Cloning of Leishmania nucleoside transporter genes by rescue of a transport-deficient mutant (purine salvageydrug resistanceyleishmaniasis)

All parasitic protozoa studied to date are incapable of purine biosynthesis and must therefore salvage purine nucleobases or nucleosides from their hosts. This salvage process is initiated by purine transporters on the parasite cell surface. We have used a mutant line (TUBA5) of Leishmania donovani that is deficient in adenosineypyrimidine nucleoside 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 significant sequence identity to the human facilitative nucleoside transporter hENT1. Hydrophobicity analysis of the LdNT1.1 and LdNT1.2 proteins predicted 11 transmembrane domains. Transfection of the adenosineypyrimidine nucleoside transport-deficient TUBA5 parasites with vectors containing the LdNT1.1 and LdNT1.2 genes confers sensitivity to the cytotoxic adenosine analog tubercidin and concurrently restores the ability of this mutant line to take up [3H]adenosine and [3H]uridine. Moreover, expression of the LdNT1.2 ORF in Xenopus oocytes significantly increases their ability to take up [3H]adenosine, confirming that this single protein is sufficient to mediate nucleoside transport. These results establish genetically and biochemically that both LdNT1 genes encode functional adenosineypyrimidine nucleoside transporters. Parasitic protozoa of the genus Leishmania are the etiological agents of leishmaniasis, a disease that affects an estimated 12 million people worldwide (1) and ranges from the disfiguring cutaneous form to fatal visceral leishmaniasis (2). Because current empirically identified drugs suffer from many deficiencies, including toxicity and resistance, it is important to identify unique biochemical targets that could be exploited for rational development of improved therapies. Perhaps the most striking metabolic discrepancy between parasites and their hosts is the purine pathway. Whereas most mammalian cells synthesize purines de novo, all parasitic protozoa studied to date are unable to synthesize purines (3) and consequently must rely on purine acquisition from their hosts for survival and growth. The first step in this salvage pathway involves the transport of these substrates across the parasite plasma membrane. Moreover, these purine transporters initiate the uptake of certain pyrazolopyrimidine analogs of hypoxanthine and inosine that are toxic to both Leishmania and Trypanosoma (4). These pyrazolopyrimidines, such as allopurinol, allopurinol riboside, and formycin B, are subsequently metabolized to the nucleotide level by the parasite metabolic machinery and incorporated into RNA, metabolic transformations that do not occur in mammalian cells (4). Both the essential nutritional function of these transporters and their roles in mediating the toxicities of well-characterized antiparasitic agents provide compelling rationale to study these membrane permeases at the molecular level. Biochemical and genetic studies have established that Leishmania donovani parasites express two distinct nucleoside transporters with nonoverlapping substrate specificities (5). One transporter mediates the uptake of adenosine and pyrimidine nucleosides and also transports tubercidin, a cytotoxic analog of adenosine, whereas the other transporter allows membrane permeation of guanosine, inosine, and formycin B (5). Parasites deficient in either or both transport activities have been isolated by mutagenesis with N-methyl-N9-nitro-N-nitrosoguanidine followed by selection in tubercidin or formycin B (6). The availability of these null mutants provided a functional strategy for cloning genes encoding each of these nucleoside permeases. In the present study, we have transfected the adenosineypyrimidine nucleoside transport-deficient TUBA5 cell line with a cosmid library containing inserts of L. donovani genomic DNA (7) and screened individual transfectants for restoration of tubercidin sensitivity. Several tubercidinsensitive transfectants were isolated and shown to contain distinct cosmids with overlapping inserts. Analysis of one of these cosmids has led to the identification of two ORFs encoding 491 amino acids, designated LdNT1.1 and LdNT1.2, that mediate restoration of tubercidin sensitivity and [3H]adenosine and [3H]uridine transport capabilities to TUBA5 cells. Furthermore, expression of this LdNT1.2 ORF in Xenopus oocytes stimulates the uptake of [3H]adenosine in this heterologous system. These results establish that the LdNT1.1 and LdNT1.2 proteins are functional nucleoside transporters. MATERIALS AND METHODS Growth of Parasites and Nucleic Acid Preparation. The DI700 (8) and TUBA5 (5) strains of L. donovani were cultured at 26°C in DMEM-L (8) containing 10% fetal calf serum. Isolation of genomic DNA and RNA and preparation of Southern and Northern blots were performed as described (9). Transfection and Screening for Tubercidin Sensitivity. To screen for cosmids containing the adenosineypyrimidine nucleoside transporter genes, 30 separate transfections were performed as described (10) on '4 3 107 TUBA5 promasThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1998 by The National Academy of Sciences 0027-8424y98y959873-6$2.00y0 PNAS is available online at www.pnas.org. This paper was submitted directly (Track II) to the Proceedings office. Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. TAF065311 and AF041473). ‡Present address: Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205. ¶To whom reprint requests should be addressed. e-mail: landfear@ ohsu.edu.