Genetic analysis of nucleoside transport in Leishmania donovani.

Abstract

Genetic dissection of nucleoside transport in Leishmiania dotnov alni indicates that the insect vector form of these parasites possesses two biochemically distinct nucleoside transport systems. The first transports inosine, guanosine, and formycin B, and the second transports pyrimidine nucleosides and the adenosine analogs, formycin A and tubercidin. Adenosine is transported by both systems. A mutant, FBD5, isolated by virtue of its resistance to growth inhibition by 5 ,uM formycin B, cannot efficiently transport inosine, guanosine, or formycin B. This cell line is also cross-resistant to growth inhibition by a spectrum of cytotoxic analogs of inosine and guanosine. A second parasite mutant, TUBA5, isolated for its resistance to 20 puM tubercidin, cannot take up from the culture medium radiolabeled tubercidin, formycin A, uridine, cytidine, or thymidine. Both the FBD5 and the TUBA5 cell lines have about a 50% reduced capacity to take up adenosine, indicating that adenosine is transported by both systems. A tubercidin-resistant clonal derivative of FBD5, FBD5-TUB, has acquired the combined biochemical phenotype of each single mutant. The wild-type and mutant cell lines transport purine bases and uracil with equal efficiency. Mutational analysis of the relative growth sensitivities to cytotoxic nucleoside analogs and the selective capacities to take up exogenous radiolabeled nucleosides from the culture medium have enabled us to define genetically the multiplicity and substrate specificities of the nucleoside transport systems in L. donovani promastigotes.The parasitic protozoa are the causative agents for a plethora of infectious diseases. Most of the major metabolic pathways in parasites are thought to be similar to those found in mammalian systems with one major exception. All the parasitic protozoa examined to date are incapable of de novo synthesis of the purine ring and are therefore auxotrophic for purines (14). Cultured forms of parasites are dependent on an exogenous source of purines for survival and growth, whereas intracellular parasites scavenge purines from their hosts. To meet their purine requirement, the parasites have evolved a series of unique purine salvage enzymes and cell surface functions for which no mammalian counterpart exists. Because nucleoside or nucleobase analogs are selectively metabolized by this unique purine salvage system, they are growth inhibitory and cytotoxic to parasitic protozoa but not to mammalian cells. For instance, allopurinol (4-hydroxypyrazolo[3,4,-d]pyrimnidine) (1, 15), allopurinol riboside (1, 19), thiopurinol (4-thiopyrazolo[3,4-d] pyrimidine) (15), thiopurinol riboside (15), and formycin B (7-hydroxy-3-,-D-ribofuranosyl-pyrazolo[4,3-d]pyrimidine)

Cloning of a Novel Inosine-Guanosine Transporter Gene fromLeishmania donovani by Functional Rescue of a Transport-deficient Mutant

“…Genetic and biochemical investigations have demonstrated that L. donovani promastigotes express two nucleoside transport activities of nonoverlapping ligand specificity (5). The first, LdNT1, transports adenosine, pyrimidine nucleosides, and the cytotoxic adenosine analog tubercidin, and the second, LdNT2, recognizes inosine, guanosine, and the cytotoxic inosine isomer formycin B (FoB) 1 (5,6).…”

“…The first, LdNT1, transports adenosine, pyrimidine nucleosides, and the cytotoxic adenosine analog tubercidin, and the second, LdNT2, recognizes inosine, guanosine, and the cytotoxic inosine isomer formycin B (FoB) 1 (5,6). Mutant L. donovani clones deficient in LdNT1 or LdNT2 activity have been isolated by virtue of their resistance to either tubercidin or FoB, respectively (5).…”

“…The first, LdNT1, transports adenosine, pyrimidine nucleosides, and the cytotoxic adenosine analog tubercidin, and the second, LdNT2, recognizes inosine, guanosine, and the cytotoxic inosine isomer formycin B (FoB) 1 (5,6). Mutant L. donovani clones deficient in LdNT1 or LdNT2 activity have been isolated by virtue of their resistance to either tubercidin or FoB, respectively (5). The availability of these transport-deficient mutants and the ability to transfect Leishmania with cosmid expression libraries (7) provided an avenue for cloning the genes encoding nucleoside transporter proteins by selecting for functional recovery of the wild type drug sensitivity phenotype and, thus, nucleoside transport capability.…”

TOR-induced resistance to toxic adenosine analogs in Leishmania brought about by the internalization and degradation of the adenosine permease

“…The toxicity of these purine based compounds has been found to be reduced in cells with high levels of TOR [9, 14 and data not shown]. Tubercidin is transported by the adenosine permease, formycin B by the guanosine permease and sinefungin by the S-adenosylmethionine transporter [4,7,9,14,28]. It is not know how puromycin enters Leishmania.…”

“…To satisfy this requirement, Leishmania possess multiple purine permeases and the necessary enzymes to convert these purines to nucleotides [3][4][5][6][7][8][9][10][11].…”