Branched tricarboxylic acid metabolism in Plasmodium falciparum

Abstract

A central hub of carbon metabolism is the tricarboxylic acid cycle, which serves to connect the processes of glycolysis, gluconeogenesis, respiration, amino acid synthesis and other biosynthetic pathways. The protozoan intracellular malaria parasites (Plasmodium spp.), however, have long been suspected of possessing a significantly streamlined carbon metabolic network in which tricarboxylic acid metabolism plays a minor role. Blood-stage Plasmodium parasites rely almost entirely on glucose fermentation for energy and consume minimal amounts of oxygen, yet the parasite genome encodes all of the enzymes necessary for a complete tricarboxylic acid cycle. Here, by tracing (13)C-labelled compounds using mass spectrometry we show that tricarboxylic acid metabolism in the human malaria parasite Plasmodium falciparum is largely disconnected from glycolysis and is organized along a fundamentally different architecture from the canonical textbook pathway. We find that this pathway is not cyclic, but rather is a branched structure in which the major carbon sources are the amino acids glutamate and glutamine. As a consequence of this branched architecture, several reactions must run in the reverse of the standard direction, thereby generating two-carbon units in the form of acetyl-coenzyme A. We further show that glutamine-derived acetyl-coenzyme A is used for histone acetylation, whereas glucose-derived acetyl-coenzyme A is used to acetylate amino sugars. Thus, the parasite has evolved two independent production mechanisms for acetyl-coenzyme A with different biological functions. These results significantly clarify our understanding of the Plasmodium metabolic network and highlight the ability of altered variants of central carbon metabolism to arise in response to unique environments.

Figure 1. Glutamine drives reverse flux through the TCA cycle

a, Relative concentrations of isotope isomers of carboxylic acids in extracts of P. falciparum-infected RBCs. Synchronized parasite were cultured in medium supplemented with either U-¹³C-glucose or U-¹³C-¹⁵N-glutamine 2 hours prior to invasion, then extracted every 8 hours post invasion (hpi) for HPLC-MS analysis. The plots to the right of the grey triangles zoom in on the profiles of the labeled metabolites. The +3 and +4 malate arises from the reductive and oxidative pathways, respectively, while +3 fumarate likely derives from interconversion of fumarate and malate by fumarate hydratase (PFI1340w). Error bars show the s.d. of n = 3 biological replicates. b, Schematic of the oxidative pathway from 2-oxoglutarate to malate. Red dots denote carbon-13 atoms. c, Schematic of the reductive carboxylation pathway from 2-oxoglutarate to malate. Ac-R represents either acetyl-CoA or acetate.

Figure 2. Acetyl groups deriving from glucose and glutamine are functionally distinct

a, Labeling of acetyl-CoA in extracts of P. falciparum-infected RBCs at t = 40 hpi as determined by HPLC-MS. b, Labeling of a singly-acetylated peptide derived from the N-terminal tail of histone H4, determined by proteomic MS. c, Labeling of UDP-GlcNAc at t = 40 hpi. Black bars: unlabeled molecule; red bars: the molecule labeled at both carbons of the acetyl group, regardless of any other labeling; dark gray bars: acetyl-CoA labeled at all 5 carbons of the ribose moiety of CoA, but not the acetyl group; white bars: UDP-GlcNAc labeled at some combination of the glucose, ribose or pyrimidine ring, but not the acetyl group; light gray bars: UDP-GlcNAc labeled at 1–3 nitrogens, but at no carbons. Error bars show the s.d. of n = 3 biological replicates.

Figure 3. Malate excretion by P. falciparum-infected RBC cultures

Parasites were grown and cultured as described above and samples of the culture medium were harvested and analyzed by HPLC-MS. The data are given as molar concentrations in the media samples. The plot at the right, indicated by grey triangles, are zoomed in on the profiles of the labeled metabolites. All error bars show the s.d. of n = 3 biological replicates.

Figure 4. An integrated model for central carbon metabolism in P. falciparum

Arrows show direction of net flux; multiple arrows depict pathways not shown in their entirety and are labeled as such. Metabolites in red are those found to be effluxed into the medium as waste products. The red arrows indicate the reductive pathway of TCA metabolism, while the blue arrows show the oxidative pathway. Asterisk (*): the specific enzyme responsible for the citrate cleavage step and its localization are unclear (see text). Double asterisk (**): there are two predicted enzymes capable of catalyzing this reaction, the cytosolic malate dehydrogenase (PFF0895w) and the putative mitochondrial malate:quinone oxidoreductase (MAL6P1.258). Abbreviations: Gln, glutamine; Glu, glutamate; OG, 2-oxoglutarate; ICT, isocitrate; Cit, citrate; Ac-R, acetate/acetyl-CoA; Ac-CoA, acetyl-CoA; OA, oxaloacetate; Mal, malate; Suc-CoA, succinyl-CoA; Fum, fumarate; Glc, glucose; Asp, aspartate; PEP, phosphoenolpyruvate; Pyr, pyruvate; Lac, lactate.