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Dissecting the Metabolism of the Malaria Parasite Plasmodium falciparum

ProQuest Dissertations and Theses, 2011
Dissertation
Author: Kellen Leonard Olszewski
Abstract:
The Plasmodium malaria parasites have been a scourge of mankind throughout the entire history of our species. The recent history of malaria control and treatment has been a series of leaps forward that are then partially or entirely reversed due to the emergence of drug resistance. The urgent need for new antimalarials requires a deep and holistic understanding of the parasite's metabolic network, as this is a broadly attractive target for drug development. Moreover, it presents a fascinating example of a metabolism highly diverged from that found in well-studied, free-living model protozoans. Elucidating the systems-level properties of this network requires the application of novel, high-throughput chemimetric technologies to characterize the architecture and dynamics of the Plasmodium metabolome. In this thesis I present the results of my metabolomic investigations into P. falciparum , the most lethal human malaria pathogen. I initially characterized the metabolic alterations induced by P. falciparum infection of human erythrocytes in an in vitro culture system. One major result was the specific depletion of extracellular arginine, which I determined to be due to the parasite's arginase enzyme. Ablation of this enzyme did not impair parasite growth, suggesting that this depletion is not growth-related but may play a role in immunomodulation and potentially cerebral malaria. Subsequent investigations focused on the nature of carbon metabolism during the Plasmodium blood stages; specifically, the architecture of mitochondrial tricarboxylic (TCA) acid metabolism. By tracing the flux of isotope-labeled nutrients I determined that the parasite lacks a "TCA cycle" as such, instead possessing a branched metabolic pathway comprising both oxidative and reductive arms. This significantly clarifies a major open question of parasite biology, and suggests a number of intriguing avenues of research. I also conducted a collaborative investigation into the genetic control of metabolism at a global level. We found an association between a major drug resistance effector and metabolites arising from hemoglobin catabolism. This suggests a functional role for this previously mysterious resistance gene, and has implications for the fitness cost associated with drug resistance.

TABLE OF CONTENTS

ACKNOWLEDGMENTS .................................................................................................... vi LIST OF FIGURES .............................................................................................................. viii LIST OF TABLES ................................................................................................................ x Chapter 1: The malaria parasite and its metabolism ............................................................. 1 Chapter 2: Host-parasite interactions revealed by Plasmodium falciparum metabolomics.. 34 Chapter 3: Branched tricarboxylic acid metabolism in Plasmodium falciparum ................. 50 Chapter 4: Metabolomic quantitative trait loci analysis implicates the Plasmodium falciparum chloroquine resistance transporter as a small peptide transporter ...................... 66 Chapter 5: Other projects, collaborations and suggested directions ..................................... 93 Appendix A: Supplementary figures .................................................................................... 113 Appendix B: Materials and Methods .................................................................................... 128 References ............................................................................................................................. 146

vi ACKNOWLEDGMENTS

First and foremost I need to thank Manuel, who has been an inexhaustible well of support and motivation. We both arrived at Princeton at roughly the same time, and for a while formed two-thirds of a three-person lab, so we’ve have had to figure all of this out side-by-side. Needless to say that none of this would have been possible without his dedicated mentorship and drive. Josh Rabinowitz has been a wonderful guide through the unfamiliar territory of metabolomics and biochemistry, and has been a signal inspiration in how I see and do science. David Botstein, as a committee member, thesis reader and reader of manuscripts, was an invaluable source of biological insight and a reminder to always keep one eye on the question at hand, and the other on the one over the horizon. I would like to acknowledge the help and friendship I experienced from everyone in the Llinás and Rabinowitz labs, but there are a few I should single out for special mention: Ilsa and Daniel, who were on board right from the beginning (or shortly thereafter) and were instrumental in laying the groundwork for a functioning lab; Tracey, Lucky, Erandi and Heather, who pushed the lab to the next level of doing world-class parasitology; Bryson, my contemporary in the Rabinowitz lab, who was always available to spitball crazy ideas with or explain any chemistry I stumbled across; and Eugene, who built the computational infrastructure that evolved data analysis from an impossible chore to a pleasure. And of course all of the other students, postdocs, undergrads and rotons that have made it such a wonderful place to work.

