Essential gene discovery in the malaria parasite Plasmodium falciparum

描述（由申请人提供）：人类疟疾是全球死亡和疾病的主要原因。每年有2亿多疟疾病例，100多万人将因感染而死亡。最严重的疟疾导致 恶性疟原虫是非洲绝大多数疟疾的病原体。疟疾造成的死亡对五岁以下儿童和孕妇的影响特别大。对现有抗疟疾药物的耐药性是有效治疗疟疾的一个经常和不断出现的障碍。对恶性疟原虫复制的基本生物学过程的分子理解将为开发新的抗疟疾疗法提供必要的工具。虽然恶性疟原虫的基因组已经完全测序，但寄生虫中5，300个基因中超过一半的功能仍然未知。许多功能未知的基因与其他生物的特征基因几乎没有同源性。因此，现有的分子遗传学和生物信息学技术不能用于有效地确定寄生虫中许多基因的功能。此外，现有技术无法预测哪些基因对寄生虫的生存至关重要。我假设这些必需基因和它们编码的蛋白质将成为合理设计新的抗疟疾疗法的有吸引力的靶点。这项建议旨在 建立一种可靠的方法来鉴定疟疾血液阶段的所有必需基因 寄生虫目前还没有一个研究必需基因功能的正向遗传学系统。我建议在恶性疟原虫中建立一个急需的正向遗传系统。正向遗传分析依赖于我设计的一个强大且严格控制的诱导表达系统。该诱导系统将用于在恶性疟原虫中进行饱和转座子介导的诱变。我将应用新一代测序技术对寄生虫进行饱和诱变以确定必需基因。此外，我将使用深度测序跟踪突变的寄生虫几代，以将相对适应性效应分配给其余非必需基因的破坏。该建议的直接目标是产生恶性疟原虫必需基因的列表。这份清单，连同基因功能的初步分子特征，将成为疟疾研究界的重要资源。这些研究的长期目标和公共卫生意义是确定新的抗疟疾疗法的新靶点。这一长期目标将作为我们在恶性疟原虫寄生虫中鉴定新的必需基因的直接结果而实现。

DESCRIPTION (provided by applicant): Human malaria is a leading cause of death and disease worldwide. Each year there are more than 200 million cases of malaria and more than one million people will die as a result of their infection. The most severe forms of malaria result from infection by the Plasmodium falciparum parasite, which causes the vast majority of malaria in Africa. Deaths from malaria disproportionately affect children under five years old and pregnant women. Resistance to existing anti-malarial medications is a constant and continually emerging hurdle to the effective treatment of malaria. A molecular understanding of the fundamental biological process of P. falciparum replication will provide the necessary tools to develop new anti-malarial therapeutics. Although the genome of P. falciparum has been fully sequenced, the function of more than half of the 5,300 genes in the parasite remains unknown. Many of the genes with unknown function have little or no homology with characterized genes from other organisms. Therefore, existing molecular genetics and bioinformatics techniques cannot be used to efficiently determine the function of many of the genes in the parasite. Furthermore, existing technologies cannot predict which genes are essential for survival of the parasite. I hypothesize that these essential genes, and the proteins that they encode, will be attractive targets for the rational design of new anti-malarial therapeutics. This proposal aims to establish a robust method to identify all of the essential genes for the blood-stage of the malaria parasite. A forward-genetic system to investigate the function of essential genes does not exist currently. I propose to establish a much needed forward-genetic system in P. falciparum. The forward-genetic analysis relies upon a robust and tightly controlled inducible expression system that I have designed. This inducible system will be used to perform saturating transposon-mediated mutagenesis in P. falciparum. I will apply next- generation sequencing to parasites following saturating mutagenesis to identify essential genes. In addition, I will follow mutated parasites for several generations using deep sequencing to assign a relative fitness effect to disruption of the remaining non-essential genes. The immediate goals of this proposal are to generate a list of essential genes in P. falciparum. This list, together with a preliminary molecular characterization of gene function, will be an important resource for the malaria research community. The long-term objectives and public health implications of these studies are to identify novel targets for new anti- malarial therapeutics. This long-term goal will be achieved as a direct result of our identification of novel essential genes in P. falciparum parasites.