Abstract
Although efforts to understand the basis for inter-strain phenotypic variation in the most virulent malaria species, Plasmodium falciparum, have benefited from advances in genomic technologies, there have to date been few metabolomic studies of this parasite. Using 1H-NMR spectroscopy, we have compared the metabolite profiles of red blood cells infected with different P. falciparum strains. These included both chloroquine-sensitive and chloroquine-resistant strains, as well as transfectant lines engineered to express different isoforms of the chloroquine-resistance-conferring pfcrt (P. falciparum chloroquine resistance transporter). Our analyses revealed strain-specific differences in a range of metabolites. There was marked variation in the levels of the membrane precursors choline and phosphocholine, with some strains having >30-fold higher choline levels and >5-fold higher phosphocholine levels than others. Chloroquine-resistant strains showed elevated levels of a number of amino acids relative to chloroquine-sensitive strains, including an approximately 2-fold increase in aspartate levels. The elevation in amino acid levels was attributable to mutations in pfcrt. Pfcrt-linked differences in amino acid abundance were confirmed using alternate extraction and detection (HPLC) methods. Mutations acquired to withstand chloroquine exposure therefore give rise to significant biochemical alterations in the parasite.
Highlights
Malaria, an ancient disease caused by protozoan Plasmodium parasites, continues to inflict a devastating toll in many parts of the world
1H-NMR estimates of metabolite amounts in uRBCs and P. falciparum-infected red blood cell (iRBC) In initial experiments, the metabolite profile of iRBCs was compared with that of co-cultured red blood cell (cRBC) cultured in the same flask, as well as with that of uRBCs cultured concurrently in a separate flask
Glucose was not detected in the 3D7 iRBCs and the amount detected in uRBCs and cRBCs was highly variable
Summary
An ancient disease caused by protozoan Plasmodium parasites, continues to inflict a devastating toll in many parts of the world. The γ -amino acid GABA (4-aminobutyrate), the phospholipid precursors phosphocholine and phosphoethanolamine, the TCA (tricarboxylic acid) cycle intermediates fumarate, α-ketoglutarate, malate and succinate, and the polyamines putrescine, spermidine and spermine, were all undetectable by 1H-NMR in uRBCs but present in iRBCs. betaine, creatine, hypoxanthine, ornithine, sarcosine and sorbitol were detected by 1H-NMR in uRBCs and cRBCs but not in iRBCs (note that ornithine was detectable in iRBCs by HPLC; below).
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