vii I must also thank my collaborators, particularly Akhil and everyone in the Vaidya lab, particularly Mike Mather and Joanne Morrissey, who I worked with throughout my graduate career. Mike Ferdig and everyone in his lab were also wonderful to work with, but Mark Wacker merits special mention as my opposite number and close collaborator.

viii LIST OF FIGURES

Figure 1. The malaria parasite and its life cycle ............................................................... 3 Figure 2. An integrated map of Plasmodium carbon metabolism ...................................... 8 Figure 3. Periodic metabolite fluctuation during the intraerythrocytic developmental cycle (IDC) ........................................................................................................................... 36 Figure 4. Amino acid levels in the culture medium ............................................................. 41 Figure 5. Arginine depletion is due to the P. falciparum arginase ...................................... 44 Figure 6. Phenotypic analysis of the P. berghei arginase knockout (argKO) strain ............ 45 Figure 7: Glutamine drives reverse flux through the TCA cycle.......................................... 53 Figure 8: Functional roles of acetyl groups deriving from glucose and glutamine ............. 55 Figure 9: Malate excretion by P. falciparum-infected RBC cultures .................................. 58 Figure 10: An integrated model for central carbon metabolism in P. falciparum ............... 62 Figure 11. Distribution of mQTL throughout the P. falciparum genome ........................... 77 Figure 12. Characteristics of three compounds linking to the locus containing pfcrt ......... 79 Figure 13. Strategy for identifying hemoglobin-derived peptides........................................ 80 Figure 14. Relative peptide levels between CQS and CQR parasites .................................. 82 Figure 15. Relative growth in rich and amino acid-restricted medium................................ 87 Figure 16. A model for PfCRT function ............................................................................... 91 Figure S1. Clustering of intracellular metabolite levels over the intraerythrocytic developmental cycle .............................................................................................................. 113 Figure S2. Clustering of extracellular metabolite levels over the intraerythrocytic developmental cycle .............................................................................................................. 114

ix Figure S3. Gene expression during the intraerythrocytic developmental cycle .................. 115 Figure S4. Tricarboxylic acid cycle intermediates and enzymes vary over the IDC ........... 116 Figure S5. Conversion of glucose into lactate, and nicotinamide into NAD+ are increased by Plasmodium infection ...................................................................................... 117 Figure S6. P. berghei arginase knockout ............................................................................. 118 Figure S7. Parasitemia in WT and argKO P. berghei (ANKA) infected BALB/c mice ..... 119 Figure S8: Cytosolic pathways generating malate and fumarate ......................................... 120 Figure S9: Aspartate is metabolized to fumarate and malate .............................................. 121 Figure S10. Glucose supplies the acetyl groups for histone acetylation .............................. 122 Figure S11: Carboxylic acid excretion by P. falciparum-infected RBC cultures ................. 123 Figure S12: Isocitrate dehydrogenase localizes to the parasite mitochondria ...................... 124 Figure S13. A citrate synthase-like protein encoded by the P. falciparum genome ............ 125 Figure S14. A divergent citrate synthase-like protein ......................................................... 126 Figure S15. ATP:citrate lyase activity in infected erythrocytes .......................................... 127

x

LIST OF TABLES

Table 1. Characteristics of the four digestive aminopeptidase enzymes predicted in the P. falciparum genome................................................................................................................ 88

1 CHAPTER 1

The malaria parasite and its metabolism

This chapter has been previously published (Olszewski and Llinas, 2010), and is presented here with significant additions.

Malaria, the “bad air” disease Malaria is one of the most ancient scourges of mankind. It afflicted our hominin ancestors prior to the existence of Homo sapiens, and has been such a constant and destructive force in the history of the species that it has, by itself, effected significant changes to our evolution. Despite a brief period of success in malaria control in the mid- 20 th century, the present-day statistics are staggering: 40% of the world’s population lives in areas at risk for malaria, roughly half a billion people contract malaria every year, and between 1 and 2 million of those will die (Sachs and Malaney, 2002). The damage is not limited to sickness and death; malaria is also indirectly an economic phenomenon, as it is not uncommon for up to 40% of a malaria-endemic country’s public health spending to go towards malaria treatment, and in such countries the disease causes an average loss of 1.3% to annual economic growth (WHO, 2007). Thus malaria is additionally an economic catastrophe, and one localized to what are already some of the poorest countries on Earth. The name “malaria” famously derives from the Medieval Italian for “bad air”, reflecting ancient beliefs that the disease was caused by the noxious miasma emanating from swamps. Recorded references to malaria-like periodic fevers date back at least

