Quantification of labile heme in live malaria parasites using a genetically encoded biosensor

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Abstract
Heme is ubiquitous, yet relatively little is known about the maintenance of labile pools of this cofactor, which likely ensures its timely bioavailability for proper cellular function. Quantitative analysis of labile heme is of fundamental importance to understanding how nature preserves access to the diverse chemistry heme enables, while minimizing cellular damage caused by its redox activity. Here, we have developed and characterized a protein-based sensor that undergoes fluorescence quenching upon heme binding. By genetically encoding this sensor in the human malarial parasite, Plasmodium falciparum, we have quantified cytosolic labile heme levels in intact, blood-stage parasites. Our findings indicate that a labile heme pool (∼1.6 µM) is stably maintained throughout parasite development within red blood cells, even during a period coincident with extensive hemoglobin degradation by the parasite. We also find that the heme-binding antimalarial drug chloroquine specifically increases labile cytosolic heme, indicative of dysregulation of this homeostatic pool that may be a relevant component of the antimalarial activity of this compound class. We propose that use of this technology under various environmental perturbations in P. falciparum can yield quantitative insights into fundamental heme biology. Significance Malaria parasites degrade substantial quantities of hemoglobin to release heme within a specialized digestive vacuole. Most of this heme is sequestered in an inert crystal. However, the concentration of bioavailable, labile heme in the parasite’s cytosol was unknown. We developed a biosensor to provide the first quantitative insights into labile heme concentrations in malaria parasites. We find that ∼1.6 µM labile cytosolic heme is maintained, including during a period coincident with intense hemoglobin degradation. The heme-binding antimalarial drug, chloroquine, which interferes with heme crystallization, specifically induces an increase in labile heme. The ability to quantify labile heme in malaria parasites opens opportunities for better understanding heme homeostasis, signaling, and metabolism, and its association with antimalarial potency.
Funding Information
  • HHS | NIH | National Institute of General Medical Sciences (5-T32-GM08334)
  • HHS | NIH | NIH Office of the Director (1DP2OD007124)
  • HHS | NIH | National Institute of General Medical Sciences (P50-GM098792)
  • Office of Extramural Research (5-P41-EB015871-27)
  • HHS | National Institutes of Health (DP3-DK101024 01)
  • HHS | National Institutes of Health (1-U01-NS090438-01)
  • HHS | National Institutes of Health (1-R01-EY017656-06A1)
  • HHS | National Institutes of Health (1-R01-HL121386-01A1)
  • Wellcome Trust (093831/Z/10/Z)

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