The proliferating cell hypothesis: a metabolic framework for Plasmodium growth and development.

We hypothesise that intraerythrocytic malaria parasite metabolism is not merely fulfilling the need for ATP generation, but is evolved to support rapid proliferation, similar to that seen in other rapidly proliferating cells such as cancer cells. Deregulated glycolytic activity coupled with impaired mitochondrial metabolism is a metabolic strategy to generate glycolytic intermediates essential for rapid biomass generation for schizogony. Further, we discuss the possibility that Plasmodium metabolism is not only a functional consequence of the 'hard-wired' genome and argue that metabolism may also have a causal role in triggering the cascade of events that leads to developmental stage transitions. This hypothesis offers a framework to rationalise the observations of aerobic glycolysis, atypical mitochondrial metabolism, and metabolic switching in nonproliferating stages.

Aerobic glycolysis drives proliferation in single-minded eukaryotes

Rapidly proliferating eukaryotes have perfected metabolic modes that efficiently convert glucose and specific amino acids into biomass (see Glossary) and energy at the required pace. The past decade has brought a change in the accepted paradigm on accelerated cell multiplication. Streamlined metabolic networks and the capacity to support anabolic reactions in a rapidly responsive manner via aerobic fermentative glycolysis and glutaminolysis, instead of pursuing thorough oxidation of the glycolytic carbons via cellular respiration, seems to be a precondition for rather than a consequence of effective proliferative signalling [1]. The corollary of this paradigm points to respiration in nonproliferating cells as the prevalent metabolic mode to generate the energy needed to perform their roles as differentiated cells.

Current concept of the Warburg effect

Although originally ascribed to anaerobic metabolism, the preference for fermentative glycolysis even under aerobic conditions was accepted long ago as a feature in cancer cells and is known as the Warburg effect [2]. Similarly, Saccharomyces cerevisiae favour fermentation over respiration when glucose is available even under oxygen abundance (Crabtree effect) [3]. In its original form, the Warburg effect also stated that the oxidation of glucose in mitochondria was ablated. However, more recent evidence points to functional mitochondrial oxidative phosphorylation in some cancer cell lines [3,4]. Under this modern version of the Warburg effect, rapidly proliferating, noncancerous cells have also been found to undergo aerobic glycolysis/fermentation [5-7].

The advantage provided to rapidly proliferating cells by increased glycolysis is attributed to the capacity of glucose to support biomass generation by redirection of glycolytic intermediates into anabolic reactions while at the same time sustaining a predominant (over 90%) fermentation flux to lactate [3,5,7,8] (Figure 1, Boxes 1 and 2). The latter is necessary for the regeneration of NAD+, an essential cofactor of glycolysis itself, but more importantly and less intuitively, to allow the cells to gauge their metabolic status. Thus, only when high levels of fermentative glycolysis are possible does the cell enter high rates of proliferation assisted by the anabolic capacity of glycolysis.

Figure 1

Proliferating cell hypothesis: similarities between cancer cells and Plasmodium falciparum. Principle end products of glucose consumption (lactate, alanine, pyruvate, glycerol-3-phosphate, and glycerol, shown in red boxes) are similar in both cancer cells [3] and asexual intraerythrocytic malaria parasites [12]. A high glycolytic flux maintains rate-limiting glycolytic intermediates to support nucleotide (via glucose-6-phosphate to 5-phosphoribosyl-α-pyrophosphate) and lipid biosynthesis (via dihydroxyacetone phosphate to glycerol-3-phosphate). Metabolic modifications (Boxes 1 and 2) allow aerobic glycolysis/fermentation to proceed rapidly whilst keeping tricarboxylic acid (TCA) flux low. Anapleorotic glutaminolysis follows past part of the TCA cycle through the five-carbon α-ketoglutarate [15]. Subsequent conversion of oxaloacetate to phosphoenolpyruvate (PEP) by phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.49) allows for further synthesis of biosynthetic intermediates (e.g., via shikimate pathway [16] and isoprenoid biosynthesis [17]). Abbreviations: GLUT-1, glucose transporter 1; PfHT1, Plasmodium falciparum hexose transporter 1; HK, hexokinase (EC 2.7.1.1); PGI, phosphoglucose isomerase (EC 5.3.1.9); PFK, phosphofructokinase (EC 2.7.1.11); G3PDH, glyceraldehyde 3 phosphate dehydrogenase (EC 1.2.1.12); PGK, phosphoglycerate kinase (EC 2.7.2.3); PK, pyruvate kinase (EC 2.7.1.40); LDH, lactate dehydrogenase (EC 1.1.1.27); PEPCase, phosphoenolpyruvate carboxylase (EC 4.1.1.31); PC, pyruvate carboxylase (EC 6.4.1.1); PDH, pyruvate dehydrogenase (EC 1.2.4.1); BCKDH, branched chain ketoacid dehydrogenase (EC 1.2.4.4); Suc-CoA, succinyl-CoA.

