A Novel Methodology for Bioenergetic Analysis of Plasmodium falciparum Reveals a Glucose-Regulated Metabolic Shift and Enables Mode of Action Analyses of Mitochondrial Inhibitors

Abstract

Given that resistance to all drugs in clinical use has arisen, discovery of new antimalarial drug targets is eagerly anticipated. The Plasmodium mitochondrion has been considered a promising drug target largely based on its significant divergence from the host organelle as well as its involvement in ATP production and pyrimidine biosynthesis. However, the functions of Plasmodium mitochondrial protein complexes and associated metabolic pathways are not fully characterized. Here, we report the development of novel and robust bioenergetic assay protocols for Plasmodium falciparum asexual parasites utilizing a Seahorse Bioscience XFe24 Extracellular Flux Analyzer. These protocols allowed us to simultaneously assess the direct effects of metabolites and inhibitors on mitochondrial respiration and glycolytic activity in real-time with the readout of oxygen consumption rate and extracellular acidification rate. Using saponin-freed parasites at the schizont stage, we found that succinate, malate, glycerol-3-phosphate, and glutamate, but not pyruvate, were able to increase the oxygen consumption rate and that glycerol-3-phosphate dehydrogenase had the largest potential as an electron donor among tested mitochondrial dehydrogenases. Furthermore, we revealed the presence of a glucose-regulated metabolic shift between oxidative phosphorylation and glycolysis. We measured proton leak and reserve capacity and found bioenergetic evidence for oxidative phosphorylation in erythrocytic stage parasites but at a level much lower than that observed in mammalian cells. Lastly, we developed an assay platform for target identification and mode of action studies of mitochondria-targeting antimalarials. This study provides new insights into the bioenergetics and metabolomics of the Plasmodium mitochondria.

Identification of Collateral Sensitivity to Dihydroorotate Dehydrogenase Inhibitors in Plasmodium falciparum

“…Using methodology developed in our group, the oxygen consumption rate (OCR) of saponin-freed schizonts can be directly measured as an output of Complexes I–IV of the electron transport chain. 24 Inhibitors of any of the enzymes along the chain result in a reduction of OCR; by varying the input energy source provided to the cells, the system allows for the functional identification of the specific target of the compound. 24 When schizonts were assayed in RPMI medium, which contains fuel molecules such as glucose and glutamine, all dehydrogenases function normally and provide electrons to the downstream Cytbc 1 complex (Complex III).…”

Human plasma plasminogen internalization route in Plasmodium falciparum-infected erythrocytes

“…As shown in Fig. 3 a, the treatment of infected erythrocytes with antimycin (oxidative phosphorylation inhibitor [ 38 , 39 ]), sodium azide (oxidative phosphorylation inhibitor [ 26 ]), brefeldin (protein transport inhibitor [ 26 , 40 ]), cycloheximide (protein synthesis inhibitor [ 41 ]), FCCP (uncoupler of oxidative phosphorylation in mitochondria [ 38 ]), 2-deoxy- d -glucose (glycolysis inhibitor [ 38 ]) and genistein (protein tyrosine kinase inhibitor [ 42 ]), did not affect the uptake of plasminogen by the parasite. The soluble parasite protein Pfaldolase was used as a loading control.…”

Gene expression to mitochondrial metabolism: Variability among cultured Trypanosoma cruzi strains

“…Results were normalized to control for number of cells in each well contributing to per well consumption of oxygen measured, using SYBR Green I fluorescent dye (Invitrogen S77563; 10,000X) staining of the amount of cellular DNA in each well. This method of controlling for cell plating variability has been used previously for Plasmodium [ 34 ]. At assay completion, the 96-well plate was centrifuged and medium was removed from each well to be replaced by 100 μl 10X SYBR Green I in lysis solution (50 mM HEPES, 125 mM KCl, 12 mM MgCl 2 and 1.6% (v/v) Triton X-100, pH 7.4).…”