Short-term metabolic adjustments in Plasmodium falciparum counter hypoxanthine deprivation at the expense of long-term viability

Shivendra G. Tewari, Krithika Rajaram, Patric Schyman, Russell Swift, Jaques Reifman, Sean T. Prigge, Anders Wallqvist

The malarial parasite Plasmodium falciparum is an auxotroph for purines, which are required for nucleic acid synthesis during the intra-erythrocytic developmental cycle (IDC) of the parasite. The capabilities of the parasite and extent to which it can use compensatory mechanisms to adapt to purine deprivation were studied by examining changes in its metabolism under sub-optimal concentrations of hypoxanthine, the primary precursor utilized by the parasite for purine-based nucleic acid synthesis. The concentration of hypoxanthine that caused a moderate growth defect over the course of one IDC was determined. At this concentration of hypoxanthine (0.5 μM), transcriptomic and metabolomic data were collected during one IDC at multiple time points. These data were integrated with a metabolic network model of the parasite embedded in a red blood cell (RBC) to interpret the metabolic adaptation of P. falciparum to hypoxanthine deprivation. At a hypoxanthine concentration of 0.5 μM, vacuole-like structures in the cytosol of many P. falciparum parasites were observed after the 24-h midpoint of the IDC. Parasites grown under these conditions experienced a slowdown in the progression of the IDC. After 72 h of deprivation, the parasite growth could not be recovered despite supplementation with 90 µM hypoxanthine. Simulations of P. falciparum metabolism suggested that alterations in ubiquinone, isoprenoid, shikimate, and mitochondrial metabolism occurred before the appearance of these vacuole-like structures. Alterations were found in metabolic reactions associated with fatty acid synthesis, the pentose phosphate pathway, methionine metabolism, and coenzyme A synthesis in the latter half of the IDC. Furthermore, gene set enrichment analysis revealed that P. falciparum activated genes associated with rosette formation, Maurer's cleft and protein export under two different nutrient-deprivation conditions (hypoxanthine and isoleucine). The metabolic network analysis presented here suggests that P. falciparum invokes specific purine-recycling pathways to compensate for hypoxanthine deprivation and maintains a hypoxanthine pool for purine-based nucleic acid synthesis. However, this compensatory mechanism is not sufficient to maintain long-term viability of the parasite. Although P. falciparum can complete a full IDC in low hypoxanthine conditions, subsequent cycles are disrupted.