2 4500 years and have been a near-constant in civilizations residing in the broad, warm band around the equator (Cox, 2002). It was not until 1880, however, that the scientific study of the disease began with Charles Laveran’s Nobel Prize-winning discovery that malaria was associated with the invasion of the host’s red blood cells (RBCs) by foreign organisms visible under a microscope (Figure 1a) (Sutherland and Hallett, 2009). This was shortly followed by Sir Ronald Ross’ demonstration that these pathogens were transmitted by the bites of infected mosquitoes which breed in the swamps historically associated with malaria (Chernin, 1988). These bites shortly give rise to blood-borne invasive cells and the manifestations of the disease. The most obvious of these is a periodic fever, typically repeating every one, two or three days. The patient also develops symptoms including fatigue, muscle and joint pain, hemolytic anemia and hypoglycemia with lactic acidosis. Complications of the disease include cerebral malaria, which leads to coma and death, and placental malaria, which can cause the death of the mother and/or child.

The malaria parasite and its life cycle The malaria pathogens are now understood to be protozoan parasites of the genus Plasmodium. The genus is resides within the family Apicomplexa, which is comprised almost entirely of intracellular parasites (such as those causing toxoplasmosis, babesiosis and cryptosporidiosis). Various Plasmodium spp. infect vertebrate hosts ranging from lizards, birds, rodents and primates, though in all cases they are transmitted by female Anopheline mosquitoes. Four Plasmodium species (P. falciparum, vivax, ovale, and malariae) infect humans, and a fifth, P. knowlesi, is a parasite of macaques apparently in

3 the process of making the evolutionary leap to a human pathogen (Singh et al., 2004). P. falciparum is the most virulent of the human malaria parasites and is the dominant species throughout sub-Saharan Africa, where most malaria deaths occur. Figure 1. The malaria parasite and its life cycle. a, Selected drawings from the first microscopic observation of malaria parasites in human blood by Charles Alphonse Laveran(Laveran, 1880). These drawings correspond to asexual schizont (left), mature male gametocyte (center) and exflagellating male gametes (right). b, Diagram of the Plasmodium life cycle, encompassing both human and insect stages(Wirth, 2002).

4 The life cycle of the parasite is broadly conserved across the Plasmodium spp. (Figure 1b) (laid out in detail in Sherman’s excellent textbook (Sherman, 1998)). Upon an infectious mosquito bite, motile sporozoites residing in the insect’s salivary glands are injected into the skin and proceed to traverse their way to the bloodstream, where they then will locate and infect the liver. A typical bite might only give rise to a single infected hepatocyte, but this then develops into a massive liver cyst containing up to 10,000 invasive cells, termed merozoites. Upon cyst rupture, these parasites enter the bloodstream and proceed to bind to and then invade RBCs, initiating the intraerythrocytic developmental cycle (IDC). The newly-invaded parasites, called the “ring stages” because of their distinctive circular nucleus, begin a tightly-regulated process of growth, modification of the host cell surface and digestion of most of the host cytosol to generate free amino acids and clear space for the developing parasite. The rings swell into larger, more metabolically active forms called trophozoites which begin the process of accumulating biomass and successively replicating the genome. Towards the end of the process the cell will undergo schizogony, subdividing into upwards of 32 new invasive merozoites, then rupturing the host and releasing these to start the cycle anew. The IDC takes 24, 48 or 72 hours to complete, depending on the species of Plasmodium, and leads to the famed periodic fevers, which are caused by inflammation associated with RBC lysis. In some species, particularly P. falciparum, trophozoite and later stage parasites also express a variety of surface antigens to the host cell membrane, rendering it adherent to blood vessels and resulting in parasite sequestration in the microvasculature. This sequestration process is central to the pathophysiology of certain complicated forms of malaria, and at least in part accounts for the high virulence of P. falciparum. For

