Phosphoenolpyruvate Carboxylase Identified as a Key Enzyme in Erythrocytic Plasmodium falciparum Carbon Metabolism

Janet Storm,Rainer Breitling,Sylke Müller,Gavin J Blackburn,Achuthanunni Chokkathukalam,Graham H Coombs,David G Watson,Sonal Sethia,Anthony A Holder

doi:10.1371/journal.ppat.1003876

Abstract

Phospoenolpyruvate carboxylase (PEPC) is absent from humans but encoded in the Plasmodium falciparum genome, suggesting that PEPC has a parasite-specific function. To investigate its importance in P. falciparum, we generated a pepc null mutant (D10Δpepc), which was only achievable when malate, a reduction product of oxaloacetate, was added to the growth medium. D10Δpepc had a severe growth defect in vitro, which was partially reversed by addition of malate or fumarate, suggesting that pepc may be essential in vivo. Targeted metabolomics using 13C-U-D-glucose and 13C-bicarbonate showed that the conversion of glycolytically-derived PEP into malate, fumarate, aspartate and citrate was abolished in D10Δpepc and that pentose phosphate pathway metabolites and glycerol 3-phosphate were present at increased levels. In contrast, metabolism of the carbon skeleton of 13C,15N-U-glutamine was similar in both parasite lines, although the flux was lower in D10Δpepc; it also confirmed the operation of a complete forward TCA cycle in the wild type parasite. Overall, these data confirm the CO2 fixing activity of PEPC and suggest that it provides metabolites essential for TCA cycle anaplerosis and the maintenance of cytosolic and mitochondrial redox balance. Moreover, these findings imply that PEPC may be an exploitable target for future drug discovery.

Highlights

Plasmodium falciparum is the causative agent of malaria, a disease claiming an estimated 660,000 lives annually, primarily among children in Africa [1]
phosphoenolpyruvate carboxylase (PEPC) is known to fix CO2 to generate metabolites used for energy metabolism in plants and bacteria, but its function in malaria parasites remained an enigma
The mutant generated (D10Dpepc) had a severe growth defect, which was rescued partially by malate or fumarate, suggesting that they feed into the same metabolic pathway

Summary

Introduction

Plasmodium falciparum is the causative agent of malaria, a disease claiming an estimated 660,000 lives annually, primarily among children in Africa [1]. Energy generation in P. falciparum asexual erythrocytic stages depends upon glucose being primarily converted to lactate by anaerobic glycolysis, which is excreted as the major metabolic end product [3] This is consistent with the finding that in P. falciparum the pyruvate dehydrogenase complex (PDH) is solely present in a plastid-like organelle, the apicoplast, where it provides acetyl-CoA for fatty acid biosynthesis and possibly other acetylating reactions [4,5]. It was shown very recently, that despite the absence of mitochondrial PDH, pyruvate can be metabolised by a PDH-like enzyme complex [6] and oxidised through a forward tricarboxylic acid (TCA) cycle in the erythrocytic stages of P. falciparum, being important in the gametocytes, and in the related apicomplexan parasite Toxoplasma gondii [3,6,7]. These findings are in contrast to previous reports that there is no link between cytosolic glucose catabolism and mitochondrial TCA metabolism [8], this report was later retracted [9]

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