New molecular techniques for T. cruzi

描述（由申请人提供）：克氏锥虫（T. cruzi）在人类中引起恰加斯病（例如，美洲锥虫病）。这种感染是拉丁美洲的地方病；然而，由于来自流行地区的移民，恰加斯病在欧洲和美国都有发现。克氏锥虫感染的惊人之处在于慢性感染的发展，在急性感染后几十年才出现疾病症状。克氏锥虫的研究一直受到基因操作困难的限制。我们使用带有配体控制的不稳定结构域（ddFKBP）的修饰的pTREX载体来调节感兴趣的基因/蛋白。该载体系统可以快速、可逆地表达克氏t细胞不同生命周期阶段的蛋白质，并对蛋白质进行高效的功能分析。利用这项技术，我们发现两个有丝分裂原活化蛋白激酶（MAPK）， TcMAPK1和TcMAPK3，是T.克氏体所必需的。我们计划在我们的ddFKBPpTREX载体系统的基础上开发一个条件基因缺失系统。此外，在ddFKBP系统中，以一种受调控的方式快速过表达致死基因应该是可行的，这可以在使用相同载体构建的多个克氏T.分离株中完成，而无需对分离株进行遗传修饰。这种可诱导致死性表型克氏锥虫对致病机制的研究非常有用，可以在感染后的不同时间点消除生物体，以解剖致病机制。这些寄生虫还将促进免疫刺激的研究，并为开发针对这种感染的新疫苗战略提供数据。因此，我们提出：(1)基于我们的ddFKBPpTREX载体，利用克氏t细胞生长必需基因TcMAPK1和TcMAPK3，开发一个强大的条件敲除载体系统。该系统应该对操纵该寄生虫的其他必需基因有用；(2)我们还将为克氏锥虫创建载体系统，允许毒素基因的调节表达，当这些基因表达时，将杀死这种寄生虫。

DESCRIPTION (provided by applicant): Trypanosoma cruzi (T. cruzi) causes Chagas Disease (e.g., American Trypanosomiasis) in humans. This infection is endemic to Latin America; however, due to immigration from endemic areas, Chagas Disease is found in both Europe and the United States. What is striking about infection with T. cruzi is the development of chronic infection with disease symptoms manifesting decades after the acute infection. Research on T. cruzi has been limited by the difficulties in genetic manipulation. We used a modified pTREX vector with a ligand-controlled destabilization domain (ddFKBP) to regulate a gene/protein of interest. This vector system allows rapid and reversible protein expression and efficient functional analysis of proteins in different T. cruzi life cycle stages. Using this technique, we found that two mitogen activated protein kinases (MAPK), TcMAPK1 and TcMAPK3, are essential for T. cruzi. We plan to develop a conditional gene deletion system based on our ddFKBPpTREX vector system. In addition, quickly over-expressing a lethal gene in a regulated fashion should be feasible in the ddFKBP system and this can be done in multiple T. cruzi isolates using the same vector construct without any need to genetically modify the isolates. Such an inducible lethal phenotype T. cruzi would be very useful for pathogenesis studies allowing elimination of the organism at various time points after infection to dissect the mechanisms of disease causation. These parasites would also facilitate studies on immune stimulation and provide data for the development of new vaccine strategies for this infection. We, therefore, propose to: (1) develop a robust conditional knockout vector system using TcMAPK1 and TcMAPK3, essential genes for T. cruzi growth, based on our ddFKBPpTREX vectors. This system should be useful for the manipulation of other essential genes in this parasite; and (2) we will also create vector systems for T. cruzi that allow the regulated expression of toxin genes that will kill this parasite when these genes are expressed.