Reconstruction and flux-balance analysis of the Plasmodium falciparum metabolic network

Abstract

Genome-scale metabolic reconstructions can serve as important tools for hypothesis generation and high-throughput data integration. Here, we present a metabolic network reconstruction and flux-balance analysis (FBA) of Plasmodium falciparum, the primary agent of malaria. The compartmentalized metabolic network accounts for 1001 reactions and 616 metabolites. Enzyme-gene associations were established for 366 genes and 75% of all enzymatic reactions. Compared with other microbes, the P. falciparum metabolic network contains a relatively high number of essential genes, suggesting little redundancy of the parasite metabolism. The model was able to reproduce phenotypes of experimental gene knockout and drug inhibition assays with up to 90% accuracy. Moreover, using constraints based on gene-expression data, the model was able to predict the direction of concentration changes for external metabolites with 70% accuracy. Using FBA of the reconstructed network, we identified 40 enzymatic drug targets (i.e. in silico essential genes), with no or very low sequence identity to human proteins. To demonstrate that the model can be used to make clinically relevant predictions, we experimentally tested one of the identified drug targets, nicotinate mononucleotide adenylyltransferase, using a recently discovered small-molecule inhibitor.

Figure 1

Annotation of reactions in the genome-scale metabolic model of P. falciparum. (A) Number of orphan (non-gene associated) reactions in P. falciparum grouped by metabolic processes. (B) Reactions grouped by Enzyme Commission (EC) classifications. (C) Reactions grouped by metabolic processes in P. falciparum and S. cerevisiae (Duarte et al, 2004).

Figure 2

Small-molecule inhibition of the parasite nicotinate mononucleotide adenylyltransferase (NMNAT). (A) Schematic of the P. falciparum NAD(P) synthesis and recycling pathway determined from the genome sequence. Nicotinamide (NM) and nicotinic acid (NA) can be scavenged from the host. Compound 1_03 is an inhibitor targeting NMNAT. (B) Compound 1_03 causes growth arrest of intraerythrocytic P. falciparum. Cultures were resuspended in niacin-free medium containing 0 or 100 μM of compound 1_03 at early ring stage and observed for 66 h (see Materials and methods). Untreated parasites undergo normal development and reinvasion, whereas drug-treated parasites arrest at the trophozoite (‘troph’) stage and do not reinvade. NM, nicotinamide; NA, nicotinic acid; NaMN, nicotinate mononucleotide; NaAD, nicotinate adenine dinucleotide; NAD(P)⁺, nicotinamide adenine dinucleotide (phosphate), reduced; NMase, nicotinamidase; NPRT, nicotinate phosphoribosyltransferase; NMNAT, nicotinate mononucleotide adenylyltransferase; NADS, NAD synthase; NADK, NAD kinase.

Figure 3

Comparison between the predicted and experimentally measured shifts in metabolite concentrations in infected red blood cells. UP/DOWN indicates direction of experimentally measured changes in metabolic concentrations in infected versus uninfected cells. Blue color indicates agreement between experiment and predictions, whereas yellow indicates disagreement. In most cases (70%, P-value=9 × 10⁻⁴), the shifts in metabolic concentrations from one stage to the next can be predicted based on changes in the P. falciparum metabolic exchange fluxes. The in silico predictions of exchange fluxes were made based on the expression-constrained flux-balance analysis (Colijn et al, 2009). Briefly, for genes with available mRNA-expression data, the maximum flux through the associated metabolic reactions was constrained proportionally to their expression level; with the highest expression value corresponding to the maximum allowed flux.