Aerobic glycolysis during the in vitro cell cycle of Plasmodium falciparum

The intraerythrocytic cycle of human falciparum malaria takes the parasites through successive rounds of mitosis every 48 h. Following erythrocyte invasion by a merozoite, but sometimes following multiple invasions, the parasite develops into a ring-shaped form in the first 24 h, and by approximately 30 h, the parasite very rapidly expands to occupy most of the space available within the erythrocyte plasma membrane, resulting in a major increase in biomass. From approximately 40 h, the vastly enlarged nucleus goes through several asynchronous and multiple segmentations that in vitro produce a number (small double figures) of next-generation merozoites [9]. Cytokinesis occurs near the end of the cycle before the new daughter cells (merozoites) emerge as free-living forms for seconds to minutes in the search for a new erythrocyte [9]. A fraction, usually less than 1% but dependent on the prevailing environment, of the newly generated intraerythrocytic parasites are programmed to differentiate as gametocytes, the sexual nondividing forms that in the natural environment continue the malaria cycle in the mosquito vector [10].

Malaria parasites committed to proliferation in the intraerythrocytic cycle are fermentative organisms [11-13] (Figure 1, Boxes 1 and 2) with an anabolic central carbon metabolism that can feed all major biomass generating pathways [14]. When directed to differentiation into gametocytes, however, these nonproliferative cells seem to follow the respiration of glucose in a manner more in line with the biology of eukaryotes in stationary phase via the canonical glucose-driven, mitochondrial tricarboxylic acid (TCA) cycle. Current evidence appears to substantiate this dichotomy of fermentation when in proliferation mode versus respiration when committed to sexual differentiation [15].

In proliferating asexual parasites, glutaminolysis feeds part of the TCA cycle through the five-carbon α-ketoglutarate. The four-carbon malate and oxaloacetate are transported to the cytoplasm. Here phosphoenolpyruvate (PEP) can be synthesised from oxaloacetate by the activity of phosphoenolpyruvate carboxykinase (PEPCK) for onward biosynthetic reactions (e.g., shikimate pathway [16] and isoprenoid biosynthesis [17]) (Figure 1). In nonproliferating gametocytes whereby a more canonical glucose TCA cycle is present, less glucose is catabolised by fermentation to lactate, and minimal glutamine is catabolised by glutaminolysis [15].

The paradigm of the rapidly proliferating eukaryote can then be applied to profile the dividing intraerythrocytic P. falciparum as an organism that in the presence of abundant glucose and glutamine, such as the levels available in human plasma, generates the required biomass by aerobic glycolysis/fermentation and glutaminolysis (Figure 1, Boxes 1–3). The rest of the macromolecular biomass is salvaged from the purine precursors, amino acids, and lipids or fatty acids of the human host. Under these conditions, a low flux glycolytic TCA cycle and a modified electron transport chain provides a further selective advantage (Boxes 1 and 2).

Are there metabolic regulatory switches controlling life cycle commitment in Plasmodium?

The established dogma states that Plasmodium metabolism is simply a functional consequence of the ‘hard-wired’ genome-wide, just-in-time regulation of expression [18,19]. However, there is increasing evidence in biology to support the notion that metabolism, in response to the environment/diet, can be causal, promoting the switch of cellular phenotypes. Examples in nature range from post-translational modifications (PTMs) of histones by constituents of royal jelly (fatty acids) causing larvae to become queens instead of worker bees [20], to PTMs of histones in the Agouti viable yellow mouse model, whereby different maternal methyl-donor supplementation (e.g., with folic acid, vitamin B12, or betaine) results in different offspring ranging from obese hyperinsulinaemic yellow to leaner nonhyperinsulinaemic pseudoagouti phenotypes [21].

