Cloning of a novel inosine-guanosine transporter gene from Leishmania donovani by functional rescue of a transport-deficient mutant

Purine transport is an indispensable nutritional function for protozoan par asites, since they are incapable of purine biosynthesis and must, therefore , acquire purines from the host milieu, Exploiting a mutant cell line (FBD5 ) of Leishmania donovani deficient in inosine and guanosine transport activ ity, the gene encoding this transporter (LdNT2) has been cloned by function al rescue of the mutant phenotype. LdNT2 encodes a polypeptide of 499 amino acids that shows substantial homology to other members of the equilibrativ e nucleoside transporter family. Molecular analysis revealed that LdNT2 is present as a single gene copy within the leishmanial genome and encodes a s ingle transcript of 3 kilobase pairs. Transfection of FBD5 parasites with L dNT2 reestablished their ability to take up inosine and guanosine with a co ncurrent restoration of sensitivity to the inosine analog formycin B, Kinet ic analyses reveal that LdNT2 is highly specific for inosine (K-m = 0.3 mu M) and guanosine (K-m = 1.7 mu M) and does not recognize other naturally oc curring nucleosides, Expression of LdNT2 cRNA in Xenopus oocytes significan tly augmented their ability to take up inosine and guanosine, establishing that LdNT2 by itself suffices to mediate nucleoside transport. These result s authenticate genetically and biochemically that LdNT2 is a novel nucleosi de transporter with an unusual and strict specificity for inosine and guano sine.