Abstract
Leishmania pyrimidine salvage is replete with opportunities for therapeutic intervention with enzyme inhibitors or antimetabolites. Their uptake into cells depends upon specific transporters; therefore it is essential to establish whether various Leishmania species possess similar pyrimidine transporters capable of drug uptake. Here, we report a comprehensive characterization of pyrimidine transport in L. major and L. mexicana. In both species, two transporters for uridine/adenosine were detected, one of which also transported uracil and the antimetabolites 5-fluoruracil (5-FU) and 5F,2′deoxyuridine (5F,2′dUrd), and was designated uridine-uracil transporter 1 (UUT1); the other transporter mediated uptake of adenosine, uridine, 5F,2′dUrd and thymidine and was designated Nucleoside Transporter 1 (NT1). To verify the reported L. donovani model of two NT1-like genes encoding uridine/adenosine transporters, and an NT2 gene encoding an inosine transporter, we cloned the corresponding L. major and L. mexicana genes, expressing each in T. brucei. Consistent with the L. donovani reports, the NT1-like genes of either species mediated the adenosine-sensitive uptake of [3H]-uridine but not of [3H]-inosine. Conversely, the NT2-like genes mediated uptake of [3H]-inosine but not [3H]-uridine. Among pyrimidine antimetabolites tested, 5-FU and 5F,2′dUrd were the most effective antileishmanials; resistance to both analogs was induced in L. major and L. mexicana. In each case it was found that the resistant cells had lost the transport capacity for the inducing drug. Metabolomics analysis found that the mechanism of action of 5-FU and 5F-2′dUrd was similar in both Leishmania species, with major changes in deoxynucleotide metabolism. We conclude that the pyrimidine salvage system is highly conserved in Leishmania species - essential information for the development of pyrimidine-based chemotherapy.
Highlights
Leishmaniasis is a neglected tropical disease caused by at least 21 species of obligate intracellular parasites of the genusK.J.H
In this study we address some of these issues and find that (1) nucleoside transport is highly similar in multiple Leishmania species; (2) that the substrate binding of the LmajNT1 transporter depends on interactions with the 2-keto and N3 positions of the pyrimidine ring and the 30 and 50 hydroxyl groups of the ribose moiety; and that (3) the antimetabolite 5-fluoro-20-deoxyuridine (5F-20dUrd) is principally converted to 5F-dUMP, by thymidine kinase, causing the inhibition of thymidylate synthase and the consequent disruption of deoxynucleotide metabolism; 5-fluorouracil is first converted to 5F-20dUrd and thence to 5F-dUMP
This is likely because the rate of metabolism of nucleosides and nucleobases is very fast in kinetoplastids and does not become rate limiting for uptake, especially over very short intervals
Summary
Leishmaniasis is a neglected tropical disease caused by at least 21 species of obligate intracellular parasites of the genusK.J.H. 5-FURes 5-Fluorouracil resistant cells 5-F20dURes 5-Fluoro-20-deoxyuridine resistant cells 5-FU 5-Fluorouracil 5F-20dUrd 5-Fluoro-20-deoxyuridine 5F-Urd 5-Fluorouridine 5F-20dCtd 5-Fluoro-20-deoxycytidine many promising therapeutic targets due to the fact that protozoan parasites are unable to synthesize the purine ring de novo and rely solely on salvage mechanisms for these important nutrients (De Koning et al, 2005). Kinetoplastid parasites are known to possess both salvage and biosynthesis routes for pyrimidines (Wilson et al, 2012; Ali et al, 2013a, 2013b). It has recently been demonstrated that both functions are important for infection, neither function is absolutely essential (Wilson et al, 2012; Ali et al, 2013b)
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