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.
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