Isolation and functional characterization of the PfNT1 nucleoside transporter gene from Plasmodium falciparum

Plasmodium falciparum, the causative agent of the most lethal form of human malaria, is incapable of de novo purine synthesis, and thus, purine acquis ition from the host is an indispensable nutritional requirement. This purin e salvage process is initiated by the transport of preformed purines into t he parasite. We have identified a gene encoding a nucleoside transporter ho m P. falciparum, PfNT1, and analyzed its function and expression during int raerythrocytic parasite development. PfNT1 predicts a polypeptide of 422 am ino 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 b ase indicate that PfNT1 is a single copy gene located on chromosome 14, Nor thern analysis of RNA from intraerythrocytic stages of the parasite demonst rates that PfNT1 is expressed throughout the asexual life cycle but is sign ificantly elevated during the early trophozoite stage. Functional expressio n of PfNT1 in Xenopus laevis oocytes significantly increases their ability to take up naturally occurring D-adenosine (K-m = 13.2 mu M) and D-inosine (K-m = 253 mu M) Significantly, PfNT1, unlike the mammalian nucleoside tran sporters, also has the capacity to transport the stereoisomer L-adenosine ( K-m > 500 mu M). Inhibition studies with a battery of purine and pyrimidine nucleosides and bases as well as their analogs indicate that PfNT1 exhibit s a broad substrate specificity for purine and pyrimidine nucleosides. Thes e data provide compelling evidence that PfNT1 encodes a functional purine/p yrimidine nucleoside transporter whose expression is strongly developmental ly regulated in the asexual stages of the P. falciparum life cycle. Moreove r, the unusual ability to transport L-adenosine and the vital contribution of purine transport to parasite survival makes PfNT1 an attractive target f or therapeutic evaluation.