弓形虫ROP18结合宿主NMI抑制IFN‐γ依赖抗感染机制的研究

Toxoplasma gondii causes serious diseases and even death in immunosuppressed patients‘ and fetal infection. The parasite achieves immune escape after infection of murine cells through phosphorylation of Immune Related GTPases by its virulence factor ROP18，while what induces immune escape in human cells remains unclear. In previous research, we found that ROP18 could bind with human N-myc and Stat1 Interactor (NMI) and phosphorylate NMI, resulted in the decrease of transcription level and protein content of NMI in the cytoplasm, also the decrease of NMI content in the nuclear. We proposed that ROP18 bound to NMI after its secretion into human cells, which resulted in decreased protein level or inhibited nuclear transportation of NMI. Consequently, the transcriptional activity of STAT1 was inhibited, which effectively inhibited the IFN-gamma dependent cellular immunity. This research intended to analyze whether ROP18 inhibited human cellular immunity after T. gondii infection through decreasing the transcription level or protein content of NMI in the cytoplasm, occupying the binding site or blocking the binding of NMI with STAT1 after it combined with NMI, or through the NMI dependent phosphorylation-ubiquitination-proteasome degradation, all of which would reduce the level of NMI combined with STAT1 in the nuclear, thereby reduce the ability of STAT1 to recruit CBP and eventually its transcriptional activity. It is a novel perspective to study the mechanism of inhibiting IFN-gamma mediated cellular immunity after Toxoplasma gondii infection in human cells based on the interaction of ROP18 with human NMI.

弓形虫在免疫抑制病人及胎儿感染中引起严重疾病甚至死亡，感染鼠细胞后通过TgROP18使宿主细胞IRGs磷酸化来实现免疫逃避，感染人细胞后的免疫逃避机制还不明了。前期我们发现ROP18可以结合人的NMI并使其磷酸化，致其转录水平及蛋白水平降低，细胞核中NMI的水平也降低。我们推断，ROP18分泌到人细胞内后与NMI结合，致其表达量下降或抑制其核转运，致使STAT1的转录活性降低，从而有效地抑制IFNγ依赖的细胞免疫。本课题拟分析ROP18是否通过降低NMI的转录水平、蛋白水平，或与NMI结合后占据或阻挡NMI与STAT1的结合位点，或使NMI发生依赖于磷酸化-泛素化-蛋白酶体的降解，致使与STAT1结合的NMI减少，进而降低STAT1招募CBP的能力，使其转录活性降低而抑制细胞免疫。本项目从ROP18与人NMI的互作出发来研究弓形虫感染后抑制IFNγ介导的细胞免疫的机制，具有理论创新的意义。

TgROP18是弓形虫在入侵宿主细胞的过程中分泌到宿主细胞内的一个关键毒力因子, 在小鼠体内通过靶定小鼠的免疫相关GTPase（immunity-related GTPases，IRGs）使其发生磷酸化而失活，来阻止纳虫泡膜的破裂，保护弓形虫的寄生。在人体细胞中几乎检测不到此IRGs系统，故其在人体细胞内的作用机制不明。本项目运用BiFC技术从18,414个人体细胞蛋白cDNA表达库中筛选出与TgROP18I/TgROP18II互作的人细胞蛋白，运用FRET及Co-IP对蛋白互作进行验证，并运用生物信息学的方法分析ROP18的人细胞互作蛋白质组。运用Co-IP及免疫荧光方法验证TgROP18I与人蛋白N-Myc互作因子(NMI)在弓形虫感染的情况下的相互作用, 鉴定出TgROP18I与NMI互作的必需氨基酸位点。通过对比在TgROP18I存在或不存在的条件下，NMI在总的细胞中、细胞质组分中、细胞核组分中、及染色质GAS区域中蛋白水平的差异，评价TgROP18I对NMI蛋白水平及NMI核转移的影响。通过虫株增殖能力评价实验确定TgROP18I对虫株IFN-γ抵抗力的影响。.从18,414个人蛋白cDNA 表达库中，共鉴定出了492个与TgROP18I相互作用的蛋白及141个与TgROP18II相互作用的蛋白。生物信息学分析结果显示大部分的TgROP18I及TgROP18II相互作用蛋白与宿主的免疫反应及细胞凋亡相关。其中NMI、IL20RB、IL21、及UBC通过FRET及Co-IP实验被证实为TgROP18I的靶标蛋白。.在感染CEP+TgROP18I虫株的细胞中，NMI可特异地被TgROP18I靶定而聚集在纳虫泡膜上。TgROP18I的激酶结构域中的439Arg为TgROP18I与NMI互作所必需，将TgROP18I的第439Arg突变为Pru将破坏TgROP18I与NMI的结合。TgROP18I可降低细胞中NMI的蛋白水平，并影响NMI向细胞核转移及结合至GAS区域上。.在人细胞中，TgROP18通过与宿主蛋白互作，尤其是与宿主免疫及细胞凋亡相关的蛋白网络互作，使弓形虫实现免疫逃避并成功寄生。弓形虫在感染过程中，TgROP18I通过其激酶结构中的关键位点Arg-439靶定NMI，并抑制NMI向细胞核转移及与GAS区域的结合，显著干扰NMI蛋白的定位及功能。