Relevance to NIAID mission: This application describes a 5-year training program for the development of an academic career in Pediatric Infectious Diseases, with a goal of independently directing research into parasite biology, pathogenesis, and therapy. Research design and methods: New antimalarial agents are urgently needed due to the spread of drug resistance in the pathogen Plasmodium falciparum. Understanding the fundamental biology of P. falciparum is key to these drug development efforts. An important metabolic pathway in all organisms is the biosynthesis of isoprenoid molecules, fundamental building blocks for diverse cellular compounds vital for cellular respiration, membrane structure, and signaling. We hypothesize that this pathway is also essential for the normal development and reproduction of Plasmodium falciparum. In malaria species, isoprenoids are made via the non-mevalonate (DXP) pathway. The parasite DXP pathway is biochemically distinct from the mevalonate pathway in humans, and evidence suggests this pathway is required for parasite survival. Research will focus on two enzymes of this pathway, deoxyxylulose phosphate reductoisomerase (DXR) and methylerythritol cyclodiphosphate synthase (IspF). To study the biological and biochemical characteristics of DXR and IspF, we propose a dual-pronged biochemical and genetic approach.
The specific aims i nclude the following: (1) Heterologous expression of DXR and IspF, development of in vitro assays suitable for high-throughput screening, and biochemical characterization of both enzymes;(2) Localization of DXR and IspF within the parasite by development of transgenic strains of P. falciparum that express GFP-fusions of DXR and IspF;(3) Generation of DXR and IspF disruption strains of P. falciparum, if possible, and detailed analysis of the phenotypic effects of inhibition of isoprenoid biosynthesis in both parasite disruption strains and strains treated with a small-molecule inhibitor of DXR, fosmidomycin. Relevance to public health: These experiments explore the basic biology of a fundamental metabolic pathway, isoprenoid biosynthesis, of Plasmodium falciparum. Isoprenoid compounds, which include quinones, photosynthetic pigments, and sterols, are vital to cellular function. This area of research is expected to provide insights into parasite pathogenesis, and ultimately therapeutics.
