Molecular mechanisms of Trypanosoma cruzi survival using host apoptosis inhibitor

克氏锥虫是一种原生动物寄生虫，在南美洲引起查加斯病。我们证明了寄生虫感染抑制了宿主细胞中死亡受体介导的细胞凋亡。这种抑制被认为是寄生虫逃避宿主免疫反应的一种防御策略。我们澄清，寄生虫显著上调细胞FLICE样抑制蛋白(c-FLIP)，这是唯一已知的哺乳动物死亡受体信号转导的特异性抑制物。我们还发现，带有小干扰RNA的c-flip下调显著恢复了感染细胞中Fas介导的细胞凋亡。为了阐明c-flip上调的机制，我们研究了克氏毛滴虫感染细胞中c-flip表达的转录后调控。有报道称，c-FLIP是肿瘤细胞中一氧化氮(NO)参与S亚硝化反应的关键靶点。用克氏毛滴虫感染HT1080或THP-1细胞，或不加供体处理，继续培养1~3天。免疫沉淀细胞裂解产物，用抗S亚硝基半胱氨酸抗体进行免疫印迹分析。通过加入NO供体硝普钠，显著提高了FliP的亚硝化水平。在感染细胞中，在培养上清液中明显检测到亚硝化的c-FLIP。这些发现表明，内源性合成的NO是克氏毛滴虫感染的媒介。FliP的S亚硝化是NO呈递Flio抵抗FasL泛素化和蛋白酶体降解的重要机制。

Trypanosoma cruzi is a protozoan parasite that causes Chagas' disease in South America. We demonstrated that the parasite infection inhibits death receptor-mediated apoptosis in host cells. This inhibition is thought to be a defense strategy for the parasite to escape from host immune responses. We clarified that the parasite dramatically up-regulates cellular FLICE-like inhibitory protein (c-FLIP), the only known mammalian inhibitor specific for death receptor signaling. We also found that c-FLIP knock-down with a small interfering RNA significantly restores Fas-mediated apoptosis in infected cells. To elucidate the mechanisms of c-FLIP up-regulation, we examined posttranscriptional regulation of c-FLIP expression in T. cruzi infected cells. It has been reported that c-FLIP is a key target for S-nitrosylation by nitric oxide (NO) in cancer cells. HT1080 or THP-1 cells were infected with T. cruzi or treated with NO donors, and further cultured for 1-3 days. Cell lysates were immunoprecipitated and analyzed by Western blot using anti-S-nitrosocysteine antibody in control and infected cells. By the addition of the NO donors, sodium nitroprusside, the nitrosylated level of FLIP was strongly increased. In infected cells nitrosylated c-FLIP was clearly detected in supernatant. These findings suggest that endogenously produced NO synthesized by NO synthase is a mediator of T. cruzi infection. S-nitrosylation of FLIP is an important mechanism utilized by NO rendering FLIP resistant to ubiquitination and proteasomal degradation by FasL.