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

Abstract

Background: 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.

Methods: 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.

Results: 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).

Conclusions: 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.

Keywords: Gene set enrichment analysis; Metabolic network model; Metabolome; Plasmodium falciparum; Purine deprivation; Stress response pathways; Transcriptome.

Fig. 1

Schematic showing hypoxanthine salvage in Plasmodium falciparum. Hypoxanthine in the cytosol of a red blood cell (RBC) can be formed by catabolism of adenosine triphosphate (ATP) or imported through hypoxanthine transporters from the environment, where it is present at 2–8 µM in human serum. The parasite takes up hypoxanthine from the RBC cytosol and uses it to make purine-based nucleic acids, whereas it synthesizes pyrimidine-based nucleic acids de novo. The parasite can produce hypoxanthine from methylthioinosine (MTI) and methylthioadenosine (MTA), which are produced during polyamine synthesis. The parasite can also produce hypoxanthine from adenosine, which it either takes up from the RBC’s cytosol or by directly cleaving S-adenosylhomocysteine (SAH). SAH is synthesized from methionine (MET), which the parasite acquires through hemoglobin digestion in the food vacuole (FV) or via plasma membrane transporters. Black dots on the RBC membrane indicate hypoxanthine and MET transporters. ADP, adenosine diphosphate; AMP, adenosine monophosphate; ATP, adenosine triphosphate; ASA, adenylosuccinic acid; GMP, guanine monophosphate; HCS, homocysteine; IMP, inosine monophosphate; SAM, S-adenosylmethionine; XMP, xanthine monophosphate

Fig. 2

Schematic diagrams showing integration of transcriptomic and metabolomic data with the metabolic network model. a The typical life cycle of P. falciparum during the blood stage, also known as the intraerythrocytic developmental cycle (IDC). The cycle begins when a merozoite infects a red blood cell (RBC: circle drawn with a solid black line). Subsequently, the parasite develops and multiplies within the RBC to form rings (R), trophozoites (T), and schizonts (S). Once the parasite completes the cycle, it ruptures the RBC to release 16–32 merozoites [2, 3], which in turn invade another RBC to begin another cycle. The typical life cycle of P. falciparum lasts about 48 h. b Upper panel: representation of gene transcription during the 48-h IDC. There is typically a time delay (denoted by τ) between the transcription of a gene and the translation of an enzyme. In the computational framework of the present study, this delay is reflected in the shift of r(t) relative to the function for gene transcription [denoted by f(g)]. Lower panel: abundance of a metabolite (m) during the 48-h IDC. In the present framework, the relative abundance of a participant metabolite is assumed to modulate the flux through a given metabolic reaction. c A metabolic network model showing integration of metabolic and transcriptomic information. In this panel, r₁(t) alters nutrient uptake of the model and affects ‘in silico growth’ r₆(t), given other metabolic reactions [r₂(t) to r₅(t)] and secretion processes in the model

Fig. 3

Effect of hypoxanthine deprivation on Plasmodium falciparum during the IDC. a Representative images of Giemsa-stained parasites at 24, 32 and 40 h in hypoxanthine-rich medium (hxan-R) and hypoxanthine-deprived medium (hxan-D). Black arrows indicate vacuole-like structures in the cytoplasm of the parasite. b Developmental-stage specific parasitaemia under the two culture conditions (hxan-R and hxan-D) at different time points. c Recovered parasite numbers shown as a percentage of parasite-infected RBCs (iRBCs) maintained in hypoxanthine-rich medium at 40 h into the IDC. The number of recovered parasites is calculated 24 h after the transfer of the deprived parasites to the rich medium. ET, early trophozoite; hxan-D, hypoxanthine-deprived; hxan-R, hypoxanthine-rich; LT, late trophozoite; R, ring; S, schizont

Fig. 4

Key purine metabolites under hypoxanthine-rich (hxan-R) and -deprived (hxan-D) conditions. a Adenosine, b inosine, c hypoxanthine, and d inosine monophosphate (IMP) levels from parasite-infected red blood cells under the two conditions. At the first two time points of hypoxanthine deprivation, each metabolite was higher than its value under the hypoxanthine-rich condition. During the second day of infection under the hypoxanthine-deprived condition, inosine and hypoxanthine levels were substantially lower than their values under the hypoxanthine-rich condition. To allow comparison of a metabolite between the two conditions, the value of a given metabolite was normalized by its initial value at time 0 h. The error bars show the standard deviations of measurements from four technical replicates

