Abstract
Glucose is an essential nutrient for Plasmodium falciparum and robust glycolytic activity is indicative of viable parasites. Using NMR spectroscopy, we show that P. falciparum infected erythrocytes consume ~20 times more glucose, and trophozoites metabolize ~6 times more glucose than ring stage parasites. The glycolytic activity, and hence parasite viability, can be measured within a period of 2 h to 5 h, using this method. This facilitates antimalarial bioactivity screening on ring and trophozoite stage parasites, exclusively. We demonstrate this using potent and mechanistically distinct antimalarial compounds such as chloroquine, atovaquone, cladosporin, DDD107498 and artemisinin. Our findings indicate that ring stage parasites are inherently more tolerant to antimalarial inhibitors, a feature which may facilitate emergence of drug resistance. Thus, there is a need to discover novel antimalarial compounds, which are potent and fast acting against ring stage parasites. The NMR method reported here can facilitate the identification of such molecules.
Highlights
Malaria is a devastating infectious disease, and the human malaria parasite P. falciparum is responsible for ~500,000 annual deaths worldwide[1]
For real time monitoring of glycolysis in P. falciparum infected red blood cell (RBC), we developed a protocol for directly tracking glycolytic activity in iRBCs by nuclear magnetic resonance (NMR)
Based on the integral value of the NMR spectra obtained for a dilution series of pure U13C-glucose standard samples (1 mM to 5 mM) (Supplementary Fig. S1d), we were able to estimate the rate at which RBCs (~108 cells) and iRBCs (~8.5 × 107 cells) metabolize glucose to lactate (Fig. 1b)
Summary
Malaria is a devastating infectious disease, and the human malaria parasite P. falciparum is responsible for ~500,000 annual deaths worldwide[1]. Prior work has shown that glycolytic activity of live P. falciparum infected RBC (iRBC) can be monitored in real time by nuclear magnetic resonance (NMR) spectroscopy, by measuring lactate accumulation over time[6]. We reasoned that this method can be used to evaluate the effect of anti-malarial compounds on distinct intra-erythrocytic stages of the malaria parasite, ring vs trophozoite stages. We have generated a high-resolution profile of glycolysis for the complete 48 h developmental cycle of the intra-erythrocytic forms of P. falciparum This was done by monitoring the conversion of U13C-glucose into U13C-lactate (Fig. 1a) by live P. falciparum infected human erythrocytes, using 13C NMR spectroscopy. By using NMR to measure parasite glycolytic activity in the presence of various mechanistically distinct inhibitor compounds, we show that ring stage parasites are inherently tolerant to potent antimalarial compounds
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