弓形虫是一种重要的细胞内原生动物，需要利用宿主的营养才能成功寄生。我们之前报道过利用慢病毒 CRISPR-Cas9-单引导 RNA (sgRNA) 文库筛选出 Casitas B 系淋巴瘤-b (Cbl-b) 作为弓形虫的宿主依赖性因子。此外，本研究还探讨了弓形虫感染所需的Cbl-b的详细机制。发现Cbl-b敲低细胞系中弓形虫的增殖受到显着抑制，并且随着弓形虫感染时间的延长，Cbl-b表达水平升高，而弓形虫感染组中MyD88水平显着降低。 Toll 样受体 (TLR)/MyD88 是一种针对病原体感染的保守的先天细胞免疫信号通路。在荧光共振能量转移和免疫共沉淀实验中发现 Cbl-b 与 MyD88 相互作用并介导 MyD88 泛素化。然后构建Cbl-b敲除（KO）C57BL/6J谱系，并与野生型（WT）小鼠一起用等量的ME49株弓形虫速殖子感染。感染后13天，WT组小鼠全部死亡，而Cbl-b KO组80%的小鼠仍然存活，直至实验结束。 Cbl-b KO 小鼠肝脏、肺和脑部的寄生虫负荷显着低于 WT 小鼠（P < 0.05）。 Cbl-b KO感染小鼠B细胞比例较高，巨噬细胞比例较低，血清中干扰素γ和白细胞介素6水平高于WT感染小鼠；差异显着（P<0.05）。此外，当宿主细胞被弓形虫感染时，发现宿主的 Cbl-b 与 MyD88 相互作用，使 MyD88 泛素化，进行核糖体依赖性降解。因此，宿主通过 TLR/MyD88 途径对抗弓形虫的先天免疫受到 Cbl-b 的负调控。重要性这是首次报道人类 E3 泛素连接酶 Casitas B 系淋巴瘤原癌基因 B (Cbl-b) ，作为细胞内原生动物弓形虫的宿主依赖性因子，以及弓形虫感染如何通过 Cbl-b 介导的 MyD88 降解抑制 TLR/MyD88 先天免疫途径的机制。这一发现对于了解宿主细胞对弓形虫感染的免疫具有重要贡献。

Toxoplasma gondii is an important intracellular protozoan, which needs to exploit host nutrition for successful parasitism. We previously reported that Casitas B-lineage lymphoma-b (Cbl-b) was screened as a host dependency factor of T. gondii by using lentiviral CRISPR-Cas9-single guide RNA (sgRNA) libraries. Furthermore, the detailed mechanism of Cbl-b being required by T. gondii infection was explored in this study. The proliferation of T. gondii was found to be significantly inhibited in Cbl-b knockdown cell lines, and the Cbl-b expression level increased with prolonged T. gondii infection, while the MyD88 level was significantly decreased in the T. gondii infection group. Toll-like receptor (TLR)/MyD88 is a conserved innate cellular immune signaling pathway against pathogens infection. Cbl-b was found to interact with MyD88 and mediate MyD88 ubiquitination in the fluorescence resonance energy transfer and co-immunoprecipitation experiments. A Cbl-b knockout (KO) C57BL/6J lineage was then constructed and, together with the wild-type (WT) mice, was infected with the same amount of T. gondii tachyzoites of ME49 strain. At 13 days post infection, all the mice in the WT group died, while 80% of the mice in the Cbl-b KO group still survived, even to the end of the experiment. The parasitic burden in the liver, lung, and brain of the Cbl-b KO mice was significantly lower than that of the WT mice (P < 0.05). In Cbl-b KO infected mice, the percentage of B cells was higher, whereas that of macrophages was lower, and the interferon-γ and interleukin-6 levels in the serum were higher than that in the WT infected mice; the difference was significant (P < 0.05). Furthermore, when host cells were infected by T. gondii, host's Cbl-b was found to interact with MyD88 to ubiquitinate MyD88 for ribosome-dependent degradation. Therefore, the host's innate immunity against T. gondii through the TLR/MyD88 pathway was negatively regulated by Cbl-b.IMPORTANCEThis is the first report that a human E3 ubiquitin ligase, Casitas B-lineage lymphoma proto-oncogene B (Cbl-b), functions as a host dependency factor for the intracellular protozoan Toxoplasma gondii and the mechanism for how T. gondii infection inhibits the TLR/MyD88 innate immunity pathway through MyD88 degradation mediated by Cbl-b. This finding is an impactful contribution for understanding the host cell immunity against T. gondii infection.