differ in virulence in mice, leading to the identification of effectors required for survival in IFNγ-activated human cells. We identify the secreted protein GRA57 and 2 other proteins, GRA70 and GRA71, that together form a complex which enhances the ability of parasites to persist in IFNγ-activated human foreskin fibroblasts (HFFs). Components of the protein machinery required for export of Toxoplasma proteins into the host cell were also found to be important for parasite resistance to IFNγ in human cells, but these export components function independently of the identified protein complex. Host-mediated ubiquitination of the parasite vacuole has previously been associated with increased parasite clearance from human cells, but we find that vacuoles from GRA57, GRA70, and GRA71 knockout strains are surprisingly less ubiquitinated by the host cell. We hypothesise that this is likely a secondary consequence of deletion of the complex, unlinked to the IFNγ resistance mediated by these effectors.
刚地弓形虫分泌蛋白效应子，足以颠覆人体免疫系统，从而建立慢性感染。相对于鼠类感染，人们对于哪些寄生虫效应子能解除人体免疫反应知之甚少。在这里，我们使用靶向 CRISPR 筛选来鉴定寄生虫在 IFNγ 激活的人细胞中存活所必需的分泌蛋白效应子。使用 2 种在小鼠中毒力不同的刚地弓形虫株进行独立筛选，导致鉴定出在 IFNγ 激活的人细胞中存活所必需的效应子。我们鉴定出分泌蛋白 GRA57 和另外 2 种蛋白 GRA70 和 GRA71，它们共同形成一个复合物，增强了寄生虫在 IFNγ 激活的人包皮成纤维细胞（HFF）中持续存在的能力。还发现将刚地弓形虫蛋白输出到宿主细胞所需的蛋白机器的组成部分对于寄生虫在人细胞中抵抗 IFNγ 也很重要，但这些输出成分独立于鉴定出的蛋白复合物发挥作用。宿主介导的寄生虫液泡泛素化以前与从人细胞中清除寄生虫的增加有关，但我们发现 GRA57、GRA70 和 GRA71 敲除株的液泡出人意料地较少被宿主细胞泛素化。我们假设这很可能是由于该复合物缺失的二级后果，与这些效应子介导的 IFNγ 抗性无关。
