MOLECULAR STRATEGIES OF TRYPANOSOMA CRUZI IN REMODELING AND CONTROLLING THE INFECTED HOST CELLS

原生动物寄生虫克氏锥虫的细胞内持久性是恰加斯病的加重原因，涉及原生动物感染特异性抑制死亡受体介导的宿主细胞凋亡。在这里，我们证明了寄生虫显著上调细胞FLICE抑制蛋白（c-FLIP），这是唯一已知的哺乳动物死亡受体信号特异性抑制剂，在感染细胞中，通过不寻常的，短寿命蛋白的转录后稳定。我们还发现c-FLIP在克氏锥虫感染的小鼠心肌细胞中蓄积。在感染细胞中刺激死亡受体Fas诱导c-FLIP的募集，从而阻断caspase-8在最上游级联的活化。用小干扰RNA敲除c-FLIP可显著恢复fas介导的感染细胞凋亡。综上所述，我们的研究结果表明，克氏锥虫转录后上调并利用宿主c-FLIP来抑制d…更多的地球诱导信号，这一机制可能允许寄生虫在宿主细胞中持续存在。T.cruzi dihydroorotate dehydrogenase（DHOD）是重新合成嘧啶生物途径的第四个酶，定位于细胞质中，利用富马酸盐作为电子受体（富马酸还原酶活性），而其他各种真核生物的酶是线粒体膜连接的。在这里，我们报道dhod敲除的T.cruzi不表达酶蛋白，即使在嘧啶核苷存在的情况下也不能存活，嘧啶核苷是潜在的活性挽救途径的底物，这表明富马酸还原酶活性在调节细胞氧化还原平衡中起着至关重要的作用。裸鳃虫DHOD基因的克隆和系统发育分析表明，裸鳃虫具有线粒体DHOD，双鞭毛骨虫具有胞质DHOD，双鞭毛骨虫是动质体中锥虫的姐妹类群。此外，Bodo saliens，一种犬齿动物，有一个ACT/DHOD基因融合编码天冬氨酸氨基甲酰转移酶（ACT），这是新生嘧啶途径的第二种酶，和DHOD。这是首次报道这种新的基因结构。这些结果与裸藻的一个古老的共同祖先具有线粒体DHOD的观点一致，该祖先的后代存在于E.gracilis中，并且动质体（齿类和锥虫）的一个共同祖先随后通过水平基因转移获得了细胞质DHOD。因此获得的胞质DHOD基因可能有助于着丝质体谱系对厌氧的适应，并进一步有助于随后在锥虫祖先中建立寄生。讨论了ACT/DHOD基因融合的进化意义。因此，T.cruzi DHOD可能是化疗的一个潜在的好靶点。少

Intracellular persistence of the protozoan parasite, Trypanosoma cruzi, is an aggravating cause of Chagas' disease, involving that the protozoan infection specifically inhibits death receptor-mediated apoptosis of host cells. Here we demonstrate that the parasite dramatically upregulates cellular FLICE inhibitory protein(c-FLIP), the only known mammalian inhibitor specific for death receptor signaling, in infected cells by an unusual, post-transcriptional stabilization of the short-lived protein. We also show that c-FLIP is accumulated in T.cruzi- infected mouse heart muscle cells in vivo. Stimulation of death receptor Fas in infected cells induces recruitment of c-FLIP to block the procaspase-8 activation at the most upstream caspase cascade. c-FLIP knock-down with a small interfering RNA significantly restores Fas-mediated apoptosis in infected cells. Taken together, our findings indicate that T.cruzi post-transcriptionally upregulates and exploits host c-FLIP for the inhibition of d … More eath-inducing signal, a mechanism that may allow parasites to persist in host cells. T.cruzi dihydroorotate dehydrogenase(DHOD), the fourth enzyme of the de novo pyrimidine biosynthetic pathway, is localized in the cytosol and utilizes fumarate as electron acceptor (fumarate reductase activity), while the enzyme from other various eukaryotes is mitochondrial membrane-linked. Here we report that DHOD-knockout T.cruzi did not express the enzyme protein and could not survive even in the presence of pyrimidine nucleosides, substrates for the potentially active salvage pathway, suggesting a vital role of fumarate reductase activity in the regulation of cellular redox balance. Cloning and phylogenetic analysis of euglenozoan DHOD genes showed that a euglenoid Euglena gracilis had a mitochondrial DHOD and that biflagellated bodonids, a sister group of trypanosomatids within kinetoplastids, possessed the cytosolic DHOD. Further, Bodo saliens, a bodonid, had an ACT/DHOD gene fusion encoding aspartate carbamoyltransferase(ACT), the second enzyme of the de novo pyrimidine pathway, and DHOD. This is the first report of this novel gene structure. These results are consistent with suggestions that an ancient common ancestor of Euglenozoa had a mitochondrial DHOD whose descendant exists in E.gracilis, and that a common ancestor of kinetoplastids (bodonids and trypanosomatids) subsequently acquired a cytosolic DHOD by horizontal gene transfer. The cytosolic DHOD gene thus acquired may have contributed to adaptation to anaerobiosis in the kinetoplastid lineage and further contributed to the subsequent establishment of parasitism in a trypanosomatid ancestor. Evolutionary implications of the ACT/DHOD gene fusion are discussed. T.cruzi DHOD would therefore be a potentially good target for chemotherapy. Less