2Malaria is caused by protozoan parasites of the genus Plasmodium. Five species of Plasmodium cause infection in humans, with the majority of lethal cases caused by Plasmodium falciparum. This species is responsible for more than 500 million clinical cases of malaria each year (59). In the past 2 decades, efforts to combat this disease have been met with the emergence of widespread resistance to most of the commonly used antimalarial drugs (68). The poor efficacy of current drugs and the lack of a promising vaccine have resulted in alarming increases in the rates of malaria morbidity and mortality worldwide, with the major toll felt in the developing world.P. falciparum is transmitted to humans via the bite of an infected female anopheline mosquito. In humans, the parasite undergoes one cycle of asexual multiplication in hepatocytes, followed by several cycles of infection and multiplication in red blood cells. Whereas the hepatocytic stage is asymptomatic, the erythrocytic stage is accompanied by the destruction of the host erythrocytes, resulting in anemia and, in the absence of treatment, death. Extensive efforts are presently under way to develop a vaccine, and advances in our understanding of the biology of the parasite and its metabolic and nutritional needs offer new routes for chemotherapy. In this regard, purine metabolism holds significant promise as a target for drug development.It has long been recognized that protozoan parasites, including Plasmodium spp., are unable to synthesize purine rings de novo (4). Consistent with this observation, the sequencing of protozoan genomes has failed to uncover any genes encoding enzymes involved in the biosynthesis of purine nucleosides or nucleobases (12). Protozoan parasites rely instead on salvage of purines from the host. The strategies used in acquiring purines vary significantly among parasite genera. This review will focus primarily on the purine salvage pathways present in the Plasmodium parasite. Recent reviews have examined the metabolic pathways for purine salvage in other protozoa (11,17,29).Initial studies on purine and pyrimidine synthesis in Plasmodium parasites were performed on erythrocytic stages of the rodent malaria species P. berghei (7,8,65), the macaque monkey malaria species P. knowlesi (46), and the avian malaria parasite P. lophurae (61,66 (46). Following on from these findings, Gutteridge and Trigg found that, in P. knowlesi, all radiolabeled purines were significantly incorporated into nucleic acids while none of the pyrimidines tested were (28). These early biochemical studies demonstrated that Plasmodium parasites lack the ability to metabolize exogenous pyrimidines and instead are entirely dependent on de novo synthesis. Conversely, Plasmodium parasites are entirely reliant upon the salvage of extracellular purines (4) and are capable of metabolizing a wide variety of exogenous purine nucleobases and nucleosides.The continuous culture of P. falciparum in serum-free media is dependent upon the supply of exogenous purines (1, 43), sug...

As the malaria parasite, Plasmodium falciparum, grows within its host erythrocyte it induces an increase in the permeability of the erythrocyte membrane to a range of low-molecular-mass solutes, including Na+ and K+ (ref. 1). This results in a progressive increase in the concentration of Na+ in the erythrocyte cytosol. The parasite cytosol has a relatively low Na+ concentration and there is therefore a large inward Na+ gradient across the parasite plasma membrane. Here we show that the parasite exploits the Na+ electrochemical gradient to energize the uptake of inorganic phosphate (P(i)), an essential nutrient. P(i) was taken up into the intracellular parasite by a Na+-dependent transporter, with a stoichiometry of 2Na+:1P(i) and with an apparent preference for the monovalent over the divalent form of P(i). A P(i) transporter (PfPiT) belonging to the PiT family was cloned from the parasite and localized to the parasite surface. Expression of PfPiT in Xenopus oocytes resulted in Na+-dependent P(i) uptake with characteristics similar to those observed for P(i) uptake in the parasite. This study provides new insight into the significance of the malaria-parasite-induced alteration of the ionic composition of its host cell.

SummaryLike all parasitic protozoa, the human malaria parasite Plasmodium falciparum lacks the enzymes required for de novo synthesis of purines and it is therefore reliant upon the salvage of these compounds from the external environment. P. falciparum equilibrative nucleoside transporter 1 (PfENT1) is a nucleoside transporter that has been localized to the plasma membrane of the intraerythrocytic form of the parasite. In this study we have characterized the transport of purine and pyrimidine nucleosides across the plasma membrane of 'isolated' trophozoite-stage P. falciparum parasites and compared the transport characteristics of the parasite with those of PfENT1 expressed in Xenopus oocytes. The transport of nucleosides into the parasite: (i) was, in the case of adenosine, inosine and thymidine, very fast, equilibrating within a few seconds; (ii) was of low affinity [ K m (adenosine) = 1.45 ± 0.25 mM; K m (thymidine) = 1.11 ± 0.09 mM]; and (iii) showed 'crosscompetition' for adenosine, inosine and thymidine, but not cytidine. The kinetic characteristics of nucleoside transport in intact parasites matched very closely those of PfENT1 expressed in Xenopus oocytes [ K m (adenosine) = 1.86 ± 0.28 mM; K m (thymidine) = 1.33 ± 0.17 mM]. Furthermore, PfENT1 transported adenosine, inosine and thymidine, with a cross-competition profile the same as that seen for isolated parasites. The data are consistent with PfENT1 serving as a major route for the uptake of nucleosides across the parasite plasma membrane.

SummaryRepeated immunizations with whole Plasmodium blood stage parasites and concomitant drug cure of infection confer protective immunity against parasite challenge in mice, monkeys and humans. Moreover, it was recently shown that infections with genetically modified rodent malaria blood stage parasites conferred sterile protection against lethal blood stage challenge. However, in these models vaccination resulted in high parasitemias and, in consequence, carries risk of vaccine-induced pathology and death. Herein, we generated a novel, completely blood stageattenuated P. yoelii rodent malaria strain by targeted deletion of parasite nucleoside transporter 1 (NT1). Immunization of inbred and outbred mouse strains with a single low dose of Pynt1 -blood stages did not induce any patent infections and conferred complete sterile protection against lethal heterologous blood stage and sporozoite challenges. Partial protection was observed against lethal challenges with another parasite species, P. berghei. Importantly, subcutaneous immunization with Pynt1 -conferred sterile protection against lethal blood stage challenges. We show that cellular and humoral immune responses are both essential for sterile protection. The study demonstrates that genetic manipulation provides a platform for the designed, complete attenuation of malaria parasite blood stages and suggests testing the safety and efficacy of P. falciparum NT1 knockout strains in humans.