A Novel Method for Inducing Amastigote-To-Trypomastigote Transformation In Vitro in Trypanosoma cruzi Reveals the Importance of Inositol 1,4,5-Trisphosphate Receptor.
A Novel Method for Inducing Amastigote-To-Trypomastigote Transformation In Vitro in Trypanosoma cruzi Reveals the Importance of Inositol 1,4,5-Trisphosphate Receptor.
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DOI：
10.1371/journal.pone.0135726
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Nara T
中科院分区：
文献类型：
--
作者：
Hashimoto M;Morales J;Uemura H;Mikoshiba K;Nara T
克氏锥虫是一种寄生原生生物，导致南美锥虫病，这是流行于拉丁美洲。由于缺乏有效的药物或疫苗，而且全世界约有800万人感染了这种寄生虫，因此迫切需要关注新药的开发。T. Cruzi感染多种哺乳动物有核细胞，优先感染心肌细胞。血流中的非分裂锥鞭毛体感染宿主细胞，在那里它们被转化成具有复制能力的无鞭毛体。无鞭毛体在从宿主细胞脱落之前回复为锥鞭毛体（锥鞭毛发生）。虽然锥鞭毛体转化是寄生虫的一个重要过程，但这一过程的分子机制尚未阐明，主要是因为缺乏体外诱导锥鞭毛体形成的测定系统。在由80%RPMI-1640和20%Grace昆虫培养基组成的转化培养基中培养无鞭毛体介导它们转化为锥鞭毛体。单独的Grace昆虫培养基也诱导锥虫鞭毛体形成。此外，在胎牛血清的存在下更有效地诱导锥虫鞭毛体形成。来自体外锥鞭虫发生的锥鞭虫能够像组织培养衍生的锥鞭虫（TCT）一样有效地感染哺乳动物宿主细胞，并表达TCT的标记蛋白。利用该检测系统，我们证明了T。cruziinositol 1，4，5-trisphosphate receptor（TcIP 3R）是一种胞内钙通道，是寄生虫体内钙信号转导的关键分子，在转化过程中起着重要作用。我们的研究结果提供了一个新的工具，以确定无鞭毛体到锥鞭毛体转化的分子机制，从而导致一个新的战略，药物开发对恰加斯病。
Trypanosoma cruzi is a parasitic protist that causes Chagas disease, which is prevalent in Latin America. Because of the unavailability of an effective drug or vaccine, and because about 8 million people are infected with the parasite worldwide, the development of novel drugs demands urgent attention. T. cruzi infects a wide variety of mammalian nucleated cells, with a preference for myocardial cells. Non-dividing trypomastigotes in the bloodstream infect host cells where they are transformed into replication-capable amastigotes. The amastigotes revert to trypomastigotes (trypomastigogenesis) before being shed out of the host cells. Although trypomastigote transformation is an essential process for the parasite, the molecular mechanisms underlying this process have not yet been clarified, mainly because of the lack of an assay system to induce trypomastigogenesis in vitro. Cultivation of amastigotes in a transformation medium composed of 80% RPMI-1640 and 20% Grace’s Insect Medium mediated their transformation into trypomastigotes. Grace’s Insect Medium alone also induced trypomastigogenesis. Furthermore, trypomastigogenesis was induced more efficiently in the presence of fetal bovine serum. Trypomastigotes derived from in vitro trypomastigogenesis were able to infect mammalian host cells as efficiently as tissue-culture-derived trypomastigotes (TCT) and expressed a marker protein for TCT. Using this assay system, we demonstrated that T. cruzi inositol 1,4,5-trisphosphate receptor (TcIP3R)—an intracellular Ca2+ channel and a key molecule involved in Ca2+ signaling in the parasite—is important for the transformation process. Our findings provide a new tool to identify the molecular mechanisms of the amastigote-to-trypomastigote transformation, leading to a new strategy for drug development against Chagas disease.