New molecular techniques for T. cruzi

描述(申请人提供)：克氏锥虫(T.ruzi)可引起查加斯病(如美国锥虫病)。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。克氏毛滴虫感染的惊人之处在于慢性感染的发展，在急性感染几十年后出现疾病症状。由于遗传操作的困难，对克氏锥虫的研究一直受到限制。我们使用带有配体控制的失稳结构域(DdFKBP)的改进的pTREX载体来调控目标基因/蛋白。该载体系统可以快速、可逆地表达蛋白质，并对克氏锥虫不同生活史阶段的蛋白质进行有效的功能分析。利用这一技术，我们发现TcMAPK1和TcMAPK3这两个丝裂原活化蛋白激酶(MAPK)是克鲁兹毛滴虫所必需的。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系统中以调控的方式快速过度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锥虫分离物中完成，而不需要对分离物进行任何遗传修改。这种可诱导致死的表型对病原学研究非常有用，可以在感染后的不同时间点消除生物体，以剖析疾病的致病机制。这些寄生虫还将促进关于免疫刺激的研究，并为开发针对这种感染的新疫苗策略提供数据。因此，我们建议：(1)利用TcMAPK1和TcMAPK3这两个克氏锥虫生长所必需的基因，在我们的ddFKBPpTREX载体的基础上，建立一个健壮的条件基因敲除载体系统。这个系统应该有助于操纵这种寄生虫中的其他基本基因；以及(2)我们还将为克氏锥虫创建载体系统，允许调节毒素基因的表达，当这些基因表达时，这些基因将杀死这种寄生虫。

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.