CLONING OF LEISHMANIA NUCLEOSIDE TRANSPORTER GENES BY RESCUE OF A TRANSPORT-DEFICIENT MUTANT

All parasitic protozoa studied to date are incapable of purine biosynt hesis and must therefore salvage purine nucleobases or nucleosides fro m their hosts. This salvage process is initiated by purine transporter s on the parasite cell surface, We have used a mutant line (TUBA5) of Leishmania donovani that is deficient in adenosine/pyrimidine nucleosi de transport activity (LdNT1) to clone genes encoding these nucleoside transporters by functional rescue. Two such genes, LdNT1.1 and LdNT1. 2, have been sequenced and shown to encode deduced polypeptides with s ignificant sequence identity to the human facilitative nucleoside tran sporter hENT1. Hydrophobicity analysis of the LdNT1.1 and LdNT1.2 prot eins predicted 11 transmembrane domains, Transfection of the adenosine /pyrimidine nucleoside transport-deficient TUBAS parasites with vector s containing the LdNT1.1 and LdNT1.2 genes confers sensitivity to the cytotoxic adenosine analog tubercidin and concurrently restores the ab ility of this mutant line to take up [H-3]adenosine and [H-3]uridine. Moreover, expression of the LdNT1.2 ORF in Xenopus oocytes significant ly increases their ability to take up [H-3]adenosine, confirming that this single protein is sufficient to mediate nucleoside transport. The se results establish genetically and biochemically that both LdNT1 gen es encode functional adenosine/pyrimidine nucleoside transporters.