New molecular techniques for T. cruzi

克氏锥虫的新分子技术

• 批准号: 8581382
• 负责人: Huan Huang
• 金额: $ 23.55万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-13 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8581382
• 关键词: Achalasia; Acute; Address; African Trypanosomiasis; Area; Biology; Cardiomyopathies; Cellular biology; Chagas Disease; Chronic; Clinical; Colon; Complication; Data; Development; Disease; Dominant-Negative Mutation; Employee Strikes; Esophagus; Essential Genes; Etiology; Europe; Gene Deletion; Gene Proteins; Genes; Genetic Techniques; Genome; Growth; Heart failure; Human; Immigration; Immune; Immune response; Infection; Kinetics; Knock-out; Latin America; Lethal Genes; Life Cycle Stages; Ligands; Methods; Mitogen-Activated Protein Kinases; Molecular; Molecular Genetics; N-terminal; Organ; Organism; Parasites; Pathogenesis; Patients; Pharmaceutical Preparations; Phenotype; Play; Polymerase; Protein Analysis; RNA Interference; Regulation; Research; Role; Symptoms; System; Techniques; Tetracyclines; Time; Toxin; Transfection; Transgenic Organisms; Trypanosoma brucei brucei; Trypanosoma cruzi; United States; Vaccine Design; Vaccines; Visceral; Work; base; design; genetic manipulation; improved; insight; interest; killings; knockout gene; mortality; novel strategies; novel vaccines; pathogen; protein expression; public health relevance; vector

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.

描述（由申请人提供）：克氏锥虫（T. cruzi）引起人类恰加斯病（例如美洲锥虫病）。这种感染在拉丁美洲流行；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，并且在急性感染数十年后才出现疾病症状。对克氏锥虫的研究因基因操作的困难而受到限制。我们使用带有配体控制去稳定结构域 (ddFKBP) 的改良 pTREX 载体来调节感兴趣的基因/蛋白质。该载体系统可实现快速、可逆的蛋白质表达，并对克氏锥虫生命周期不同阶段的蛋白质进行有效的功能分析。利用这项技术，我们发现两种丝裂原激活蛋白激酶 (MAPK)，TcMAPK1 和 TcMAPK3，对于克氏锥虫至关重要。我们计划开发基于我们的 ddFKBPpTREX 载体系统的条件基因删除系统。此外，以受控方式快速过表达致死基因在 ddFKBP 系统中应该是可行的，并且这可以使用相同的载体构建体在多个克氏锥虫分离株中完成，而不需要对分离株进行遗传修饰。这种诱导致死表型克氏锥虫对于发病机制研究非常有用，可以在感染后的不同时间点消除该生物体，以剖析疾病因果机制。这些寄生虫还将促进免疫刺激研究，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) 基于我们的 ddFKBPpTREX 载体，使用 TcMAPK1 和 TcMAPK3（T. cruzi 生长的必需基因）开发强大的条件敲除载体系统。该系统对于操纵这种寄生虫中的其他必需基因应该很有用。 (2)我们还将为克氏锥虫创建载体系统，该系统允许毒素基因的表达得到调节，当这些基因表达时，这些基因将杀死这种寄生虫。