Molecular mechanisms of Trypanosoma cruzi survival using host apoptosis inhibitor

克氏锥虫（Trypanosoma cruzi）是一种原生动物寄生虫，在南美洲引起恰加斯病。我们证明了寄生虫感染抑制宿主细胞中死亡受体介导的凋亡。这种抑制被认为是寄生虫逃避宿主免疫反应的防御策略。我们阐明了寄生虫显著上调细胞FLICE样抑制蛋白（c-FLIP），这是唯一已知的对死亡受体信号传导特异性的哺乳动物抑制剂。我们还发现，c-FLIP敲低与小干扰RNA显着恢复Fas介导的细胞凋亡在感染的细胞。为了阐明c-FLIP上调的机制，我们检测了T.克鲁兹感染的细胞。据报道，c-FLIP是癌细胞中一氧化氮（NO）S-亚硝基化的关键靶标。用T. cruzi或用NO供体处理，并进一步培养1-3天。将细胞裂解物免疫沉淀，并在对照和感染的细胞中使用抗S-亚硝基半胱氨酸抗体通过Western印迹进行分析。通过添加NO供体硝普钠，FLIP的亚硝基化水平强烈增加。在感染的细胞中，在上清液中清楚地检测到亚硝基化的c-FLIP。这些结果表明，由NO合成酶合成的内源性NO是T.克氏感染FLIP的S-亚硝基化是NO使FLIP抵抗FasL的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.