Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California Davis, Davis, California, USA.
Apicolipid Team, Institute for Advanced Biosciences, CNRS UMR5309, INSERM U1209, Université Grenoble Alpes, Batiment Jean Roget, Grenoble, France.
mBio. 2023 Apr 25;14(2):e0006023. doi: 10.1128/mbio.00060-23. Epub 2023 Mar 14.
virulence depends on its ability to evade or survive the toxoplasmacidal mechanisms induced by interferon gamma (IFNγ). While many genes involved in the evasion of the murine IFNγ response have been identified, genes required to survive the human IFNγ response are largely unknown. In this study, we used a genome-wide loss-of-function screen to identify genes important for parasite fitness in IFNγ-stimulated primary human fibroblasts. We generated gene knockouts for the top six hits from the screen and confirmed their importance for parasite growth in IFNγ-stimulated human fibroblasts. Of these six genes, three have homology to GRA32, localize to dense granules, and coimmunoprecipitate with each other and GRA32, suggesting they might form a complex. Deletion of individual members of this complex leads to early parasite egress in IFNγ-stimulated cells. Thus, prevention of early egress is an important fitness determinant in IFNγ-stimulated human cells. infection causes serious complications in immunocompromised individuals and in the developing fetus. During infection, certain immune cells release a protein called interferon gamma that activates cells to destroy the parasite or inhibit its growth. While most parasites are cleared by this immune response, some can survive by blocking or evading the IFNγ-induced restrictive environment. Many genes that determine parasite survival in IFNγ-activated murine cells are known but parasite genes conferring fitness in IFNγ-activated human cells are largely unknown. Using a adapted genome-wide loss-of-function screen, we identified many genes that determine parasite fitness in IFNγ-activated human cells. The gene products of four top hits play a role in preventing early parasite egress in IFNγ-stimulated human cells. Understanding how IFNγ-stimulated human cells inhibit growth and how counteracts this, could lead to the development of novel therapeutics.
毒力取决于其逃避或存活干扰素 γ（IFNγ）诱导的弓形虫杀伤机制的能力。虽然已经鉴定出许多参与逃避鼠 IFNγ 反应的基因，但对于存活人 IFNγ 反应所需的基因知之甚少。在这项研究中，我们使用全基因组功能丧失筛选来鉴定在 IFNγ 刺激的原代人成纤维细胞中对寄生虫适应性至关重要的基因。我们生成了筛选中排名前六的基因敲除体，并证实了它们在 IFNγ 刺激的人成纤维细胞中对寄生虫生长的重要性。在这六个基因中，有三个与 GRA32 同源，定位于致密颗粒，并与彼此和 GRA32 共免疫沉淀，表明它们可能形成一个复合物。该复合物的单个成员缺失会导致寄生虫在 IFNγ 刺激的细胞中早期出芽。因此，在 IFNγ 刺激的人细胞中，阻止早期出芽是一个重要的适应性决定因素。弓形虫感染会导致免疫功能低下的个体和发育中的胎儿出现严重并发症。在感染过程中，某些免疫细胞会释放一种称为干扰素 γ 的蛋白质，该蛋白质会激活细胞以破坏寄生虫或抑制其生长。虽然大多数寄生虫可以通过这种免疫反应清除，但有些寄生虫可以通过阻断或逃避 IFNγ 诱导的限制环境而存活。许多决定寄生虫在 IFNγ 激活的鼠细胞中存活的基因是已知的，但赋予寄生虫在 IFNγ 激活的人细胞中适应性的基因在很大程度上是未知的。使用一种适应的全基因组功能丧失筛选，我们鉴定出许多决定寄生虫在 IFNγ 激活的人细胞中适应性的基因。四个顶级命中的基因产物在 IFNγ 刺激的人细胞中阻止寄生虫早期出芽中发挥作用。了解 IFNγ 刺激的人细胞如何抑制寄生虫生长以及寄生虫如何对抗这种情况，可能会导致新疗法的开发。