5 example, cerebral and placental malaria are a consequence of parasites accumulating near the blood-brain barrier and within the placenta, respectively. These blood stages are haploid, asexual and not transmissible to a mosquito. At some low frequency, parasites exit the IDC and commit to forming either male or female gametocytes. It is unknown what triggers this developmental switch, but it seems to be induced by stress conditions (pH changes, low glucose, heat shock) that might indicate the host is soon to perish (Talman et al., 2004). Mature gametocytes freely circulate in the blood and are ingested during the mosquito blood meal, where male and female cells mate to produce an ookinete, the sole diploid stage of the life cycle. The ookinete traverses the midgut epithelium and forms an oocyst on the gut wall. When the oocyst ruptures, it releases haploid sporozoites that migrate to the salivary glands and the cycle begins anew. Since Sir Ronald Ross’ investigations into malaria transmission in birds (Chernin, 1988), malaria has been studied in model organisms as diverse as chickens (P. gallinaceum), macaque monkeys (P. knowlesi), and lab rodents, using species that in the wild infect bush rats (P. yoelii, P. berghei, P. chabaudi, P. vinckei). A landmark in malariology occurred in 1976, when Trager and Jensen developed the first in vitro continuous culture system for human malaria (Trager and Jensen, 1976). This basically involves incubating parasites with freshly collected RBCs in a rich tissue culture medium supplemented with serum, and maintained at 37° C in an oxygen-depleted atmosphere, such as from a candle jar (currently standardized to 5% CO 2 and 6% O 2 ). This initiated decades of fruitful research into the molecular biology of the parasite that was

6 instrumental in defining its metabolic capabilities, invasion processes, antigenic variation and, crucially, the mechanisms of various antimalarial drugs.

Antimalarial drugs There are numerous antimalarial drugs that have been developed and seen at least limited clinical use, but these fall into a few broad classes (Na-Bangchang and Karbwang, 2009). The 4-aminoquinolines (chloroquine, amodiaquine) and derivatives of quinine (mefloquine), a natural product of the cinchona tree used since antiquity as an antimalarial by the Quechua Indians of Peru, bind free heme or hemozoin crystals produced during the digestion of host cell hemoglobin, preventing their detoxification and resulting in the formation of free radicals (Kaur et al.). Other drugs, such as atovaquone, have been developed to target the parasite’s mitochondrial electron transport chain (Kessl et al., 2007). Antifolates (pyrimethamine, proguanil, the sulfa drugs) inhibit the parasite’s folate metabolism at either the dihydrofolate reductase or dihydropteroate synthase steps (Hyde, 2005). Certain antibiotics that inhibit bacterial ribosomes (doxycycline, clindamycin) also affect translation in the Plasmodium apicoplast, a non- photosynthetic plastid-like organelle of prokaryotic origin (Wiesner et al., 2008). And finally, artemisinin, a natural product of the herb Sweet Wormwood (Artemisia annua) used in ancient Chinese malaria remedies, and its derivatives (artemether, artesunate) are potent antimalarials with an as-yet-undetermined mechanism of action (Price, 2000). The critical caveat underlying these drugs is that in all cases the Plasmodia have evolved protective resistance. Single and multi-drug resistant parasite populations have been reported in the field for all these drugs, including the current frontline antimalarial

7 artemisinin (Noedl et al., 2008). Some drugs, such as chloroquine, have effectively been lost entirely due to resistant mutants (Ginsburg, 2005), and resistance is developing at a pace far outstripping our current drug discovery efforts.

