Thursday, April 3, 2025
4/3/2025
Heme Homeostasis in the Malaria Parasite Plasmodium falciparum - PROJECT SUMMARY Malaria is the deadliest vector born disease, responsible for 249 million cases and 608,000 deaths in 2022. Malaria parasites replicate asexually in red blood cells where they digest up to 80% of host hemoglobin into globin peptides and heme. Heme is an essential cofactor, but is redox active in its free form. Parasites detoxify the majority of hemoglobin-derived heme by sequestering into inert hemozoin. Heme biosynthesis is inactive in blood stages, suggesting that blood stage parasites scavenge a portion of hemoglobin-derived heme for heme-requiring processes. As blood stage parasites mature, hemoglobin digestion increases, yet cytosolic heme concentrations remain constant at approximately 1.6 µM. How do parasites maintain cytosolic heme concentrations? To test the hypothesis that Plasmodium utilizes heme binding proteins to maintain heme homeostasis and that these heme-binding proteins will be essential for parasite viability, two orthogonal approaches will be taken: one unbiased approach and one directed approach. For the unbiased approach, parasite heme-binding proteins will be identified using a cutting-edge click-chemistry based method. Tagged conditional knockdown parasites will be generated to determine essentiality, expression, and localization of candidate proteins. To assess the involvement of candidate proteins in parasite heme handling, dose response assays will be used to assess the susceptibility of knockdown parasites to antimalarials that are known to disrupt heme homeostasis. In addition, heme species will be quantified under wild type and knockdown conditions using a pyridine-based heme fractionation assay and a genetically encoded heme biosensor. For the direct approach, the role of Heme Detoxification Protein (PfHDP; PF3D7_1446800) in heme homeostasis will be investigated. Recombinant PfHDP can bind heme and promote hemozoin formation in vitro. While PfHDP knockdown parasites have a severe growth defect, these parasites display no discernable defect in hemozoin formation. To investigate the hypothesis that PfHDP is involved in parasite heme handling independent of hemozoin formation, it will first be determined if PfHDP binds heme within the parasite basally or when heme homeostasis is disrupted. Next, sensitivity of PfHDP knockdown parasites will be assessed to quinoline antimalarials that are known to increase cytosolic heme concentrations. Finally, levels of cytosolic heme, hemozoin, and hemoglobin will be quantified under wildtype and PfHDP knockdown conditions using the pyridine-based heme fractionation assay and a genetically encoded heme biosensor. Interfering with heme detoxification has proven an effective antimalarial strategy. However, resistance to the traditional hemozoin biosynthesis inhibitors has limited their therapeutic use. Results from the experiments proposed here will provide insight into plasmodial heme homeostasis mechanisms. These results will reveal novel parasite proteins that can be exploited for future drug development and may provide insight into novel heme handling mechanisms in other organisms.
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