Cloning of Leishmania nucleoside transporter genes by rescue of a transport-deficient mutant

Abstract

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 adenosine͞pyrimi-dine nucleoside transport activity (LdNT1) to clone genes encoding these nucleoside transporters by functional rescue. 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-NЈ-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 adenosine͞pyrimidine 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...

Identification of the Intracellular Gate for a Member of the Equilibrative Nucleoside Transporter (ENT) Family

“…The adenosine transport measurements (1 M, 2.5 Ci/ml, 40s) depicted in the figures were performed at least three times (n Ն 3), and values within a given experiment were the mean of triplicate determinations using 100-l aliquots by the previously described oil-stop method (33). For each mutant, two independent clones were isolated and tested in parallel to confirm each result (data not shown).…”

Functional characterization of a H+/nucleoside co‐transporter (CaCNT) from Candida albicans, a fungal member of the concentrative nucleoside transporter (CNT) family of membrane proteins

“…A number of ENT family members have recently been identified and functionally characterized from parasitic protozoa, including TgAT from Toxoplasma gondii (Chiang et al, 1999), the P1-and P2-type transporters TbNT2 and TbAT1 from Trypanosoma brucei (Maser et al, 1999;Sanchez et al, 1999), LdNT1.1 from Leishmania donovani (Vasudevan et al, 1998) and PfENT1 from Plasmodium falciparum (Carter et al, 2000;Parker et al, 2000). In contrast to their mammalian counterparts, at least some of the protozoan ENT family members appear to function as active transporters, catalysing the symport of nucleosides with protons (de Koning and Diallinas, 2000;Carter et al, 2000).…”

Second-site Suppression of a Nonfunctional Mutation within the Leishmania donovani Inosine-Guanosine Transporter

“…LdNT1 transports adenosine and pyrimidine nucleosides, whereas LdNT2 is selective for inosine and guanosine (2,3). Essentially nothing is known about the permeation mechanism of the ENTs, and only a few amino acids that govern ligand recognition have been identified.…”