Metabolic pathways in the malaria parasites The central role of metabolic perturbation to the pathology of malaria, the promise of antimetabolites as antimalarial drugs and a basic scientific interest in understanding this fascinating example of highly divergent microbial metabolism has spurred a major and concerted research effort towards elucidating the metabolic network of the Plasmodium parasites. Decades of painstaking efforts have significantly clarified our understanding of the pathways of parasite metabolic flux, and this foundational knowledge, coupled with the advent of advanced analytical technologies, have set the stage for the development of a holistic, network-level model of plasmodial metabolism. Most of this research has focused on the blood stages of parasite development, as this is where Plasmodium infection truly becomes the disease malaria. Our models of plasmodial metabolism now derive primarily from studies of Plasmodium falciparum, the most lethal of the human malaria parasites, but also integrate results from simian, avian and rodent models of malaria that were a major focus of early investigations (Figure 2). While the process of evolving into a parasitic niche seems to have resulted in a 'paring down' of many Plasmodium metabolic pathways, including the wholesale loss of de novo amino acid and purine biosynthesis, pioneering early work suggested that most of the core conserved components of carbon metabolism – glycolysis (Scheibel and Pflaum, 1970; Sherman et al., 1969), the pentose phosphate pathway (Shakespeare et al.,

8 1979), lipid biogenesis (Rock, 1971a), glycosylation (Schmidt-Ullrich et al., 1980; Udeinya and Van Dyke, 1980) and at least some components of citric acid metabolism (Sherman and Ting, 1966; Sherman et al., 1970) – were present in some form. However, efforts to precisely map the metabolic network of the malaria parasite were often stymied Figure 2. An integrated map of carbon flow through the metabolic network of Plasmodium falciparum. Arrows show the proposed direction of flux through the corresponding enzymatic reaction as suggested by experimental evidence; note that this is only intended to indicate net flux, and that the reaction in question might be reversible. Cofactors (ATP, NADH, etc.) are not shown for the sake of clarity. Text in circles represent major biomass components; the circled question mark indicates uncertainty about the existence of the enzyme transaldolase. Abbreviations: Glc, glucose; G6P, glucose-6- phosphate; F6P, fructose-6-phosphate; F1,6BP, fructose-1,6-bisphosphate; DHAP, dihydroxyacetone phosphate; GADP, glyceraldehyde-3-phosphate; 1,3BPG, 1,3- bisphosphoglycerate; 3PG, 3-phosphoglycerate; 2PG, 2-phosphoglycerate; PEP, phosphoenolpyruvate; Pyr, pyruvate; Lac, lactate; Ac-CoA, acetyl-CoA; Ac-R, either acetate or acetyl-CoA; GlycP, glycerol-3-phosphate; Glyc, glycerol; Man6P, mannose-6-phosphate; Man1P, mannose-1-phosphate; GDP-Man, GDP-mannose; GlcN6P, glucosamine-6- phosphate; GlcNAc6P, N-acetyl-glucosamine-6-phosphate; GlcNAc1P, N-acetyl-glucosamine- 1-phosphate; UDP-GlcNAc, UDP-N-acetyl-glucosamine; 6PGL, 6-phosphoglucono-δ-lactone; 6PGa, 6-phosphogluconate; Ru5P, ribulose-5-phosphate; R5P, ribose-5-phosphate; X5P, xylulose-5-phosphate; S7P, sedoheptulose-7-phosphate; E4P, erythrose-4-phosphate; Asp, aspartate; Gln, glutamine; Glu, glutamate; 2OG, 2-oxoglutarate; ICT, isocitrate; Cit, citrate; OAA, oxaloacetate; Mal, malate; Suc-CoA, succinyl-CoA; Suc, succinate; Fum, fumarate; GPI, glycophosphatidylinositol.

9 by difficulties pertaining to the isolation and culturing of parasites, complications arising from the interconnected nature of parasite and host cell metabolism, technical limitations of metabolic tracing using classical methodologies and in some cases marked divergence between pathways in Plasmodium spp. and the model organisms in which they were first elucidated (reviewed in (Homewood and Neame, 1980; Roth, 1990; Scheibel, 1988)). Dedicated efforts by a host of researchers, however, have resolved most of these technical problems and filled in many of the blank spots in the metabolic map. The picture that arises from these studies is of a network that is both more streamlined and more modular than that found in free-living protozoa. This seems to be a consequence of dispensing with the flexibility that a free-living microbe must maintain in order to dynamically regulate its metabolic network to consume any of a wide variety of possible combinations of nutrients. Within the nutrient-rich and homeostatic environment of the blood stages, the parasite appears to consume a defined set of nutrients (glucose, amino acids, free fatty acids, etc.) via several discrete pathways (glycolysis/pentose phosphate pathway, carboxylic acid metabolism, fatty acid elongation and modification) with a low degree of interconnectivity. A fine-grained understanding of plasmodial metabolism will hopefully aid in exploiting this metabolic rigidity when selecting drug targets and designing antimalarials. The broad outline of the metabolic network is likely conserved between the Plasmodium spp. due to the markedly similar life cycles, comparable drug sensitivities and low levels of divergence in metabolic enzymes at the genome level. However, such extrapolations must be made with caution, since the metabolism of the rodent and avian malaria parasites in particular may well be more complicated given their proclivity to

