Purine transport in plasmodium falciparum

[摘要] Purine transport in malaria parasites has become an important potential drug target because the malaria parasite is unable to synthesis purine de novo and has to salvage it from the host milieu through transporter(s). Knowledge of the number, selectivity and kinetic parameters of the transporters expressed by the parasite would therefore facilitate a rational purine-based chemotherapy of malaria.The rates of transport of radiolabeled purines into Plasmodium falciparum-infected erythrocytes were measured using classical uptake techniques. We found that the uptake of purine into intact parasite-infected erythrocytes was mediated by the endogenous human erythrocyte nucleoside and nucleobase transporters (hENT1 and hFNT1) rather than the parasite-induced New Permeation Pathways (NPP). The overall rate of purine uptake was observed to be approximately doubled in parasite-infected cells compared to uninfected cells and the rate of adenosine uptake was seen to be faster than hypoxanthine in parasite-infected cells. It was observed that transport of hypoxanthine and adenine through the hFNT1 was unexpectedly inhibited by furosemide. This inhibition by furosemide of [3H]-hypoxanthine and [3H]-adenine uptake was not through inhibition of the NPP, as it was observed equally in infected and uninfected hRBC.To gain further understanding of purine transport in the malaria parasite, uptake was measured in saponin-freed P. falciparum trophozoites. Treatment of parasite infected erythrocytes with saponin render both the erythrocyte membrane and the parasitophorous vacuole membrane permeable to solutes, allowing transport to be measured across the parasite membrane. The data obtained from the uptake assays were analysed using Michaelis Menten plots to obtain the kinetic properties (Km and Vmax) of the parasite’s purine transporters. Three separate and novel transport activities were identified in saponin-freed P. falciparum trophozoites: (i) a high affinity hypoxanthine transporter with a secondary capacity for purine nucleosides, named PfNT1, (ii) a separate high affinity transporter for uptake of adenine (named PfADET1) and (iii) a low affinity/high capacity adenine carrier (denoted PfADET2). Additionally, the presence in the parasite of a low affinity adenosine transporter designated PfLAAT, with kinetic properties similar to that previously reported by other research groups was also confirmed. At room temperature, uptake of hypoxanthine through PfNT1 was observed to be 12-fold more efficient than adenosine. The gene encoding the high affinity hypoxanthine/nucleoside transporter PfNT1 was disrupted by a single crossover event and the parasite clones obtained were designated 3D7DPfNT1. The characteristics of purine uptake into parasites lacking a functional PfNT1 gene were then compared to uptake in wild-type parasites. The high affinity uptake of hypoxanthine or adenosine was completely abolished in 3D7DPfNT1, whereas uptake of 25µM [3H]-adenosine (through the low affinity transporter, PfLAAT) was similar in both 3D7DPfNT1 and wild-type P. falciparum parasites. Adenine transport was observed to increase in 3D7DPfNT1, presumably to partly compensate for the loss of the high affinity hypoxanthine transporter. An improved microfluorometric technique for determining parasite’s sensitivity to antimalarial drugs was developed and the method was used to evaluate antiplasmodial potential of a small number of purine analogues. A ‘hypoxanthine-like’ purine analogue, JA-32, was found to inhibit the uptake of hypoxanthine by PfNT1, supporting the potential of purine transporters as targets for antimalarial drug development. The findings from this study provide evidence that purine salvage in P. falciparum is predominantly based on the highly efficient uptake of hypoxanthine by PfNT1 and secondarily on the high capacity uptake of nucleosides by a lower affinity carrier. The exact contribution to the overall purine salvage in parasite by the two transporters of adenine is not clear. Indeed, the issue of whether Plasmodium can utilise adenine as a purine source is controversial. These findings re-emphasise the importance of purine transporters as targets for novel antimalarial drugs and open the gateway for a systematic purine-based chemotherapy of malaria.

[发布机构] University:University of Glasgow;Department:Institute of Infection Immunity and Inflammation

[关键词] Plasmodium falciparum, malaria, nucleoside transport, equilibrative nucleoside transporter family, purine salvage, PfNT1, drug target, gene disruption, purine analogue