Isolation and Functional Characterization of the PfNT1 Nucleoside Transporter Gene from Plasmodium falciparum

Abstract

Plasmodium falciparum, the causative agent of the most lethal form of human malaria, is incapable of de novo purine synthesis, and thus, purine acquisition from the host is an indispensable nutritional requirement. This purine salvage process is initiated by the transport of preformed purines into the parasite. We have identified a gene encoding a nucleoside transporter from P. falciparum, PfNT1, and analyzed its function and expression during intraerythrocytic parasite development. PfNT1 predicts a polypeptide of 422 amino acids with 11 transmembrane domains that is homologous to other members of the equilibrative nucleoside transporter family. Southern analysis and BLAST searching of The Institute for Genomic Research (TIGR) malaria data base indicate that PfNT1 is a single copy gene located on chromosome 14. Northern analysis of RNA from intraerythrocytic stages of the parasite demonstrates that PfNT1 is expressed throughout the asexual life cycle but is significantly elevated during the early trophozoite stage. Functional expression of PfNT1 in Xenopus laevis oocytes significantly increases their ability to take up naturally occurring D-adenosine (K m ‫؍‬ 13.2 M) and D-inosine (K m ‫؍‬ 253 M). Significantly, PfNT1, unlike the mammalian nucleoside transporters, also has the capacity to transport the stereoisomer L-adenosine (K m > 500 M). Inhibition studies with a battery of purine and pyrimidine nucleosides and bases as well as their analogs indicate that PfNT1 exhibits a broad substrate specificity for purine and pyrimidine nucleosides. These data provide compelling evidence that PfNT1 encodes a functional purine/pyrimidine nucleoside transporter whose expression is strongly developmentally regulated in the asexual stages of the P. falciparum life cycle. Moreover, the unusual ability to transport L-adenosine and the vital contribution of purine transport to parasite survival makes PfNT1 an attractive target for therapeutic evaluation.

Transport of nucleosides across the Plasmodium falciparum parasite plasma membrane has characteristics of PfENT1

“…4) is consistent with similar results obtained by Parker et al (2000), and the cross-competition results obtained here for the effect of thymidine, adenosine, inosine and cytidine on the uptake of [ 3 H]-adenosine by PfENT1-expressing oocytes (Fig. 6D) are consistent with those obtained in similar experiments by Carter et al (2000).…”

“…Both K m values are indicative of a 'low-affinity' transport process and we would place no particular significance on the apparent difference between them. By contrast, both values are substantially (≥25-fold) higher than the 13 µM estimate reported earlier by Carter et al (2000) for the transport of adenosine by PfENT1. Whether the discrepancy is due to the single amino acid difference between the form of PfENT1 used both here and in the study by Parker et al (2000), on the one hand, and the form used in the study by Carter et al (2000), on the other, is unclear.…”

“…The P. falciparum genome is now known to have genes encoding four members of the equilibrative nucleoside transporter (ENT) family, all of which are expressed during the intraerythrocytic stage of the life cycle (Martin et al ., 2005). In two independent studies, one of these proteins, designated either PfNT1 (Carter et al ., 2000) or PfENT1 ( P. falciparum equilibrative nucleoside transporter 1) (Parker et al ., 2000), was cloned and expressed in Xenopus oocytes and shown to transport a range of nucleosides. There are significant differences between the results of the two studies with regard to the reported characteristics of the transporter.…”

Functional characterization of a H+/nucleoside co‐transporter (CaCNT) from Candida albicans, a fungal member of the concentrative nucleoside transporter (CNT) family of membrane proteins

“…A number of ENT family members have recently been identified and functionally characterized from parasitic protozoa, including TgAT from Toxoplasma gondii (Chiang et al, 1999), the P1-and P2-type transporters TbNT2 and TbAT1 from Trypanosoma brucei (Maser et al, 1999;Sanchez et al, 1999), LdNT1.1 from Leishmania donovani (Vasudevan et al, 1998) and PfENT1 from Plasmodium falciparum (Carter et al, 2000;Parker et al, 2000). In contrast to their mammalian counterparts, at least some of the protozoan ENT family members appear to function as active transporters, catalysing the symport of nucleosides with protons (de Koning and Diallinas, 2000;Carter et al, 2000). PfENT1, like human and rat ENT2, also functions as a nucleobase transporter (Parker et al, 2000).…”

Purine Salvage Pathways in the Apicomplexan Parasite Toxoplasma gondii

“…Measurement of adenine uptake and conversion by APRT is described under "Materials and Methods." (ENTs) have previously been reported for both T. gondii (31) and P. falciparum (32). TgAT1 transports adenosine, inosine, and guanosine nucleosides (31), and there is biochemical data for additional adenosine, inosine, and nucleobase transporters (33,34).…”