10 invade immature, nucleated erythrocytes (reticulocytes) that are more metabolically complex than the mature erythrocytes favored by simian parasites. Below, I summarize the extent of our knowledge of the major metabolic pathways in malaria parasites.

Glycolysis Based on numerous classical experiments, carbon metabolism of the malaria parasites has been considered largely synonymous with carbohydrate metabolism, principally the Embden-Meyerhof-Parnas (EMP) pathway of glycolysis. It has long been clear from in vitro studies of blood-stage Plasmodium parasites that they are voracious consumers of glucose (blood sugar). The host cell, at least in the human parasites, is the mature erythrocyte (red blood cell, or RBC), whose metabolism has been exhaustively studied and is comparatively simple enough to be simulated by comprehensive kinetic models (Jamshidi et al., 2001; Joshi and Palsson, 1989). The RBC lacks a mitochondrion and therefore is entirely dependent on glucose fermentation. As a nonproliferative cell with modest energetic needs, RBC glucose consumption is relatively low, on the order of 5 μmol glucose / 24 hours / 10 9 RBCs (Jensen et al., 1983). Upon invasion by a Plasmodium parasite, however, the glucose consumption rate is estimated to increase up to 100-fold at the most metabolically active trophozoite and schizont stages (Roth, 1990). In vitro culture experiments using several other sugars have established that, besides glucose, only fructose can support continuous growth (albeit at a reduced rate) (Geary et al., 1985; Woodrow et al., 2000). Most of the glucose consumed (60-70%) by P. falciparum is incompletely oxidized to lactic acid and excreted (Jensen et al., 1983), although the exact percentage varies between different Plasmodium species and the

11 atmospheric culture conditions used. This glucose consumption contrasts with the >90% glucose-to-lactate conversion observed in uninfected RBCs and reflects the increased flux of glucose carbon into biomass (nucleic acids, lipids, glycosylated proteins) required for proliferating parasites. Only a very small fraction of the total glucose is completely oxidized to carbon dioxide, at least in mammalian malaria parasites (Bowman et al., 1961; Scheibel and Miller, 1969; Scheibel and Pflaum, 1970), which has generally been taken to indicate the absence of a functional citric acid cycle contributing to energy generation. This is in keeping with the observation that in vitro cultures of P. falciparum exhibit only minimal oxygen consumption (Krungkrai et al., 1999) (and in fact prefer microaerophilic conditions of ~5% oxygen, being growth-inhibited by normal atmospheric oxygen concentrations (Scheibel et al., 1979).) These results strongly suggest that blood-stage Plasmodium rely primarily upon glucose fermentation for their energetic needs. Accordingly, inhibitors of mitochondrial respiration have only a small effect on parasite ATP pools (Fry et al., 1990). The parasite accommodates this vastly increased need for glucose by expressing at least one essential hexose transporter to the surface of the infected cell (Slavic et al.), increasing the hexose permeability of the erythrocyte membrane (Kirk et al., 1996). Such modifications of the host cell raise the question of precisely how the host glycolytic machinery interacts with that of the parasite. All the enzymes required for a complete EMP pathway are (1) encoded in the parasite genome (Gardner et al., 2002), (2) expressed during the blood stages (Bozdech et al., 2003), and (3) detected in infected cells, in some cases substantially increasing the activity normally observed in RBCs (reviewed in (Sherman, 1998)). Free glucose should be quickly phosphorylated to