Fig. 5

Model-predicted effect of hypoxanthine deprivation on major biomass components of Plasmodium falciparum. a DNA synthesis rate, b RNA synthesis rate, c protein synthesis rate, d cofactor synthesis rate, e polyamine synthesis rate, and f inorganic ion generation rate. The error bars show standard deviation of 50 simulations computed after adding random Gaussian noise with a mean of zero and a standard deviation of 5% to the transcriptomic data obtained under hypoxanthine-rich and hypoxanthine-deprived conditions. hxan-D, hypoxanthine-deprived; hxan-R, hypoxanthine-rich

Fig. 6

Gene set enrichment analysis of stress response pathways (SRPs) in Plasmodium falciparum. Results for transcriptomic data obtained under a hypoxanthine and b isoleucine deprivation. The figure shows gene ontologies associated with the SRPs of P. falciparum. Here each circle represents a gene ontology associated with the SRPs, where the size of an ontology circle is proportional to the number of genes in it. A black line drawn from a colored circle to the SRP indicates that this gene set directly influences the SRP. Similar gene sets are clustered together and grey lines are used to indicate interaction between them. The cluster named ‘interaction with host’ contains proteins associated with Maurer’s cleft and parasitic protein export. ‘RBC adhesion’ contains P. falciparum red blood cell (RBC) membrane protein 1 (PfEMP1) and associated proteins, which are inserted in the RBC plasma membrane. The colour of a circle indicates whether or not a given gene set was enriched (dark red, p < 0.01; light red, 0.01 ≤ p < 0.05; yellow, 0.05 ≤ p < 0.10; green, p ≥ 0.10)

Fig. 7

Plasmodium falciparum metabolic enzymes involved in the hypoxanthine salvage and recycling process. a Hypoxanthine phosphoribosyltransferase (HXPRT) catalyzes the formation of inosine monophosphate (IMP) from hypoxanthine. b IMP dehydrogenase (IMPD) catalyzes the formation of xanthine monophosphate (XMP) from IMP. c Adenylosuccinate synthase (ADSS) catalyzes the formation of adenylosuccinic acid from IMP. d Compensatory mechanism involved in maintenance of the hypoxanthine pool under deprivation conditions. e Purine nucleoside phosphorylase (PNP) catalyzes the formation of hypoxanthine from inosine (INS). In a–c and e, the ordinate shows the reaction rate of the metabolic enzyme. The error bars show the standard deviations of 50 simulations computed after adding random Gaussian noise with a mean of zero and a standard deviation of 5% to the transcriptomic data obtained under hypoxanthine-rich and hypoxanthine-deprived conditions. ADN, adenosine; ETH, ethanolamine; HCS, homocysteine; hxan-D, hypoxanthine-deprived; hxan-R, hypoxanthine-rich; MET, l-methionine; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; SER, l-serine

Fig. 8

Consequences of hypoxanthine deprivation on parasite metabolism, as captured in model simulations. These are most clearly delineated in model simulations of parasite metabolism during the first 24 h of hypoxanthine deprivation. The simulations indicated that the parasite supplies phosphoenolpyruvate (PEP) to synthesize chorismate, which is a precursor for ubiquinone synthesis as part of shikimate synthesis. PEP is also utilized to synthesize oxaloacetate (OAA), which in turn leads to enhanced porphyrin metabolism. The red arrows indicate the metabolic flow occurring under hypoxanthine-deprivation conditions. A dotted arrow indicates more than one reaction step. AKG, alpha-ketoglutarate; ETC, electron transport chain; FUM, fumarate; MAL, l-malate; nad, oxidized nicotinamide adenine dinucleotide; nadp, oxidized nicotinamide adenine dinucleotide phosphate; nadph, reduced nadp; PPP, pentose phosphate pathway; SUC, succinate; q8, ubiquinone