The malaria parasite controls vital virulence processes such as host cell invasion and cytoadherence, at least in part, by epigenetic mechanisms [22]. With this in mind, and given that in vitro and in vivo nutrient/stress conditions have been linked with life cycle commitment in Plasmodium [23-25], it is not inconceivable that parasite metabolism may promote changes in phenotype via one or more of the many metabolites that are known to influence epigenetic gene regulation in other cell types.

In cancer cells and yeast, for example, nutrient availability and metabolic status, including the yeast metabolic cycle (YMC) fluctuating from oxidative phosphorylation and fermentation, is coupled to the control of gene expression via key metabolites such as NAD+, acetyl Co-A, FAD, and folates [26-28].

The influence of metabolism on parasite epigenetics is certainly an exciting area for future research, and some evidence, although circumstantial, exists to link nutrient levels to parasite development. Environmental stress has been consistently correlated with enhanced gametocyte production both in vitro and in vivo. The methodology applied to enrich in vitro cultures of P. falciparum with sexual forms has the common denominator of nutrient deprivation: low haematocrit, haemoglobin depletion, lysed erythrocytes, and recycling of spent media, among others [23,29]. Antimalarials that act as antimetabolites such as antifolates have long been known to increase gametocyte production in vivo [24]. In vivo transcriptional profiles of P. falciparum blood stages show that a proportion of the parasite population appears to be in states similar to what is known as either a starvation response or environmental stress in yeast [25]. Therefore, natural variability of substrate levels in the human host, perhaps not surprisingly, seems to be a selective force for life cycle commitment pathways in field populations of Plasmodium. Unfortunately, cellular metabolism of malaria parasites under variable nutrient availability has been poorly investigated, a situation not helped by the routine use of highly enriched media normally used for the in vitro culture of P. falciparum [30].

The decision of a parasite to commit to a sexual lineage is believed to take place in the first 20 h (the ‘ring’ stage) of the preceding erythrocytic cycle [29]. Interestingly, the early ring stages of P. falciparum have less compact histone cores (nucleosomes) than in later stages [9], and usually this ‘open’ conformation is reflective of, and conducive to, transcriptional regulation. As in other organisms and cell types it is therefore possible that in Plasmodium there exists a metabolic component that controls, via an epigenetic mechanism, the commitment to replicate or to differentiate.

A further, metabolically controlled, decision-making option open to the parasite in the early hours of intracellular parasite life is the possibility of reversible cell cycle arrest. As part of their parasitic lifestyle, P. falciparum become dependent on the extracellular supply of isoleucine due to an absence of this amino acid in human haemoglobin. Media that lacks isoleucine induce reversible cell cycle arrest with parasites not progressing beyond the first half, the ring stage, of their asexual intraerythrocytic life cycle unless the missing nutrient is provided [31]. In malaria, the phenomenon of reversible cell cycle arrest is poorly understood. Nonetheless, there is a new interest in studying malaria dormancy in the intraerythrocytic stages of the parasite life cycle due to the potential role of reversible cell cycle arrest in the slow clearance and/or ring stage survival (RSA0–3h) phenotypes seen in clinical failures with artemisinins [32-35].

Concluding remarks

Glucose and glutamine contribute to malaria parasite biomass for the biosynthesis of nucleotides and lipids via aerobic glycolysis/fermentation and glutaminolysis. Together with salvaged amino acids, fatty acids, and purines, these are the main biochemical resources used to assemble the macromolecular structure of the plasmodial cell. However, there are two further options available: (i) differentiation into a sexual lineage as gametocytes and (ii) cell cycle arrest. The first half of the intraerythrocytic cycle of P. falciparum, particularly within the initial 10 h, seems to be the stage at which quorum sensing and decision making is most relevant. As seen with other organisms and cell types, we have discussed the possibility that this occurs via nutrient/metabolite-dependent epigenetic mechanisms. Deconvolution of these regulatory processes offers a new and exciting chapter in our understanding of Plasmodium biology (Box 4).