12 glucose-6-phosphate by the host cell hexokinase upon entry into the RBC cytosol, rendering it membrane-impermeant and effectively trapping it within the host compartment. Its import into the parasite may depend on dephosphorylation by an acid phosphatase (PFI0880c) that is trafficked to the erythrocyte and cleaves phosphate from a diversity of small molecules (Muller et al.). This nonspecific cleavage of high-energy phosphate bonds might have the effect of draining energy from the host cytosol, despite the fact that the parasite seems to require the host cell to remain “viable” (maintaining its redox state, etc.) until lysis occurs in order to successfully complete its developmental cycle. Malaria parasites may circumvent this difficulty by actively supplying the host with metabolically useful molecules such as ATP (Kanaani and Ginsburg, 1989) and glutathione (Atamna and Ginsburg, 1997). This secreted phosphatase may also help explain the decline observed in infected cells in the levels of 2,3-diphosphoglycerate (Ali et al., 1971; Deslauriers et al., 1982; Mehta et al., 2005), an allosteric regulator of hemoglobin oxygen affinity produced by an enzyme (diphosphoglycerate mutase) present in the erythrocyte but not the parasite (Gardner et al., 2002; Roth et al., 1988).

Other carbon sources Many microbes that prefer sugars as a carbon source maintain the ability to metabolize other compounds such as acetate, pyruvate, ethanol or amino acids depending on their availability. Generating the 5- and 6-carbon sugars necessary for growth alternatively depends on gluconeogenesis, in which the catabolic reactions of glycolysis are essentially reversed through the use of different enzymes at the regulated thermodynamic control steps (reviewed in (Exton, 1972)). However, the complete

13 sequencing of the P. falciparum genome revealed no homologs of fructose bisphosphatase, an enzyme normally required for gluconeogenesis (Gardner et al., 2002). Strangely, the parasite does possess phosphoenolpyruvate carboxykinase (PEPCK, PF13_0234) (Hayward, 2000), an enzyme converting oxaloacetate and ATP to PEP and carbon dioxide that usually functions in supplying citric acid cycle or amino acid-derived carbon to gluconeogenesis. The parasites also encode a PEP carboxylase (PEPC, PF14_0246), which has almost the opposite gene expression profile (peak expression at 18 and 47 hours post invasion for PEPCK and PEPC, respectively) (Bozdech et al., 2003). PEPC has been purified from P. berghei (McDaniel and Siu, 1972), and essentially runs the reverse reaction: PEP and carbon dioxide converted to oxaloacetate and inorganic phosphate. The role of this enzyme remains unclear, but would suggest a regulated carbon fixation step. It is well-established through 14 CO 2 incorporation experiments that all studied Plasmodium spp. possess the ability to fix carbon, although the nature of the end products differs between species (Scheibel, 1988). Interestingly, the up-regulation of PEPCK in P. falciparum gametocytes and zygotes has prompted the hypothesis that there is a switch to gluconeogenic metabolism at these stages (Hayward, 2000). How this can be achieved in the absence of fructose bisphosphatase remains unclear. Several early reports suggested that various Plasmodium spp. possess at least a limited ability to metabolize a number of other substrates, such as glycolytic end- products or tricarboxylic acid cycle intermediates (Ali and Fletcher, 1985; Maier and Coggeshall, 1941; Nagarajan, 1968; Wendel, 1943). However, most of these experiments rely on the stimulation of oxygen uptake as a measure of nutrient consumption, which

14 can be problematic given the unclear relationship between this metric and parasite growth. Possible contaminating sources for this activity have been extensively discussed elsewhere (Homewood and Neame, 1980; Roth, 1990; Scheibel, 1988). Other experiments using simian and avian malaria parasites directly demonstrated the conversion of radioactively labeled lactate and pyruvate to glycolytic intermediates, organic acids and volatiles such as formate and acetate (Ali and Fletcher, 1985; Scheibel and Pflaum, 1970; Sherman et al., 1970). However, I caution that since these experiments used non-physiological glucose-free conditions and/or involved erythrocyte- free parasite preparations, special care should be taken in weighing their relationship to in vivo parasite metabolism, where (1) the parasite resides within a selectively permeable host erythrocyte, (2) serum glucose levels are robustly maintained by the host, and (3) lactate is rapidly excreted. Plasmodium spp. also lack the ability to generate carbohydrate stores in the form of polysaccharides such as glycogen (Bowman et al., 1961; Dasgupta, 1960; Scheibel and Miller, 1969). Thus the blood-stage parasites are obligately dependent on the fermentation of a constant and abundant supply of glucose. This adaptation is sensible given the parasite’s adaptation to a peculiar evolutionary niche: glucose is the most abundant nutrient in human serum and its homeostasis is maintained by a powerful regulatory system. The availability of carbohydrates or other potential carbon sources in the other stages of the life cycle is difficult to study given the general intractability of culturing these stages, but it has been shown that the hemolymph of Anopheles stephensi is rich in glucose and the storage carbohydrate trehalose (Mack et al., 1979) and the

15 essential P. berghei hexose transporter (PB000562.01.0) is expressed throughout development in the mosquito (Slavic et al.).

Pentose phosphate pathway The pentose phosphate pathway (PPP, also known as the hexose monophosphate shunt), a critical conserved pathway in virtually all cells capable of metabolizing carbohydrates as a carbon source, is composed of two interconnected branches. The oxidative arm, in which glucose-6-phosphate is ultimately oxidized to ribose-5- phosphate, generates both the riboses needed for nucleic acid synthesis, as well as NADPH, which is used for redox control and as a cofactor for biosynthetic reactions. The non-oxidative arm, comprising a series of reversible reactions interconverting 3, 4, 5, 6 and 7-carbon sugar phosphates can either recycle ribose-5-phosphate generated by the oxidative arm back into glycolytic intermediates (when NADPH is required but nucleotide synthesis is not) or else converts glycolytic intermediates into ribose-5- phosphate without concomitant NADPH production. It is one of the major metabolic pathways in human erythrocytes, consuming 3-11% of the glucose metabolized under normal conditions (Yunis and Yasmineh, 1969), as it is the only source of the NADPH required to reduce glutathione in response to oxidative stress. Though counterintuitive given its indispensable nature in proliferating cells, the existence of a complete PPP in the malaria parasites was for decades a point of controversy due to difficulties in detecting the necessary enzymes in parasite extracts (Scheibel, 1988) and early reports of very slight increases in pathway activity in RBCs infected with simian, avian and rodent malaria parasites (Bowman et al., 1961; Shakespeare et al., 1979; Sherman et al., 1970).

Full document contains 179 pages
Abstract: The Plasmodium malaria parasites have been a scourge of mankind throughout the entire history of our species. The recent history of malaria control and treatment has been a series of leaps forward that are then partially or entirely reversed due to the emergence of drug resistance. The urgent need for new antimalarials requires a deep and holistic understanding of the parasite's metabolic network, as this is a broadly attractive target for drug development. Moreover, it presents a fascinating example of a metabolism highly diverged from that found in well-studied, free-living model protozoans. Elucidating the systems-level properties of this network requires the application of novel, high-throughput chemimetric technologies to characterize the architecture and dynamics of the Plasmodium metabolome. In this thesis I present the results of my metabolomic investigations into P. falciparum , the most lethal human malaria pathogen. I initially characterized the metabolic alterations induced by P. falciparum infection of human erythrocytes in an in vitro culture system. One major result was the specific depletion of extracellular arginine, which I determined to be due to the parasite's arginase enzyme. Ablation of this enzyme did not impair parasite growth, suggesting that this depletion is not growth-related but may play a role in immunomodulation and potentially cerebral malaria. Subsequent investigations focused on the nature of carbon metabolism during the Plasmodium blood stages; specifically, the architecture of mitochondrial tricarboxylic (TCA) acid metabolism. By tracing the flux of isotope-labeled nutrients I determined that the parasite lacks a "TCA cycle" as such, instead possessing a branched metabolic pathway comprising both oxidative and reductive arms. This significantly clarifies a major open question of parasite biology, and suggests a number of intriguing avenues of research. I also conducted a collaborative investigation into the genetic control of metabolism at a global level. We found an association between a major drug resistance effector and metabolites arising from hemoglobin catabolism. This suggests a functional role for this previously mysterious resistance gene, and has implications for the fitness cost associated with drug resistance.